Ornithine transcarbamylase from Salmonella typhimurium: purification, subunit composition, kinetic analysis, and immunological cross-reactivity. Ornithine transcarbamylase (OTCase) was purified to hemogeneity from a derepressed strain of Salmonella typhimurium. The optimal pH for enzyme activity is 8.0. The molecular weight of the enzyme was calculated to be 116,000, based on measurements of the sedimentation coefficient by sucrose gradient ultracentrifugation and the Stokes radius by gel filtration. Polyacrylamide gel electrophoresis of cross-linked OTCase in the presence of sodium dodecyl sulfate showed that the enzyme is composed of three identical subunits. The molecular weight of the monomer was determined to be 39,000. Steady-state kinetics indicate that the reaction mechanism is sequential. The limiting Michealis constants for carbamylphosphate and ornithine were determined to be 0.06 and 0.2 mM, respectively. The dissociation constant for carbamylphosphate was 0.02 mM. Product and dead-end inhibition patterns are consistent with an ordered Bi Bi mechanism, in which carbamylphosphate is the first substrate added and phosphate is the last product released. OTCase activity was inhibited by arginine, but relatively high concentrations were required for significant inhibition. The inhibition by arginine might be physiologically significant in the regulation of carbamlphosphate utilization; a single carbamylphosphate synthetase is responsible for the synthesis of carbamylphosphate for both arginine and pyrimidines in S. typhimurium and the inhibition by argine might serve to divert carbamlphosphate to the synthesis of pyrimidines when arginine is present at high concentrations. The crossreaction of OTCases from different microorganisms with purified antibodies raised against the homogeneous OTCase from S. typhimurium was investigated. The results of immunotitration and immunodiffusion experiments revealed a high degree of identity between the enzymes form S. typhimurium and Esherichia coli B and W. In these three cases, a single gen (argl) encodes OTCase. Wild-type E. coli K-12 and strain 3000 X 111, which carry two OTCase genes (argI, argF), also revealed similar cross-reactivity, supporting the hypothesis that argF is the product of a relatively recent duplication. The activity of OTCase from Bacillus subtilis was partially inhibited by antibodies against the enzyme from S. typhimurium, indicating unusual conservation of primary structure among widely different taxonomic groups. OTCase from Saccharomyces cerevisiae, whose molecular weight and primary structure are similar to those of the enzyme from S. typhimurium, was without detectable cross-reactivity.


Properties of an inducible extracellular neuraminidase from an Arthrobacter isolate. The elective isolation of a soil microorganism, tentatively assigned to the genus Arthrobacter, which produced an extracellular neuraminidase is described. The secretion of neuraminidase from washed cells in minimal medium required the presence of sialo-containing glycoproteins, whereas free N-acetyl-neuraminic asid of N-acetylmannosamine were poor inducers. No enzyme could be dected in the induction fitrated of cells, in the absence of inducer or in the culture filtrate of cells grown in a complete medium. The routine enzyme inducer was a hot-water extract of "edible bird's nest." Mild acid treatment (0.05 N H2SO4) of this extract increased enzyme activity two--to threefold and the specific activity about eightfold. Neuraminidase induction with acid-treated bird's nest was manifested at a linear rate for 6 h without increase in cell number. No other anticipated glycohydrolase or protease activities were foud. The amount of enzyme located within the cells was barely detectable as compared to that found in the induction filtrate. Experiments with chloramphenicol or chlortetracycline indicate that de novo protein synthesis was required for neuraminidase production and that this exoenzyme was not released from a preformed pool. Neuraminidase from this source has an apparent molecular weight of 87,000, a pH optimum of 5 to 6, and an apparent Km of 2.08 mg/ml for collocalia mucoid and 3.3 X 10(-3) M for N-acetylneuraminlactose and is insensitive both to Ca2+ ions and ethylenediaminetetraacetic acid. Preliminary studies indicate that the enzyme can hydrolyze alpha-2,3-, alpha-2,6-, or alph-2-8-N-acetylneuraminylglycosidic linkages. From total activity data and purification criteria, it would appear that this isolate can produce about 5 mg of enzyme per liter of induction medium.


Motility of Mycoplasma pneumoniae.

Motility of Mycoplasma pneumoniae. Cell of Mycoplasma pneumoniae FH gliding on a glass surface in liquid medium were examined by microscopic observation and quantitatively by microcinematography (30 frames per min). Comparisons were made only within the individual experiments. The cells moved in an irregular pattern with numerous narrow bends and circles. They never changed their leading end. The average speed (without pauses) was relatively constant between o.2 and 0.5 mum/s. The maximum speed was about 1.5 to 2.0 mum/s. The movements were interrupted by resting periods  of different lengths and frequency. Temperature, viscosity, pH, and the presence of yeast extract in the medium influenced the motility significantly; changes in glucose, calcium ions, and serum content were less effective. The movements were affected by iodoacetate, p-mercuribenzoate, and mitomycin C at inhibitory or subinhibitory concentrations. Sodium fluoride, sodium cyanide, dinitrophenol, chloramphenicol, puromycin, cholchicin, and cytochalasin B at minimal inhibitory concentrations did not affect motility. The movements were effectively inhibited by anti-M. pneumoniae antiserum. Studies with absorbed antiserum suggested that the surface  components involved in motility are heat labile. The gliding of M. pneumoniae cells required an intact energy metabolism and the proteins involved seemed to have a low turnover.


Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus. A medium  was developed to obtain maximum yields of extracellular amylase from Bacteroides amylophilus 70. Crude enzyme preparation, obtained by ammonium sulfate precipitation of cell-free broth, contained six amylolytic isoenzymes that were detected by isoelectric focusing and polyacrylamide gel electrophoresis. One of these amylases was purified by diethylaminoethyl-Sephadex A-50 ion-exchange chromatography and Sephadex G-200 gel filtration techniques. Some properties of the purified extracellular alpha-amylase were: optimum pH, 6.3; optimum temperature, 43 degrees C: PH stability range, 5.8 to 7.5; isoelectric point, pH 4.6; molecular weight, 92,000 (by sodium dodecyl sulfatedisc gel electrophoresis); and sugars causing inhibition, cyclomaltoheptaose, cyclomaltohexaose, and alpha-d-phenylglucoside. In addition, Ca2+ and Co2+ were strong activators,and Hg2+ was a strong inhibitior; all other cations were slightly stimulatory. Dialysis against 0.01 M ethylenediaminetetraacetic acid caused a 58% loss of activity that was restored to 92% of the original by the addition of 0.04 M Ca2+. The enzyme affected a blue-value-reducing-value curve characteristic of alpha-type amylases. The relative rates of hydrolysis of amylose, soluble starch, amylopectin, and dextrin were 100, 97, 92, and 60%, respectively; Michaelis constants for these substrates were 18.2, 18.7, 18.2, and 16.7 mumol of d-glucosidic bond/liter, respectively. The enzyme degraded maize (corn) starch granules to some extent and had relatively little activity on potato starch granules.


Formation of glutamine from [13n]ammonia, [13n]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica. A method is described for the isolation of metabolically active heterocysts from Anabaena cylindrica. These isolated heterocysts accounted for up to 34% of the acetylene-reducing activity of whole filaments and had a specific activity of up to 1,560 nmol of C2H4 formed per mg of heterocyst chlorphyll per min. Activity of glutamine synthetase was coupled to activity of nitrogenase in isolated heterocysts as shown by acetylene-inhibitable formation of [13N]NH3 and of amidelabeled [13N]glutamine form [13N]N2. A method is also described for the production of 6-mCi amounts of [13N]NH3. Isolated heterocysts formed [13N]glutamine from [13N]NH3 and glutamate, and [14C]glutamine from NH3 and [14C]glutamate, in the presence of magnesium adenosine 5'-triphosphate. Methionine sulfoximine strongly inhibited these syntheses. Glutamate synthase is, after nitrogenase and glutamine synthetase, the third sequential enzyme involved in the assimilation of N2 by intact filaments. However, the kinetics of solubilization of the activity of glutamate synthase during cavitation of suspensions of A. cylindrica indicated that very little, if any, of the activity of that enzyme was located in heterocysts. Concordantly, isolated heterocysts failed to form substantial amounts of radioactive glutamate from either [13N]glutamine or alph-[14C]ketoglutarate in the presence of other substrates and cofactors of the glutamate synthase reaction. However, they formed [14C]glutamate rapidly from alpha-[14C]ketoglutarate by aminotransferase reactions, with various amino acids as the nitrogen donor. The implication of these findings with regard to the identities of the substances moving between heterocysts and vegetative cells are discussed.


Measurement of the pH of frozen buffer solutions by using pH indicators. A method was established to estimate the pH change of several buffers solutions on freezing by using a combination of pH indicators. Among more than 30 buffers solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220 degrees K; the results were tabulated. Glycerol was found to suppress the pH changes because of its "salt buffer" effect.


Mathematical analysis of ligand-induced monomerization and dimerization in the monomer dimer equilibrium of proteins. Application to D-amino acid oxidase. We have mathematically analyzed ligand-induced monomerization and dimerization in a protein monomer-dimer equilibrium system, in which the monomer has one and the dimer two binding sites. These dimer sites have the same binding constants for the first ligand but may cooperatively interact when one of them is occupied by a ligand molecule. In this system, the apparent dimerization constant and the apparent molecular weight are functions of free ligand concentration, and depend on the intrinsic binding constants of the ligand molecule to the monomer and the dimer. The behavior of these functions is classified into 17 cases according to the values of the three intrinsic binding constants, and some calculated examples are shown graphically for selected parameters. The theory was also applied to D-amino acid oxidase [EC 1.4.3.3], a flavoprotein, and the pH dependence of the apparent dimerization constant and the apparent molecular weight in the presence of ligand, p-aminobenzoate, were studied theoretically using parameters obtained in our previous experiments (5).


Studies on the microsomal electron-transport system of anaerobically grown yeast. IV. Purification and characterization of NADH-cytochrome b5 reductase. The presence of NADH-cytochrome b5 reductase [EC 1.6.2.2] in microsomes from anaerobically grown yeast was confirmed by its isolation and purification. The purified preparation of the reductase showed an apparent molecular weight of 27,000 daltons. The reductase appeared to contain loosely-bound FAD as a prosthetic group. The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5. The reductase, however, could not reduce cytochrome c. Michaelis constants of the reductase for NADH and calf liver cytochrome b5 were 6.3 and 1.5 micron M, respectively, and optimal pH for cytochrome b5 reduction was 5.6. Although some differences exist between the properties of NADH-cytochrome b5 reductase from yeast and from mammalia, the results indicate a functional similarity of the present enzyme to mammalian NADH-cytochrome b5 reductase in the microsomal electron-transport system.


Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].


I-protein, a new regulatory protein from vertebrate skeletal muscle. III. Function. I-protein inhibited theMg-activated ATPase [EC 3.6.1.3] activity of actinomyosin by approximately 50% at low ionic strength. Concomitantly, the onset of superprecipitation was retarded. I-protein was found to bind to myosin, but not to F-actin. The inhibitory action of I-protein occurred only in the absence of Ca ions in the troponintropomyosin-actin myosin system. Addition of Ca ions abolished the effect. Thus, it is very likely that I-protein prevents unnecessary hydrolysis of ATP in the relaxed state of muscle.


Kinetic studies on the ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase. Evidence for the K.S.T. mechanism with two enzyme-ATP complexes and two phosphorylated intermediates of high-energy type. The kinetic properties of the [3H]ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3.6.1,3] were investigated, using NaI-treated microsomes from bovine brain, and the following results were obtained. 1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45 micronM free Mg2+, 100 mMNaCl, 80 micronM ATP, and 160 micronM ADP at pH 6.5 and 4-5 degrees. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction parameters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 micronM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM. The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants Km, for both were 43 micronM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 micronM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 micronM ATP. 2. The N+-dependent [3H]ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+-concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H]ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration. From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP + ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP (formula: see text) produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step (formula: see text), determined by a method previously reported by us.


Spontaneous deamidation of a protein antibiotic, neocarzinostatin, at weakly acidic pH. Conversion to a homologous inactive preneocarzinostatin due to change of asparagine 83 to aspartic acid 83 accompanied by conformational and biological alterations. The amide content of neocarzinostatin (NCS), an antitumor protein, has been determined by analysing asparagine and glutamine in the Pronase-aminopeptidase M digests of tetra-S-carboxymethyl-NCS and carboxyl-modified NCS (modified with a water-soluble carbodiimide and [14C]glycine methyl ester). Preneocarzinostatin (PRE) was separated and purified from a crude NCS preparation by CM-cellulose column chromatography. PRE was found to contain one mole less asparagine than NCS, and asparagine was deamidated to aspartic acid in PRE. A time-dependent conversion of NCS to PRE at pH 3.2 at 4 degrees or in 0.1 M acetic acid at 26 degrees was studied in two ways; first, by quantitative determination of NCS and PRE by CM-cellulose column chromatography and second, by following the release of free NH3 during dialysis in an air-tight container. Within experimental error, PRE was indistinguishable from NCS in amino acid content after acid hydrolysis, as well as in apparent molecular weight as determined by SDS-disc gel electrophoresis (10% acrylamide), and N- and C-terminal amino acid residues. Both NCS and PRE shared a common antigenicity as determined by Ouchterlony's agar diffusion method. Only a slight difference between the two in electrophoresis on a cellulose acetate membrane and on a peptide map of the tryptic digest was demonstrated. PRE, however, was completely devoid of biological activity. In addition to the chromatographic difference, a conformational difference was observed by CD spectroscopy, namely, an apparently looser structure of PRE was indicated by the shallowness of the trough in the 240-265 nm region. This interpretation was supported by the finding that digestions by Pronase were more extensive with PRE than with NCS. These results indicate an important role of the single asparagine residue (Asn 83) of NCS in the biological activity, which is evidently governed by the conformation.


Kallikrein inhibitors in rat plasma.

Kallikrein inhibitors in rat plasma. The kallikrein inhibitor contents of human and animal plasma were determined with glandular kallikreins [EC 3.4.21.8]. One ml of plasma could inactivate 20-700 kallikrein units (KU). Rat plasma was the most potent and inactivated 230-700 KU. However, no enzyme capable of inactivating kallikrein could be found in this plasma. Two fractions which inhibited hog pancreatic kallikrein, a fraction corresponding to alpha2-macroglobulin and a fraction which was eluted prior to albumin, were separated from rat plasma by Sephadex G-200 gel filtration. The former inhibitor could inhibit hog pancreatic kallikrein action on Nalpha-benzoyl-L-arginine ethyl ester (BAEE) as well as in the dog vasodilator assay. The other inhibitor was partially purified from rat plasma. One mg of the preparation inhibited 67 KU and the hydrolysis of 5.8 micronmoles/min of BAEE by hog pancreatic kallikrein [EC 3.4.21.8]. The inhibitor also inhibited other glandular and plasma kallikreins, trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1], etc. The optimal pH of the inhibitor was 7.5-8. The inhibitor was unstable below pH 5, and was destroyed by heating at temperature above 60 degrees. The isoelectric point of the inhibitor was determined by Ampholine focusing to be 4.4, and its molecular weight was estimated to be 73,000 by Sephadex G-100 and G-150 filtrations. Several experimental results suggested that this inhibitor differed from alpha1-antitrypsin.


Studies on the catalytic action of poly-alpha-amino acids. VII. Stereospecificity in the enzyme-like hydrolysis of benzoyl-L-(D)-arginine-p-nitroanilides by copoly (Cys, Glu). The substrate specificity in the hydrolysis of L-, DL-, and D-BAPA (benzoylarginine-p-nitro-anilide) by copoly (L-Cys, L-Glu) and copoly (D-Cys, D-Glu) was studied, and enzyme-like stereospecific hydrolyses by poly-alpha-amino acids were identified for the first time. The L-type copolymer hydrolyzed L-BAPA faster than D-BAPA and the rates (v) of BAPA hydrolyses by L-type copolymer were found to be in the order vL greater than vDL greater than vD. On the other hand, the D-type copolymer hydrolysed D-BAPA faster than L-BAPA and the rates of BAPA hydrolyses by D-type copolymer were in the order vD greater than vDL greater than vL. In all cases, the reaction followed Michaelis-Menten kinetics when the substrate concentration was corrected, and the optimum conditions of the reaction were pH 6.0 and 40 degrees. The activity appeared after a certain amount of BAPA had combined with the polymer. D- and L-substrates combine competitively with the polymer and the different rates of hydrolysis are presumably due to the different substrate configurations in relation to the conformation of the active site in the polymer. The polymer shows activity near the range of random coil conformation, where some alpha-helical conformation is still present. Only some of the cysteine residues in the copolymer are involved in the hydrolytic activity.


DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography. A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the alpha-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65 degrees was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.


Kinetics of conformational change of troponin-C induced by proton binding or removal in the absence of calcium ions. The kinetics of the conformational change of troponin-C induced by binding or removal of protons was studied by a stopped-flow pH-jump spectrofluorometric method. In the pH-down experiment (to investigate the kinetics of conformational change from the deprotonated state to the protonated state), a single first-order reaction with a rate constant and amplitude of 1.75-2.4 sec-1 and around 10% respectively, was observed. On the other hand, two first-order reactions with rate constants of 0.84-1.6 sec-1 and 0.08-0.4 sec-1 were observed in the pH-up experiment, the total amplitudes of these reactions being around 10-20%. The pH dependences of the rate constants of these reactions were analyzed in terms of a three-species mechanism.


Self-assembly of myosin in vitro caused by rapid dilution. Effects of hydrogen ion, potassium chloride, and protein concentrations. The in vitro assembly of rabbit skeletal myosin was studied by flow birefringence. Filaments were obtained from a solution of myosin in 0.5 M KCl by rapid dilution to lower ionic strength. In most cases, the filament length as determined from extinction angle measurements increased or decreased gradually for about 1 h after dilution, depending on pH, KCl concentration and the previous history. The filament length (l) immediately after dilution also showed a marked dependence on pH, KCl concentration and protein concentration (c) at the moment of assembly. The general characteristics obtained from our limited study (0.04-6.0 mg/ml) show three distinctive modes of effect of the protein concentration on the filament length: d logl/d log c is positive (0.1-1) at small c, negative (from -1 to -0.2) at intermediate c, and zero or slightly positive (0.0-0.3) at large c. Lowering of the KCl concentration (75-250 mM) as well as increase of the hydrogen ion concentration (pH 6-8) influenced the filament length in qualitatively the same manner as increase of the protein concentration. A model of the assembly reaction of myosin in which the polarity of filaments is crucial was constructed and shown to give qualitatively the experimental dependence of the filament length on the protein concentration.


Temperature induced transition of the pH-activity curve of heavy meromyosin adenosine triphosphatase and inosine triphosphatase. The pH-activity curve of heavy meromyosin ATPase [EC 3.6.1.3] was measured at various temperatures. The pH-activity curve at higher temperatures showed a maximum at low pH and a minimum at pH 7 to 8 as has been already reported. At lower temperatures it was sigmoidal in shape, similar to a simple dissociation curve of pKa 6 to 7. The pH-activity curve at intermediate temperatures appeared to be inbetween the two extreme shapes. These changes in pH-activity curve with temperature were found to be common in the presence of divalent cations such as Mg2+, Mn2+, and Ca2+. The ATPase mechanism may be identical in the presence of any divalent cation, and the rate determining step revealing the steady state rate alters by changing the temperature. The transition temperatures estimated at pH 8 were 10 degrees, 8 degrees, and about 5 degrees in the presence of MnCl2, CaCl2, and MgCl2, respectively. The difference in the temperature coefficients above and below the transition temperature was most distinct in the presence of MnCl2, and vague in the presence of CaCl2. A similar change of pH-activity curve with temperature was found with heavy meromyosin ITPase in the presence of MgCl2.


Temperature dependence of the decay of the UV absorption difference spectrum of heavy meromyosin induced by adenosine triphosphate and inosine triphosphate. The UV absorption difference spectrum of heavy meromyosin induced by ATP was measured at various temperatures. At higher temperatures, the difference spectrum formed rapidly after adding ATP and continued steadily during the steady state which we have called the ATP-form of difference spectrum. At lower temperatures, the ATP-form of difference spectrum decayed into the other form before the steady state was attained. This was identical to the difference spectrum obtained by adding ADP and has been called the ADP-form of difference spectrum. At intermediate temperatures, biphasic decay was observed. The results indicate that the dominant intermediate at the steady state is altered from the one showing the ATP-form of difference spectrum at higher temperatures to that showing the ADP-form at lower temperatures. The population of the two intermediates depends on the temperature between the two extremes. This temperature-induced transition was observed in the presence of any divalent cation such as Mg2+, Mn2+, or Ca2+. A similar transition was observed with the difference spectrum induced by ITP in the presence of MgCl2. The pH dependence of the single early decay of the ATP-induced difference spectrum was measured in the presence of MnCl2 at 1 degree. The apparent rate constant of the decay showed a biphasic pH dependence, having the same shape as the pH activity curve of ATPase [EC 3.6.1.3] observed at higher temperatures. The rate determining step for the steady state ATPase at higher temperatures is thought to be the step of changing from the intermediate complex showing the ATP-form of difference spectrum to that showing the ADP-form. This is inconsistent with our previous mechanism (Yazawa, M. et al. (1973) J. Biochem. 74, 1107-1117). The rate determining step at lower temperatures was assigned as a step of ADP dissociation.


Acceptor activity of subcellular membranes for two terminal sugars, galactose and sialic acid. The acceptor activities of subcellular membrane preparations for the terminal sugars, galactose and sialic acid, were compared using a Golgi fraction purified from rat liver as an exogenous emzymes source for sugar transfer. Data are presented which strongly suggest that completion of carbohydrate chains of membrane glycoproteins and glycolipids occurs in the Golgi apparatus. Significant differences of acceptability of galactose and sialic acid were found between plasma membranes of rat liver and hepatoma cells (AH-130), indicating "incompleteness" of sugar chains in the latter.


A novel and simple colorimetric assay for human serum lipase. A new and simple colorimetric method for human serum lipase [EC 3.1.1.3] assay has been developed, using 2,3-dimercaptopropan-1-ol tributyroate as a substrate, 5,5'-dithiobis(2-nitro-benzoic acid) as a chromogenic reagent, phenylmethylsulfonyl fluoride as an inhibitor of serum esterases, and sodium dodecylsulfate as a lipase activator. The method requires only 50 micron1X2 of serum sample and a reaction time of less than 30 min. The method is reproducible and sensitive enough to measure low levels of lipase activity in normal and abnormal sera. The gel filtration of serum samples on a Sephadex G-200 column gave one peak of lipase activity, when measured by the present method, and the molecular weight of the enzyme was identical with that of lipase of human pancreatic origin, confirming the specificity of this new method for the serum lipase.


Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus. Nucleoside triphosphate phosphohydrolase [EC 3.6.1.15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2.7.7.6] using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.


Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus. The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C) greater than poly(A) greater than poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.


The reactions of phenylglyoxal and related reagents with amino acids. 1. The reaction of phenylglyoxal (PGO), glyoxal (GO), and methylglyoxal (MGO) with amino acids were investigated at mild pH values at 25 degrees. These aldehydes reacted most rapidly with arginine and the rate of reaction increased with increasing pH values. Histidine, cystine, glycine, tryptophan, asparagine, glutamine, and lysine reacted with these aldehydes at significant but various rates, depending on the pH and the kind of the reagent used. The reactions with these amino acids seemed to involve both the alpha-amino groups and the side chain groups, and no significant reaction appeared to occur with the side chain alone except with those of arginine, lysine, and cysteine. These reagents were similarly reactive with the guanidinium group of arginine, but PGO appeared to be much less reactive with the epsilone-amino group of lysine than MGO and GO. The other ordinary amino acids were very much less reactive or did not react at all with these reagents, with the exception of cysteine. 2. Di-PGO-L-arginine was prepared from Nalpha-benzyloxycarbonyl-L-arginine, and di-PGO-methylguanidine from methylguanidine, and the stoichiometry of the reaction of two PGO molecules with one guanidino group was confirmed. A glyoxal derivative of L-arginine (GO-arginine) was prepared by reaction of glyoxal with arginine. GO-arginine was fairly unstable, especially at higher pH values. A similar derivative (MGO-arginine) was also found to be formed by reaction of MGO with L-arginine, and was similarly unstable. These derivatives, however, did not regenerate arginine upon acid hydrolysis.


The structure and function of acid proteases. VII. Distribution and some properties of acid proteases in monkey tissues. 1. The distribution of acid protease activity in various tissues of Japanese monkey (Macaca fuscata fuscata) was investigated with hemoglobin as a substrate at pH 3.0. The activity per protein weight in crude extracts was highest in spleen and lung, and decreased in the order: spleen, lung greater than kidney, testis greater than brain greater than liver, placenta greater than thyroid gland, muscle. The activity in crude muscle extract was about one-tenth those of spleen and lung. The activity per wet tissue weight was in roughly the same order except for a lower activity per wet weight of brain. 2. Upon chromatography of each crude extract on a Sephadex G-100 column, one major activity peak was eluted at a position corresponding to a molecular weight of about 41,000. This enzyme activity is attributed to cathepsin D [EC 3.4.23.5]. In addition, a minor activity peak was eluted in the case of spleen, lung and kidney at the break-through position, corresponding to a molecular weight of more than 100,000. This activity peak is presumably due to cathepsin E. These acid protease activities were, in most cases, strongly inhibited by pepstatin, an acid protease-specific peptide inhibitor. 3. The distribution of acid protease activity was investigated in the brain of crab-eating monkey (Macaca fascicularis). The activity was fairly evenly distributed among several regions of the brain, and its distribution was similar to those of other acid hydrolases, especially N-acetyl-beta-D-glucosaminidase [EC 3.2.1.30] and acid phosphatase [EC 3.1.3.2], which are marker enzymes of lysosomes.


Studies on the heterotropic interaction of hemoglobin. I. Mass spectrometric method for determination of the pKa of the beta-146 histidine residue in human hemoglobin. A mass spectrometric method was developed to determine pH-dependent hydrogen-deuterium exchange at the C-2 position of the imidazole ring of histidine, after converting the amino acid to the methylthiohydantoin derivative. The amount of deuterium exchange in N-acetyl-histidine estimated by the present method was confirmed to be in good agreement with that determined by NMR spectrometry. N-Acetylhistidine was deuterated at various pH's. From the amount of deuterium exchange, a pseudo-first order rate constant (kpsi) was calculated. A pKa value of 7.2 for the amino acid was obtained from the relation between kpsi and pH. This method was applied to estimate the pKa value of beta-146 histidine in human hemoglobin. Human hemoglobin deuterated at various pH's was digested with carboxypeptidase A [EC 3.4.12.2] to release the beta-146 histidine. The amount of deuterium exchange in the isolated histidine was determined to obtain kpsi. From these measurements pKa values of 7.0 for the histidine in oxyhemoglobin and of 8.2 for that in deoxyhemoglobin were found at 36.5 degrees, respectively.


Glutamate dehydrogenase from Bacillus subtilis PCI 219. I. Purification and properties. Bacillus subtilis PCI 219 has a single glutamate dehydrogenase (GDH) [EC 1.4.1.3] with dual coenzyme specificity [for NAD(H) and NADP(H)]. The enzyme was purified 800-fold from crude extracts of B. subtilis from the post-exponential phase of growth and showed one significant protein band on gel electrophoresis. This band was determined, by activity staining, to have all the GDH nucleotide specificities. Its molecular weight was estimated to be 250,000+/-20,000 by gel filtration, and 270,000+/-30,000 by zone centrifugation in a sucrose density gradient. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that GDH has a subunit size of about 57,000. The pI of GDH was found to bepH 3.7 by isoelectric focusing. GDH exhibited nonlinear kinetics in the reduction of NAD+, and in the reverse direction, the substrate, NH4+, was strongly inhibitory at high concentrations. Purine nucleotides did not affect the activity. The oxidative demination of glutamate was significantly inhibited by the metabolites oxaloacetate and citrate, which acted as allosteric effectors of this enzyme,inhibiting the reaction in one direction. The pH optimum of each of the activities of GDH and the stability of GDH are also reported.


Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans. An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mg2+.


Purification of hyaluronidase from human placenta. Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.


Reaction of chlorocruorin with heme iron ligands and carbonyl reagents. Chlorocruorin was purified from Potamilla leptochaeta and the spectral properties of its derivatives wwere investigated. Ferri- or ferrochlorocruorin did not exhibits a ferrihemochrome or ferrohemochrome spectrum, respectively. Oxy- and carbonmonoxy-ferrochlorocruorin did show ferrohemochrome-type spectra. Ferrihemochromes were formed, however, when oxy-or ferrichlorocruorin was treated with 0.02-0.05% SDS, and they were transformed to ferrohemochromes by reduction with sodium dithionite. Ferrihemochrome formation was also brought about by increasing the pH of a ferrichlorocruorin solution to 9, or by liganding of extrinsic imidazole or cyanide to the ferric pigment. Therefore, it is apparent that at least one of the coordination positions on the heme iron in ferri-and ferrochlorocruorin is vacant or occupied by a weak-field ligand. Titration studies of ferrichlorocruorin with imidazole indicated that this supposedly vacant coordination position was occupied first by the imidazole, and that the intrinsic ligand of protein orgin was replaced finally at higher concentrations. The extrinsic ligands in the cyanide and imidazole complexes of ferrichlorocruorin were excluded from their coordination positions as the protein moiety assumed conformations inherent to the reduced pigment. Spectral analyses indicated that the intrinsic ligand is an imidazole moiety of a histidyl residue. When chlorocruorin was intact, carbonyl reagents such as cyanide and sodium bisulfite did not add to the formyl group of chlorocruoreheme. When the protein conformation was perturbed by SDS, addition to ferrichlorocruorin occurred appreciably. This addition was accelerated if the heme iron coordination position had been occupied by strong field ligands,and was reversed to some extent as the chlorocruorin complexes were reduced.


Molecular architecture of cytochrome oxidase and its transition on treatment with alkali or sodium dodecyl sulfate. In dimeric cytochrome oxidase [EC 1.9.3.1], one of the two heme a molecules of one monomeric unit has been proposed to be converted by the other unit, thus becoming latent in terms of catalytic functions (1). As the dimer was split into two monomers by treatment with alkali or sodium dodecyl sulfate (SDS), it was shown that the intensity of circular dichroism (CD) in the Soret region due to heme a decreased, probably reflecting release of the strain on the latent heme. On the other hand, the profile of magnetic circular dichroism (MCD) was nearly unchanged during this conversion, except for a weakening of the signal due to deprotonation of the heme during the alkali treatment. When the monomer was further dissociated into constituent subunits in strong alkali or at high concentrations of SDS, the CD spectrum disappeared almost completely, indicating loss of the asymmetric interactions of the chromophoric heme a with its immediate environments, consisting of the subunit assembly. The MCD pattern also suffered a small change as the dissociation proceeded, and a specific pattern appeared as the Schiff base was finally formed. The Schiff base formation of cytochrome oxidase in strong alkali proceeded in two steps whether the heme iron was in the oxidized or reduced state. As a consequence of the initial rapid reaction, the enzyme was suggested to have been disintegrated into constituent subunits with heme a being attached nonspecifically to either one, and structural characteristics dependent on the redox state were completely lost. The Arrehenius plot for this rapid change showed a break, indicating a transition in the structure of the cytochrome oxidase assembly, although no such phenomenon was observed during the slow reaction. Activation parameters in the rapid and slow reactions for the oxidized and reduced oxidase are given. Based on these findings, as well as other considerations, a molecular architecture of this enzyme is proposed; the role of heme a in anchoring four 14,000-dalton polypeptides into the minimal functional unit catalyzing the aerobic oxidation of ferrocytochrome c is emphasized.


Reconstitution of vesicles capable of energy transformation from phospholipids and adenosine triphosphatase of a thermophilic bacterium. 1. A stable ATPase [EC 3.6.1.3] complex (TF0-F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation,measured as ATP-dependent enhancement of fluorescence of 8-anilinonoaphthalene-1-sulfonate. 2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0-F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5 mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30 degrees with soybean P-lipids and 45 degree with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 3. The vesicles obtained under these conditions were mainly 100-200 nm in diameter, covered with 9.5 nm spheres, and had a bouyant density of 1.06 in sucrose andan internal volume of about 0.5 mul per mg of P-lipids.


Proteases from purulent sputum. Purification and properties of the elastase and chymotrypsin-like enzymes. A procedure is described for the purification of the elastase and chymotrypsin-like enzymes from purulent sputum. This procedure permitted the isolation of 132 mg and 120 mg of the elastase and chymotrypsin-like enzymes, respectively, from 230 g of purulent sputum. The elastase enzymes consist of a family of five isozymes, and at least three isozymes comprise the chymotrypsin-like enzyme system. The elastases proved to be immunologically identical with the corresponding enzyme of human leukocytes. These enzymes were characterized with respect to molecular weight, amino acid and carbohydrate composition, several kinetic parameters, and inhibition by various synthetic and natural inhibitors. The properties so found were comparable to those which had been previously reported by others for the elastase and chymotrypsin-like enzymes isolated directly from leukocytic granules.


Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains. Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.


Asparagine transaminase from rat liver.

Asparagine transaminase from rat liver. Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.


Quantitative determination of carbamino adducts of alpha and beta chains in human adult hemoglobin in presence and absence of carbon monoxide and 2,3-diphosphoglycerate. The principal component of normal adult human hemoglobin was equilibrated under various conditions with 13CO2. Quantitative analysis of the carbamino resonance intensities over the pH range of 6.5 to 9.0 shows that the effects of conversion from the deoxy to the liganded state in reducing the carbamino adduct formation occur predominantly at Val-1beta. Analysis of the pH dependence of carbamino formation at constant total carbonates yields values of pKz and pKc for Val-1beta and Val-1alpha in the deoxy and liganded conditions. In contrast to the Val-1beta as the allosteric site for CO2, the Val-1alpha site is shown to be primarily an alkaline Bohr group. 2,3-Diphosphoglycerate is shown to reduce substantially the Val-1beta carbamino resonance intensity in deoxyhemoglobin. Evidence for 2,3-diphosphoglycerate effects in carbon monoxide hemoglobin at both Val-1alpha and Val-1beta sites is presented. Enhanced carbamino formation in carbon monoxide hemoglobin at Val-1beta is observed at pH values less than 7.8. Finally, chemical exchange analysis of the spectra shows the release rate of the deoxy Val-1alpha carbamino adduct to be greater than that for deoxy Val-1beta. At pH 7.47 k-1obs,beta congruent to 1.0 and k-1obs, alpha congruent to 11.0 s-1.


Distribution of sulfated mucopolysaccharides in invertebrates. The sulfated mucopolysaccharide composition of 22 species of invertebrates belonging to the phyla Arthropoda, Mollusca, Annelida, Tunicata, Echinodermata, Coelenterata, and Porifera was analyzed. It is shown that all the species contain variable amounts of one or more types of sulfated mocopolysaccharides, most of which similar to the ones found in vertebrates. It is shown also that each species has a characteristic composition, differing from each other regarding the relative amount and type of chondroitin sulfates A, B, and C, heparitin sulfate, and heparin. The possible biological role of the sulfated mucopolysaccharides in cell recognition or aggregation or both is discussed in view of the present findings.


Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes. Nuclear magnetic relaxation methods were used to investigate the interaction of the inhibitor succinate with aspartate transcarbamylase from Escherichia coli. Over the pH range 7 to 9, the dissociation constant for succinate remains less than the inhibitor concentration used for most of this work (0.05 M). As a result, the enzyme predominantly exists in a single "gross" conformational state. Succinate binding to this enzyme state (generally known as the R form) parallels the behavior seen previously with the isolated catalytic subunit (Beard, C. B., and Schmidt, P.G. (1973) Biochemistry 12, 2255-2264). The pH and temperature dependence of succinate proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl phosphate, is interpreted in terms of a binding mechanism involving three forms of the enzyme, differing by their states of protonation. The least protonated form of the enzyme does not interact with succinate, the singly protonated species binds succinate to form a rapidly dissociating complex, and the doubly protonated species undergoes a conformational isomerization upon succinate binding, yielding a slow exchange complex. Relaxation data provide sufficient information to determine pKa values of 7.2 and 8.9 for two ionizing groups, as well as the dissociation constant for succinate in the fast exchange complex, Kd =1.6 X 10(-2) M. Rate constants for the forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1, respectively, indicate a significantly slower reverse rate from that obtained in the earlier NMR study of the isolated catalytic subunit. In experiments where the succinate concentration was varied, the relaxation rates showed sigmoidal binding of that ligand to the fast exchange complex above pH 9.1, (a) indicating cooperative binding of succinate, and (b) suggesting that above pH 9.1, the system cannot be characterized by a single dissociation constant, ionization constant, or relaxation effect. CTP and ATP were tested for their ability to affect succinate binding to the fast exchange complex. Heterotropic interactions were observed for CTP but not for ATP. Addition of low concentrations of the transition state analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl phosphate-succinate complex sharply decreased the relaxation rate, indicating that the measurements are sensitive only to succinate bound specifically to the active site.


Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. R-Plasmids from a number of trimethoprim-resistant Escherichia coli and Citrobacter sp. were studied after transfer to E. coli K12 hosts. Each was found to specify a dihydrofolate reductase which was resistant to trimethoprim and Methotrexate, and which could be completely separated from the host chromosomal enzyme by gel filtration. Two distinct types of R-plasmid dihydrofolate reductases were identified. Type I enzymes, typified by the R483 enzyme previously described (Sköld, O., and Widh, A. (1974) J. Biol. Chem. 249, 4324-4325), are synthesized in amounts severalfold higher than the chromosomal enzyme. The 50% inhibitory concentrations (I50) of trimethoprim, Methotrexate, and aminopterin are increased several thousandfold over the corresponding values for the chromosomal enzyme. Type II R-plasmid dihydrofolate reductases are synthesized in about the same amount, or less, as the chromosomal enzyme, but are practically several hundredfold higher than those for the type I enzymes. Both types of R-plasmid dihydrofolate reductase showed little difference from the chromosomal enzyme in the binding of dihydrofolate, NADPH, folic acid, and 2,4-diaminopyrimidine.


Porcine A blood group-specific N-acetylgalactosaminyltransferase. I. Purification from porcine submaxillary glands. The membrane-bound N-acetylgalactosaminyltransferase from porcine submaxillary glands which provides A blood group specificity to mucin has been purified 38,000-fold by affinity chromatography on UDP-hesanolamine-agarose in aqueous Triton X-100. Design of a suitable purification procedure was developed by assessing the strength of interaction between enzyme and affinity adsorbent using batch desorption. The pure transferase has an apparent molecular weight of 100,000 as judged by zonal centrifugation and by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of a reducing agent. The reduced and carboxymethylated protein has an apparent molecular weight of 46,000 and 57,000 as judged by sedimentation equilibrium and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting that the native enzyme contains two subunits. It is a glycoprotein with a specific activity of 30 micronmol/min/mg of enzyme, which is 55,000 times that reported for the same enzyme isolated from human serum.


Porcine A blood group-specific N-acetylgalactosaminyltransferase. Porcine A blood group-specific N-acetylgalactosaminyl-transferase required either Mn2+, Cd2+, or Zn2+ for activity and 2'-O-alpha-fucosylgalactosides as acceptor substrates. The presence of detergent stabilizes the enzyme but is not essential for catalysis. To obtain information about the kinetic mechanism of the transferase reaction, initial rate parameters have been determined using 2'-fucosyllactose or A--mucin as acceptors, and Mn2+ or Cd2+ as cosubstrates. 2'-Fucosyllactose is a competitive inhibitor with respect to A--mucin and a noncompetitive inhibitor with respect to UDP-N-acetylgalactosamine. UDP inhibits noncompetively with respect to acceptor; thus UDP-N-acetylgalactosamine or acceptor can bind to the transferase via an equilibrium random pathway. The transferase converts human O blood type erythrocytes of A blood types. After exhaustive glycosylation, 3 X 10(6) N-acetylgalactosaminyl residues were incorporated per cell. Gel electrophoretic analysis of the labeled erythrocyte membranes indicates that glycoproteins with apparents molecular weights from 30,000 to 100,000 have been glycosylated; glycolipids account for only 15% of the labeled material, although pure H-glycolipid is a good acceptor. The transferase, with its strict acceptor specificity, can thus be used as a tool to study the biosynthesis and function of glycolipids and glycoproteins.


Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity. Reaction of phospholipase A2 (Naja naja naja) with p-bromophenacyl bromidine leads to almost complete loss of enzymatic activity. The rate of inactivation is pH-dependent with pKa equals 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidine/mol of enzyme as judged by amino acid analysis and incorporation studies with 14C-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase A2, by acylation of the two amino groups, a tyrosine, and 0.5 mol of histidine/mol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidine/mol of enzyme, can be further inactivated by treatment with p-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the 14C-labeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A2 with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.


Functional characterization of anion transport system isolated from human erythrocyte membranes. The anion transport system of human red blood cells was isolated in vesicles containing the original lipids of the membrane and predominantly the 95,000-dalton polypeptides (Band 3) associated with intralipid particles. The vesicles display various characteristic properties of anion permeation closely resembling those of the native system. The properties include energy of activation, pH dependence, anion sleectivity, sensitivity to specific inhibitors, and exchange and net rates of sulfate transport. Based on these and other criteria, the functional properties of isolated vesicles could be equated with those of the intact cell system. Direct support for the involvement of 95,000-dalton polypeptides in permeation functions is provided.


Phosphoglycolate phosphatase in human erythrocytes. Human erythrocytes were found to contain an enzyme capable of dephosphorylating phosphoglycolate. The rates of hydrolysis of 14 other metabolites by the enzyme preparation were less than 6% of the rate with phosphoglycolate. The pH optimum is in the range of 6 to 7 and the Km for phosphoglycolate is 0.76 mM. The molecular weight appears to be about 79,000. Such an activity has previously been reported only for plant cells.


The reactivity of alpha-chymotrypsin immobilized on radiation-grafted hydrogel surfaces. The enzymatic activity of alpha-chymotrypsin (CT), immobilized on hydrogel-coated polymer film supports, has been investigated. The support was prepared by radiation-graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAAc) on silicone rubber films. The enzyme was covalently coupled to the carboxylic group of MAAc via the N-hydroxysuccinimide (NHS) ester active intermediate. Increasing MAAc contents of the hydrogel resulted in increased attachment of CT. The integrity of the CT active site after attachment was assessed by an active site titration with diisopropyl fluorophosphate (DFP). As the MAAc content of the hydrogel was increased, an increasing fraction of the attached CT retained its activity to DFP. A greater fraction of CT was active towards DFP when adsorbed than when coupled. The rates of hydrolysis of some synthetic model substrates by the immobilized CT were also measured. The negative charge on the hydrogel had a large effect on the rates of these hydrolyses. The pH optimum for the hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) by immobilized CT was higher than that of free CT. Increasing MAAc content of the hydrogel resulted in larger shifts in the pH optimum. The maximum rates of ATEE hydroylsis per mg CT declined sharply with increasing MAAc content of the hydrogel. This is probably related to the increasing repulsive force between the ATEE (negatively charged above congruent to pH 9.5) and the hydrogel with increasing MAAc content. The activity of immobilized CT to ATEE is small compared to that of free CT, partly due to this charge effect. Conversely, the rate of hydrolysis of BAEE, a positively charged substrate, by immobilized CT at pH 11, is almost fourfold greater than that by free CT at its pH optimum.


Mechanochemical studies of enzymatic degradation of insoluble collagen fibers. A mechanochemical method was developed for studying the enzymatic degradation of insoluble collagen fibers. The method involves stretching the collagen fiber to a fixed extension in the presence of a solution of collagenase and measuring the rate of relaxation of the force induced in the fiber. In this work, bacterial collagenase was used for reasons of availability. We observed invariably an exponential decrease in force with respect to ttime. The slope of the linear plot of logarithm of the force versus time was taken as a measure of the rate of enzymatic degradation. This rate was found a) to vary linearly with collagenase concentration; b) to be maximal at pH 7-8; c) to vary with temperature according to the Arrhenius relationship in the range 10-56 degrees C; d) to be reduced to varying extent by addition of EDTA omicron-phenanthroline, 2,3-dimercaptopropanolol, and D,L-cysteine; e) to be minimal when the strain on the fiber was ca. 4%; f) to be increased dramatically by denaturation of the collagen fiber; and g) to be reduced by an increase in the crosslink density of the collagen fiber. Except for the effect of strain, which can not be conveniently studied by existing methods these results are consistent with those observed by other methods for the study of the enzymatic degradation of collagen. The mechanochemical method is, however, uniquely suited to monitor continuously the enzymatically induced decay in the stress-bearing ability of collagen fibers. It has also been found useful in the design of collagenous implants with specified resistance to enzymatic degradation in vivo.


Basal and stimulated serum growth hormone concentrations in inflammatory bowel disease. Patients with inflammatory bowel disease (IBD) manifest growth failure which may antecede abdominal symptoms by some years. Eight of ten children with documented IBD had records of decreasing growth velocities. Investigation of growth hormone reserves showed excessive rather than impaired responses. Mean basal GH level was 6.2 +/- 0.75 (SEM) ng/ml. During sleep, the mean GH level rose to 26.0 +/- 4.7 ng/ml and following propranolol-glucagon stimulation, to 46.0 +/- 4.5 ng/ml. All values were significantly higher than levels obtained in a control population of 25 children investigated for short stature who were not GH deficient. The mean peak GH response following insulin in the IBD group (10.8 +/- 3.8 ng/ml), however, did not differ from the mean peak response in the control group (13.5 +/- 3.3 ng/ml). Growth failure in patients with IBD is not the result of GH deficiency and is not an irreversible phenomenon. On the contrary, judicious use of glucocorticoids aimed at the control of the disease usually produces compensatory growth acceleration ("catch-up growth").


Insulin, proinsulin, glucagon and gastrin in pancreatic tumors and in plasma of patients with organic hyperinsulinism. Insulin, proinsulin, glucagon and gastrin were determined in extracts of tumors of 27 patients with pancreatic islet cell neoplasia of pancreas, in one patient with nesidioblastosis, in extracts of uninvolved portions of the pancreas in 11 of the tumor patients and of 15 control pancreases. Mean insulin concentration in solitary adenomas and in adenomas of patients with adenomatosis was higher than in control pancreases; however, in all but 1 patient the insulin concentration in neoplastic islet tissue was lower than in islet tissue of control pancreas, assuming islet volume is 1% of pancreas. The percentage of proinsulin was elevated in 52% of tumors. Adenoma insulin content correlated with increments of plasma insulin after tolbutamide administration. Insulin and proinsulin concentrations in pancreas uninvolved by tumor were not suppressed. Fasting plasma glucagon was elevated in patients with islet cell adenomatosis and in patients with islet cell carcinoma some of whom had multiple endocrine adenomatosis. The mean concentration of glucagon in tumors was lower than in control pancreases. Elevated concentration of gastrin was found in some adenomas. The data indicate: 1) insulin-secreting islet cell tumors have decreased storage capacity for insulin, 2) elevated concentration of proinsulin in tumors may be due to decreased capacity to store insulin and in some to decreased conversion of proinsulin to insulin as well, 3) tolbutamide stimulates the exaggerated release of a relatively constant fraction of insulin stored in adenomas. 4) solitary adenomas may contain excess amounts of pancreatic hormones in addition to insulin, 5) elevated plasma glucagon in patients with organic hyperinsulinism may indicate malignancy, microadenomatosis or multiple endocrine adenoma syndrome, and 6) chronic hyperinsulinism and hypoglycemia due to adenoma do not suppress insulin and proinsulin content of uninvolved pancreas.


Changes in occurrence of capsular serotypes of Streptococcus pneumoniae at Boston City Hospital during selected years between 1935 and 1974. The number of patients with pneumococcal bacteremia, empyema, and meningitis at Boston City Hospital during selected years between 1935 and 1974 is reported. The distribution of specific types in the bacteremic patients during each of the selected years and in the various focal infections in all the years is also detailed. The numbers and rates per 1,000 admissions of bacteremic pneumococcal infections and the numbers of cases of pneumococcal meningitis and empyema varied independently over these years and differed from those previously reported for 1929 to 1936. The types most frequent in pneumococcal bacteremias varied over the years, and the distribution of types among them differed markedly from that among the patients with focal infections. Variations in the distribution of pneumococcal types at different times in the same place, in different places, and in various sites of infection may be important in selecting types to include in pneumococcal vaccines for different populations.


Treatment of aplastic anemia by marrow transplantation from HLA identical siblings. Prognostic factors associated with graft versus host disease and survival. 73 consecutive patients with severe aplastic anemia were treated by marrow transplantation from hematologically normal HLA identical siblings. 68 patients lived long enough to document marrow engraftment. 21 rejected the graft and 19 of these died. 47 sustained engraftment and 18 of these died. In 16 patients, death was associated with graft versus host disease. 29 patients with sustained engraftment are alive with complete hematologic restoration between 8 mo and 5 yr. This analysis, by using a proportional hazards regression model, was directed at identifying factors that predicted survival (and absence of graft versus host disease). Of the 24 factors entered into the analysis only two strongly correlated with survival: (a) sex match of donor and recipient (P less than 0.01), and (b) absence of refractoriness to random donor platelets at the time of transplantation (P less than 0.05). Refractoriness adversely influenced the survival of the sex mismatched patients, These data suggest that X and Y-associated transplantation antigen systems are important determinants of the outcome of marrow grafts between HLA identical siblings for the treatment of aplastic anemia. The machanism by which refractoriness to random donor platelets influences survival is currently unclear.


Postsynthetic deamidation of hemoglobin Providence (beta 82 Lys replaced by Asn, Asp) and its effect on oxygen transport. Carriers of hemoglobin Providence have three types of beta chain in their hemolysates. The two abnormal chains have asparagine (Providence N, Prov N) or aspartic acid (Providence D) at position beta 82, instead of lysine. In vitro, only two beta chains are synthesized by reticulocytes of carriers, betaA and betaProv N. In vivo studies showed that the specific activity of Providence N was initially 10-fold higher than that of Providence D; the specific activities of the two labeled hemoglobins were approximately equal 5 wk after injection of isotope. Oxygen affinity of carriers' blood was somewhat increased, but they were not polycythemic. The affinity of the purified hemoglobins Providence was decreased. Addition of 2, 3 diphosphoglycerate had little effect on the affinity of either hemoglobin component, and addition of inositol hexaphosphate produced no change in the affinity of Providence D. These studies demonstrate that Providence N is deamidated to Providence D during the life span of the erythrocyte, and suggest this finding may represent only an easily observed prototype of posttranslational modification of proteins in general. Despite and abnormal P50 of the blood, oxygen transport is probably normal in carriers of the abnormal hemoglobins.


Mechanism of resistance to the phosphaturic effect of the parathyroid hormone in the hamster. The effect of parathyroid hormone and calcitonin on the renal excretion of phosphate, calcium, and cyclic AMP was evaluated in the thyroparathyroidectomized hamster, a mammal apparently reisstant to the phosphaturic effect of parathyroid hormone. Parathyroid hormone did not increase phosphate excretion, although it decreased excretion of calcium and increased urinary excretion of cyclic AMP. This lack of a phosphaturic response to parathyroid hormone was not reversed by administration of 25-OH vitamin D or infusions of calcium or phosphate. Calcitonin, another potentially phosphaturic hormone, also vailed to increase phosphate excretion but markedly elevated urinary excretion of cyclic AMP. In hamsters pretreated with infusion of urinary ammonium chloride, which decreased plasma and urinary pH, both parathyroid hormone and calcitonin increased excretion of phosphate as well as that of cyclic AMP. Acetazolamide had no phosphaturic effect in ammonium chloride-loaded hamsters, and it decreased cyclic AMP and calcium excretion. Alkalinization of urine by acetazolamide did not prevent the phosphaturic effect of parathyroid hormone in ammonium chloride-loaded hamsters, but it blocked the increase in urinary cyclic AMP excretion. Parathyroid hormone and calcitonin both stimulated adenylate cyclase in a cell-free system (600-g pellet) from hamster renal cortex, elevated tissue cyclic AMP levels, and activated protein kinase in tissue slices from hamster renal cortex. In acid medium, the increase in cyclic AMP and activation of protein kinase in response to parathyroid hormone was diminished, but addition of acetazolamide restored responsiveness of both parameters to control values. Acetazolamide, on the other hand, did not influence adenylate cyclase or its response to parathyroid hormone or cyclic AMP phosphodiesterase activity. We conclude that the lack of a phosphaturic effect of parathyroid hormone and calcitonin in the hamster depends on steps in the cellular action of these hormones, steps that are sensitive to pH subsequent to cyclic AMP generation and protein kinase activation. In addition, acetazolamide may potentiate the phosphaturic effect of parathyroid hormone by promoting accumulation of cyclic AMP in tissue. Thus, the hamster is a particularly useful model for studies of syndromes in which there is renal resistance to phosphaturic hormones.


Serological grouping of streptococci by a slide coagglutination method. A new method for the serological grouping of streptococci by coagglutination with specific antibodies absorbed to protein A-containing staphylococci has been assessed. A total of 242 strains of streptococci, including beta-haemolytic streptococci of groups A, B, C, F, and G, Streptococcus pneumoniae and Strep. faecalis were studied. All streptococci of groups A, B, C, and G, groupable by standard methods, were correctly grouped by coagglutination, although 7-3%  showed varying degrees of cross-agglutination. Two beta-haemolytic strains of Strep. faecalis produced coagglutination with group C streptococcal reagent. The method appears to be quick, accurate, reproducible, and simple to perform.


Plasma level studies of penbutolol after oral dose in man. Plasma levels of penbutolol (HOE 893d) were determined in eight healthy adult male subjects after oral administration of 50-mg capsules. Fast absorpiton of the drug from the gastrointestinal tract was indicated by the rapid increase in plasma levels during the absorption phase, with a peak time at about 1 hour after dosing in all subjects. After the peak level, plasma concentrations declined biexponentially, with an average half-life of 2.5 and 27 hours for the fast and slow disposition phases, respectively. These values were in good agreement with data previously found for this drug. Cumulative excretion of intact drug in the urine of the eight subjects during 72 hours after dosing was less than 4 per cent, except for one subject who excreted 9.82 per cent of the dose. Large individual variations were found for area under the plasma level curves, disposition rates, and amounts of intact drug excreted in the urine. Significant pharmacologic effects were noted in all eight subjects at the 50-mg dose level, and mild side effects were evident in one half of these subjects. The average drop in blood pressure and pulse rate for all subjects was 26/18 mm Hg and 19 beats per minute, respectively.


Penfluridol, a peroral long-acting neuroleptic, for the maintenance treatment of schizophrenic patients who relapse. In a multicenter collaborative study, 28 newly readmitted schizophrenic patients, stabilized for one week on short-acting neuroleptic drugs, had their medication abruptly changed to penfluridol given once a week on an outpatient basis. The average dose required for maintenance was approximately 40 mg weekly. Analysis of BPRS evaluations carried out during the 16-week trial revealed a significant linear trend toward further improvement. Social functioning, as measured by the KAS questionnaire in the outpatient period of the trial, also revealed a significant linear trend toward improvement. Significant worsening was not found with any psychometric evaluation. Side effects, when observed, were neither frequent nor severe. Three laboratory and vital sign values showed significant changes: increase in BUN concentrations, decrease in pulse rate, and increase in body weight. The changes in weight and pulse appeared to be within relatively normal ranges, and the increase in BUN concentrations did not appear to be clinically significant. During the first part of a long-term study, penfluridol received a high degree of patient acceptability and is a welcome addition to the maintenance treatment of schizophrenia.


Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity.


The effect of adenylate cyclase inhibitor (ACI) on guanylate cyclase, phosphodiesterase and other enzymes in heart. The effect of an inhibitor of adenylate cyclase (ACI) was measured on some enzymes associated with cyclic nucleotide-regulated metabolism. Soluble guanylate cyclase was inhibited; both soluble and particulate cyclic GMP-phosphodiesterases were stimulated. Cyclic AMP phosphodiesterases were unaffected. In contrast, the activities of Na, K-ATPase, protein kinase, phosphorylase kinase, glycogen synthetase and a number of glycosidases were not altered by equipotent amounts of the inhibitor. It is concluded that this substance acts as a modulator of both cyclic AMP and cyclic GMP metabolism in heart and other tissues.


Clinical evaluation of intranasal topical flunisolide therapy in allergic rhinitis. A double-blind, vehicle control, parallel clinical trial evaluated the effectiveness and safety of the local application of flunisolide, a potent new topical steroid, in the treatment of ragweed hay fever. Fifty patients with well-defined, poorly controlled ragweed allergy were studied during the 1974 ragweed season. Analysis of the data showed that the active drug group had a significant decrease in individual symptoms of sneezing, nasal stuffiness, and nasal secretions, compared with the placebo group. Antihistamine usage was statistically decreased in the active drug over placebo group. There was no evidence of adrenal suppression. This study indicates that intranasal administration of flunisolide in adult patients is both efficacious and safe in the treatment of seasonal allergic rhinitis.


Adverse effects of the drugs most frequently administered to the elderly--Part II. The uncertainties associated with identificatin of adverse drug reactions are numerous. These effects which involve the eyes and vision constitute an important group but they are inadequately described and classified in most reports. Vague statements of visual disturbances, blur, changes in color vision, or difficulties in reading are indicated in many articles. Both parts of this report provide a listing of potential adverse effects, systemic and ocular. The drug groups included in part II are the antipsychotics, antianxiety drugs, antidepressants, antiparkinson drug and a miscellaneous group.


Lipopolysaccharide-induced suppression of the primary immune response to a thymus-dependent antigen. The immune response to a thymus-dependent antigen was depressed in vivo and in vitro in spleen cells from mice injected with LPS i.p. a few days before challenge with the antigen. Spleen cells from LPS-injected mice could, however, respond with increase DNA synthesis after activation with polyclonal B and T cell activators in vitro. The LPS-activated spleen cells could actively suppress normal cells in their response to the antigen sheep red blood cells. The suppressor cells contained in the LPS-activated spleens were most likely B lymphocytes, and the possible mechanism for their inhibitory function is discussed.


Heterologous antigenic stimulation in induction of delayed hypersensitivity. An influence of a delayed hypersensitive reaction to a primary antigen on the induction of delayed hypersensitivity to a second unrelated antigen was observed in guinea pigs immunized with azobenzenearsonate-N-acetyl-L-tyrosine (ABAT), and injected intradermally 3 weeks later with a mixture of ABAT and secondary antigen. Animals so treated developed delayed hypersensitivity to sheep red blood cells (SRBC) or Type II pneumococcal polysaccharide as secondary antigens, as measured by skin test reactivity and inhibition of macrophage migration, whereas ABAT unsensitized control groups did not. However, attempts to induce delayed reactivity to proteins as secondary antigens were unsuccessful. The injection of secondary antigen into a mineral oil-induced inflammatory lesion did not induce delayed hypersensitivity, suggesting that specific reactivity to ABAT is a prerequisite for heterologous induction. Possible mechanisms for the observed phenomenon, including a role for macrophages, are discussed.


Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide I. Comparison of the suppression induced by con A and low dose paralysis. Concanavalin A (Con A) administered at the time of immunization induces suppression of the in vivo splenic plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III). As with low dose paralysis of the PFC response to SSS-III, Con A-induced suppression could not be demonstrated in congenitally athymic (nu/nu) mice and could be eliminated partially by treatment with anti-lymphocyte serum (ALS). The kinetics for Con A-induced suppression paralleled those for low dose paralysis of the antibody response to SSS-III. These findings support the view that Con A-induced suppression is produced in vivo by suppressor T cells and that this form of suppression shares with low dose paralysis a common pathway through which suppression is mediated.


Cell lines from old immunodeficient donors give normal responses in young recipients. Two different immune responses were compared in spleen cells obtained from old and young CBA/HT6J mice. Spleen cells from old mice (23 to 33 months) responded about half as well as did spleen cells from young mice (4 to 10 months) in the adoptive transfer anti-sheep red blood cell (SRBC) plague-forming assay, and caused slightly less than half the uptake of tritiated thymidine in response to phytohemagglutinin (PHA) in vitro. Marrow stem cell from some of the old and young mice whose splenic immune responses were tested were transplanted into irradiated young CBA/CaJ recipients. Seven to 17 weeks later these same immune responses were tested in the spleen cells of these young recipients, and the T6 chromosome marker was used to identify donor cells. Old animals' responses varied greatly, perhaps due to suppressing cells or factors in some individuals. Therefore, cells were never pooled and the responses of receipients were compared to the responses of the donor whose marrow had populated them. The response for a particular old donor, or for the recipients of its stem cells, was divided by the response for the young control used with that donor, or for its stem cell recipients. This was called the old/young ratio. With original donors with an old/young ratio for the SRBC response of (mean  +/- S.D.) 0.35 +/- 0.14, The old/young ratio for that same response in the recipients was significantly improved to 1.26 +/- 0.71. In original donors with an old/young ratio for the PHA response of 0.44 +/- 0.17, the old/young ratio in the recipients improved significantly to 0.86 +/- 0.27. Thus, little or none of the decline with age in these immune responses was intrinsic to the old lymphoid stem cells.


The allogeneic effect on tumor growth. I. Inhibition of a murine plasmacytoma, MOPC 315, by the graft-vs-host reaction. The allogeneic effect has been employed as a potent immunopotentiator in preventing the growth of a murine plasmacytoma and prolonging host survival. Parental BALB/c spleen cells were passively transferred to (BALB/c x A/H)F1 hybrid mice, who were then given a highly lethal dose of MOPC 315 plasmacytoma, a tumor of BALB/c origin. The resultant graft-vs-host reaction protected the recipient mice against growth of the tumor and significantly prolonged survival. This phenomenon was dependent upon the dose of BALB/c lymphoid cells employed, the route of administration, and the time interval between lymphoid cell transfer and tumor inoculation. A wide range of lymphoid cell doses and time intervals were effective, and repeated doses of allogeneic cells provided better protection than a single dose.


The allogeneic effect on tumor growth. II. Suppression of both ascitic and solid MOPC 315 plasmacytoma by the graft-vs-host reaction, with pathologic correlation. The growth of an ascitic murine plasmacytoma, MOPC 315, can be retarded in CAF1 hybrid host mice by the i.p. injection of donor lymphoid cells. The graft-vs-host reaction can be established by a variety of donor cells, including parental BALB/c and A/J and congenic inbred B10.D2 which share the major histocompatibility locus with BALB/c(H-2d). Optimal results are consistently obtained when parental BALB/c spleen cells are injected before tumor inoculation, and a second dose of donor spleen cells injected 1 week later. This aloogeneic effect on tumor growth is manifested by delayed appearance of the tumor and prolonged host survival. Pathologic studies on the ascites tumor indicated that the allogeneic effect suppresses the initial appearance and early growth of the plasmacytoma. However, once established, MOPC 315 grows rapidly and fatally in both control mice and recipients of donor lymphoid cells. Further, a subcutaneous implant of MOPC 315 is suppressed by an allogeneic effect established either i.v. with BALB/c spleen cells before tumor inoculation or by BALB/c spleen cells administered subcutaneously at the time of MOPC 315 implant. Thirty percent of mice treated by i.v. or subcutaneous donor lymphoid cells were tumor free at 150 days after tumor inoculation.


Mouse lymphocytes with and without surface immunoglobulin: preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin. Mouse spleen cells could be preparatively separated into immunoglobulin positive (Ig+) and immunoglobulin-netative (Ig-)populations by incubating as many as 2 X 10(8) cells per 100 mm diameter petri plate coated with specifically purified goat anti-mouse immunoglobulin. The non-adherent population was 95% or more Ig-, and possessed graft versus host and cytotoxic effector activities, as would be expected for T cells. They could also give a mixed lymphocyte reaction and generate cytotoxic effector activity on culture in vitro. The adherent cells could not be released undamaged from plates coated with undiluted anti-Ig, but they could be released from plates coated with a 1/4 or 1/10 dilution of anti-Ig in an irrelevant antibody. The released cells were over 90% viable by trypan-blue staining, and 94% or more of the viable cells were Ig+.


Immunochemical and morphological studies of hepatitis B core antigen isolated from the nuclei of hepatocytes. Immunochemical and morphological properties of hepatitis B core antigen (HBc Ag) were studied in intranuclear particles isolated from human liver. Immunochemical integrity of the purified particles was indicated in the production by guinea pigs of antibody to HBc Ag (anti-HBc) that was immunochemically identical to human anti-HBc. The HBc Ag particles were 27-30 nm in diameter, displayed apparent icosahedral symmetry, and consisted of distinct subunits. The susceptibility of HBc Ag particles to proteolytic and glycolytic enzymes indicated the presence of proteins and glycoproteins. The structural integrity of core particles depended on disulfide, hydrophobic, and hydrogen bonds, and immunological activity relied on intact sulfhydryl groups. Agents active against lipids did not affect immunological reactivity or core structure, as seen by electron microscopy.


Effect of atherosclerosis on lysosomal cholesterol esterase activity in rabbit aorta. Radiolabeled cholesteryl oleate, when incorporated into phospholipid vesicles, was hydrolyzed at acid pH by an enzyme present in rabbit aortic homogenates. In contrast, cholesteryl oleate presented as an acetone dispersion was not effectively hydrolyzed at acid pH under identical conditions. Using the vesicle preparation as substrate, a sensitive assay system for the acid hydrolase was developed in which hydrolysis was proportional to protein concentration and incubation time, and was independent of substrate concentration. The physical state of the vesicles was apparently not altered by the assay conditions, and no hydrolysis of the vesicle-associated phospholipid was detected. Acid cholesterol esterase activity in atherosclerotic aortic tissue was 2.5-fold greater than that of control tissue, and even greater increases were observed in the activities of other lysosomal enzymes (N-acetyl-beta-d-glucosaminidase and beta-glucuronidase). Glucose-6-phosphatase activity was also increased in aortas from cholesterol-fed animals while 5' nucleotidase activity remained unchanged. Labeled triolein also was incorporated into phospholipid vesicles and was hydrolyzed by an acid lipase in aortic tissue. Similarities between triolein and cholesteryl oleate hydrolysis existed with respect to pH optimum and the effect of cholesterol feeding on activity, suggesting that a single enzyme may hydrolyze both lipids.


[Mathematical simulation of the respiratory system (author's transl)]. The respiratory system is described as a feedback control system. The controller consists of the peripheral chemoreceptors and the central chemosensitive structures, the respiratory centre in the medulla oblongata and the thorax-lung pump which they drive. The controlled system is comprised of three compartments (lung, brain and the remaining tissue) connected by the blood circulation. The controlled values are arterial pH and arterial O2 partial pressure and cerebral extracellular pH. Earlier models have been improved by: (1) the dead space description, (2) the thermodynamic formulation of the CO2 dissociation equation and the simple but accurate O2 dissociation equation of the blood, (3) the alteration of the CO2 dissociation equation for the brain and the remaining tissue to accommodate recent results, (4) the application of the one-receptor-theory of central chemosensitivity, (5) the pH dependence of brain circulation, (6) the bicarbonate exchange between blood and extracellular fluid of the brain and (7) the introduction of variable circulation times. Respiratory and metabolic disturbances of the respiratory system are analyzed. The mathematical formulation of the respiratory system is a differential difference equation system. In the steady state the experimental results are reproduced fairly well. A slight discrepancy is found in the simulation of metabolic acidosis. Apparently we have assumed the sensitivity of the peripheral chemoreceptors to be too large so that the respiratory response is not correctly predicted. In the numerical solution there is an overshoot in the on-transient and a damped oscillation in the off-transient of the alveolar CO2 partial pressure during respiratory acidosis. We have varied the parameters to make deviations small. The best agreement seems to result, if the central threshold is near the normal extracellular pH of the brain. A further deviation from experimental findings is that the cerebral CO2 and H+ concentration, the blood circulation of the brain, the alveolar O2 partial tension and the ventilation show a slight oscillation in the off-transient. Except for these discrepancies the experimental results, especially the stability of the extracellular pH of the brain, are reproduced fairly well. During hypoxia there are deviations form the experimental results if the central residual activity is constant and the central threshold deviates from the normal extracellular pH of the brain. But if the central residual activity is pH dependent and if the central threshold is equal to the normal extracellular pH of the brain, then the time course of VE and the other variables agree fairly well with experimental results. There is also a good correspondence between the theoretical and experimental data during hyperoxia. During metabolic acidosis the time constant of the bicarbonate exchange between blood and extracellular fluid of the brain is important. If a time constant of one minute is assumed, then the predicted and the experimental results correspond sufficiently well.


Enzymic changes in the cervix of the rat and hamster during the oestrous cycle and the effect of steroids. Changes in four hydrolytic enzymes, namely acid phosphatase, alkaline phosphatase, arylsulphatase A and B, of the cervix of the rat and hamster have been studied during the 4-day oestrous cycle. All four enzymes showed maximal activity on the day of oestrus and least activity on day 2 of dioestrus. All the enzymes showed significant reduction of activity after ovariectomy, arylsulphatase A and B showing the earliest changes in specific activity. A single subcutaneous injection of 0-02 microng oestradiol-17beta/rat increased the especific activity of arylsulphatase A and B from the low ovariectomized level to that observed in control oestrous animals within 18 and 6 h respectively. A higher concentration of oestradiol 17beta (2-0 microng) had an inhibitory effect. Progesterone was without effect on arylsulphatase B activity, but when given (2-0 mg) with 0-02 microng oestradiol-17beta, it inhibited the response to oestrogen. Cycloheximide prevented the rise in arylsulphatase B activity occurring after oestrogen injection, suggesting a regulation of cervical arylsulphatase B at the level of protein biosynthesis. These results suggest that arylsulphatase B activity may be induced by oestrogen in the cervix of the rat.


Developmental expression and biochemical characterization Nassarius obsoleta. Electrophoretic analysis of catalase isozyme patterns of Nassarius obsoleta indicates that these isozymes are products of two separate loci. Five aminopeptidase isozymes have also been detected in N. obsoleta and the data suggest that at least two loci encode these aminopetidase isozymes. Experiments designed to test for the interconvertibility of the isozymes indicated that the two catalase isozymes and that two of the five aminopeptidase isozymes tested were not conformational artifacts but distinct genetic products. No change in catalase isozyme expression, but considerable quantitative variation in catalase specific activity was noted during early developmental stages of N. obsoleta. Both qualitative and quantitative changes were noted in aminopeptidase expression during early developmental stages. This paper also describes several physicochemical parameters for each of the two enzymes under study.