Pyridine-2, 6-dicarboxylic acid (dipicolinic acid) formation in Bacillus subtilis. II Non-enzymatic and enzymatic formations of dipicolinic acid from alpha, epsilon-diketopimelic acid and ammonia. Non-enzymatic formation of dipicolinic acid (DPA) from diketopimelic acid and ammonia was clearly demonstrated using a new method for DPA analysis. The reaction rates of DPA formation were almost the same under aerobic and anaerobic conditions. Nearly equimolecular quantities of DPA and tetrahydrodipicolinic acid were detected in spontaneous reaction mixture. The spontaneous reaction seemed to be due to dismutation of dihydrodipicolinic acid, resulting in DPA and tetrahydrodipicolinic acid. The apparent optimum pH of the spontaneous reaction was 8.2 and the maximal rate of DPA formation was observed with a 1 : 4 molar ratio of diketopimelic acid to ammonia. The rate of the spontaneous reaction was stimulated by ferrous sulfate, FMN, and riboflavin. Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate and aspartic beta-semialdehyde, with NADPH as reductant. The reductase was isolated from Bacillus subtilis, and found to stimulate DPA formation from diketopimelic acid and ammonia. The enzymatic DPA formation was absolutely dependent on oxygen, and optimum pH was 6.4. The catalytic action of the enzyme was similar to that of the oxidase. Possible mechanisms of DPA formation from diketopimelic acid and ammonia are proposed.


Trinitrophenylation of nucleic acids and their constituents. 1. Under relatively mild conditions, nucleic acids and their constituents were trinitrophenylated with 2,4,6-trinitrobenzenesulfonate (TNBS) in aqueous solution (pH 8-11), yielding reddish-orange trinitrophenyl (TNP) derivatives. Guanine residues were trinitrophenylated on the base residues at the 2-amino group (N2-TNP derivatives), and in addition, 2'- and 3'-hydroxyl groups of the ribose moieties of nucleosides or nucleotides were trinitrophenylated to form Meisenheimer complexes. 2. The preparation of TNP derivatives (N2-TNP-guanine, -guanosine, N2, O-bis-TNP-guanosine, O-TNP-guanosine, -adenosine, -cytidine , and -uridine), their rates of formation, absorption spectra (UV, visible, and infrared), molar extinction coefficients, Rf value, electrophoretic mobilities, and stability in acid or alkaline solution, are presented. 3. Trinitrophenylation of several kinds of nucleic acid was investigated. Calf thymus DNA and yeast transfer RNA showed a resistance to trinitrophenylation compared to guanosine 3'(2')-phosphate, yeast RNA or denatured calf thymus DNA. TNP-RNA showed resistance to the action of ribonucleases T1 and T2 [EC 3.1.4.8 and 3.1.4.23]. 4. Trinitrophenylation reactions using 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrofluorobenzene were compared with that using TNBS as regards specificity and reaction rate.


Activation of mouse splenic lymphocyte guanylate cyclase by calcium ion. The guanosine 3',5'-cyclic monophosphate (cGMP) level in the mouse splenic lymphocytes was increased about 2- to 3-fold by concanavalin A. This increase was completely dependent on the presence of Ca2+ in the medium. Homogenates of mouse splenic lymphocytes contained significant guanylate cyclase [EC 4.6.1.2] activity in both the 105,000 X g (60 min) particulate and supernatant fractions and both fractions required Mn2+ for full activity. Calcium ion (3mM) activated soluble guanylate cyclase 3-fold at a relatively low concentration of Mn2+ (less than 1mM) but inhibited the particulate enzyme slightly at all Mn2+ concentrations tested. Concanavalin A itself did not stimulate either fraction of guanylate cyclase. Thus these results suggest that elevation of the cGMP level in lymphocytes by concanavalin A might be brought about by stimulation of Ca2+ uptake and activation of soluble guanylate cyclase by the latter.


Rat intestinal brush border membrane peptidases. II. Enzymatic properties, immunochemistry, and interactions with lectins of two different forms of the enzyme. The properties of two purified peptidases derived from the intestinal brush border membrane of the rat have been investigated. The pH optima, heat stabilities, substrate specificities, and metal ion requirements of the two enzymes and the effects of inhibitors on their activities were nearly identical. The isoenzymes catalyzed the hydrolysis of a wide range of peptides containing from 2 to 8 amino acid residues. The enzymes are aminopeptidases; no evidence for carboxypeptidase or endopeptidase activity was found. For hydrolysis, there appears to be an absolute requirement for an L-amino acid at the NH2-terminus of the peptide substrate. There was a similar but less absolute requirement for the penultimate NH2-terminal amino acid. Thus, although peptides of the type L-aminoacyl-L-proline, L-aminoacyl-L-prolyl-(L-amino acid)n, or L-aminoacyl-D-amino acid were not hydrolyzed, L-leucyl-beta-naphthylamide could be utilized as a substrate. The enzymes appeared to be metalloenzymes in that metal ion-chelating agents could inhibit their activities. Co2+ partially restored the activities lost by chelation. Immunodiffusion studies showed that the two enzymes were immunologically identical. The antipeptidase antisera were specific for the enzymes and did not react with other constituents of the intestinal cell. Both enzymes have binding sites for the lectin phytohemagglutinin which recognizes N-acetylgalactosamine residues located at or near the terminal positions of glycoprotein carbohydrate chains. Both the lectin and the antibodies inhibited enzyme activities, but the mechanisms of inhibition appeared to be different.


Transport of riboflavin into yeast cells. Riboflavin-requiring mutants of Saccharomyces cerevisiae are able to transport 14C-labeled riboflavin into the cell, although no significant transport is seen in commercial yeast or in the parent strain from which the mutants were derived. Transport activity is greatest in the early to mid-log phase of anaerobic growth and declines sharply in the late log phase. In aerobically grown cells activity is substantially lower at all stages of growth. In the assay devised for its measurement, transport activity shows a sharp pH optimum at pH 7.5, a strong temperature dependence (EA = 23,100 cal/mol), and saturation kinetics with respect to riboflavin (Km = 15 muM), characteristics consistent with a carrier-mediated mechanism. Monovalent inorganic cations, particularly K+ and Rb+, stimulate riboflavin uptake, while certain organic cations are inhibitory. Besides riboflavin only 7-methylriboflavin, 8-methylriboflavin, and 5-deazaflavin have been found to serve as substrates, while lumiflavin, tetraacetylriboflavin, and N10-[4'-carboxybutyl]-7,8-dimethylisoalloxazine do not, although a number of flavin analogs in which the ribityl side chain is modified are good competitive inhibitors of riboflavin uptake. Compounds resembling the ribityl side chain, such as sugars and sugar alcohols, do not inhibit. An apparent inhibition of uptake by D-glucose, D-mannose, and D-fructose, which develops in the course of assay, proved to result from stimulation of an opposing process, the release of riboflavin from the cells.


Soluble enzyme system for vitamin K-dependent carboxylation. The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.


Glutamate synthase. Properties of the glutamine-dependent activity. Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.


Properties of apoglutamate synthase and comparison with glutamate dehydrogenase. Glutamate synthase from Escherichia coli K-12 exhibits NH3-dependent activity. NH3-dependent activity is increased approximately 5-fold in apoglutamate synthase lacking flavin and non-heme iron. Whereas glutamine plus 2-oxoglutarate have the capacity to reoxidize the chemically reduced flavoenzyme, no such reoxidation is obtained with 2-oxoglutarate plus NH3. These results establish that the glutamine- and NH3-dependent syntheses of glutamate occur by different pathways of electron transfer from NADPH. The NH3-dependent activity of native and apoglutamate synthase exhibits similar catalytic properties. Some properties of apoglutamate synthase are similar to those of glutamate dehydrogenase. These properties include pH optima for synthesis and oxidative deamination of glutamate, inactivation by alkylating reagents and p-mercuribenzoate, an enhanced rate of inactivation by alkylating reagents and p-mercuribenzoate at low pH, 2-oxoglutarate protection against inactivation by p-mercuribenzoate, and reactivation of p-mercuribenzoate-treated enzyme by 2-mercaptoethanol. 2-Oxoglutarate protects against alkylation of glutamate synthase by iodo [1-14C]acetamide and reduces incorporation of methyl [1-14C]carboxamide into the small subunit of the enzyme.


Mechanism of the irreversible inhibition of aspartate aminotransferase by the bacterial toxin L-2-amino-4-methoxy-trans-3-butenoic acid. The naturally occurring toxin L-2-amino-4-methoxy-trans-3-butenoic (AMB) acid irreversibly inhibits pyridoxal phosphate-linked aspartate aminotransferase. The inhibitor is a substrate for the enzyme, and as such is converted into a highly reactive intermediate which chemically reacts with an active site residue, thus irreversibly inactivating the enzyme. Enzymological and model studies on AMB are presented which enable one to determine the precise mechanism of action of this toxin. The mechanism involves Schiff base formation between the enzyme and toxin followed by alpha-C--H bond cleavage and aldimine isomerization to generate a bifunctional Michael acceptor. This molecule alkylates an active site residue by an addition and elimination route.


The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor. The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.


Accleration of autooxidation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalyzed aniline hydroxylation. Changes in the ultraviolet/visible spectrum of human oxyferrohemoglobin upon addition of aniline were indicative of a concentration-dependent interaction of aniline with hemoglobin, resulting in accelerated autooxidation of the hemoprotein. Oxygen was found to markedly inhibit this interaction of aniline with oxyhemoglobin. The dependence of the rate of autooxidation on aniline concentration followed saturation kinetics and showed a half-maximal response at 8 mM aniline. This value is equal to the value of Km for aniline as substrate for the O2-dependent, hemoglobin-catalyzed hydroxylation reaction which yields p-aminophenol (Mieyal, J. J., Ackerman, R.S., Blumer, J.L., and Freeman, L.S. (1976) J. Biol. Chem. 241, 3436-3441). Thus, an aniline-oxyhemoglobin complex is implicated in the overall catalytic reaction. No detectable p-aminophenol was formed when aniline was combined with oxyhemoglobin in the absence of an electron donor, but hydroxylation of aniline does occur when NADPH, NADPH plus P-450 reductase, or Na2S2O4 are also added.


Regulation of glutaminase B in Escherichia coli. I. Purification, properties, and cold lability. Escherichia coli contains two glutaminases, A and B, with pH optima below pH 5 and above pH 7, respectively. Neither glutaminase A nor B is released from E. coli by osmotic shock. Glutaminase B has been purified 6,000-fold and the purified preparation is estimated to contain about 40% glutaminase B. The enzyme has a molecular weight of 90,000 and an isoelectric point of 5.4. Glutaminase B exhibits a broad pH optimum between 7.1 and 9.0. Only L-glutamine is deamidated by glutaminase B, L-asparagine and D-glutamine are not deamidated. The substrate saturation curve for glutaminase B shows an intermediary plateau region. Like many regulatory enzymes, glutaminase B is cold-labile. The enzyme is inactivated by cooling and activated by warming; both processes are first order with respect to time. The activation energy for activation by warming was calculated to be 5900 cal/mol. Activation by warming increased the Vmax and decreased the S0.5 for L-glutamine, but did not alter the molecular weight of the catalytically active enzyme. Borate and glutamate protected glutaminase B from inactivation by cold.


Effects of divalent cation ionophore A23187 on potassium permeability of rat erythrocytes. A23187 transports calcium rapidly into rat erythrocytes, apparently by an electroneutral exchange for intracellular magnesium and protons. When red cells are incubated in the absence of any added divalent cations, A23187 transports internal magnesium out of the cells, in exchange for extracellular protons. Magnesium uptake into erythrocytes is produced by A23187, providing the extracellular concentration of this cation exceeds intracellular levels, and the ionophore also transports strontium, but not barium, into red cells. A23187 produces a rapid and extensive loss of intracellular potassium from erythrocytes during uptake of calcium or strontium, but not magnesium. When red cells are incubated in the absence of any exogenous divalent cations, A23187 still produces a potassium efflux and this is inhibited completely by small amounts of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and restored by the addition of calcium in excess of the chelator. Although EDTA enhances the extent of magnesium release from erythrocytes incubated with A23187, it prevents the potassium efflux. Dipyridamole and 4-acetamid-4'-isothiocyano-stilbene-2,5'-disulfonic acid, which decrease chloride premeability of erythrocytes, inhibit the A23187-induced potassium loss from red cells. Rutamycin, peliomycin, venturicidin, and A23668B also inhibit potassium efflux from intact cells incubated with A23187, but this effect is not correlated with their abilities to inhibit various ATPases in red cell membrane preparations. It is concluded that A23187 does not transport potassium directly across the erythrocyte plasma membrane, but permits small amounts of endogenous calcium to interact with some membrane component to enhance potassium permeability of the cell.


Yeast alpha-isopropylmalate isomerase. Factors affecting stability and enzyme activity. Yeast alpha-isopropylmalate isomerase was found to be markedly stabilized by high concentrations of glycerol and (NH4)2SO4. Such conditions of high ionic strength inhibited the enzyme, stabilized the enzyme to heat, and affected kinetic parameters. The isomerase was found to exhibit ionic strength-dependent hysteresis when enzyme, totally but reversibly inhibited by storage under conditions of high ionic strength of (NH4)2SO4, was transferred to a lower concentration of (NH4)2SO4. Alpha-Isopropylmalate isomerase was found to be sensitive to KCN and certain other chelators. The inactivation by KCN was prevented by high concentrations of (NH4)2SO4. These observations implicated a metal involvement but the nature of the metal was not revealed. The metal involvement and some of the other properties of alpha-isopropylmalate isomerase reveal a similarity to aconitase. The similarities in properties between the isomerase and aconitase are summarized. Studies of yeast alpha-isopropylmalate isomerase indicated that it is a single polypeptide of about Mr = 90,000.


Differential reactivity of the two active site cysteine residues generated on reduction of pig heart lipoamide dehydrogenase. Reduction of the active center disulfide bond in the flavoprotein pig heart lipoamide dehydrogenase generates two sulfur moieties which are chemically inequivalent in the 2-electron reduced form of the enzyme. Thus 1 cysteine residue is at least 13-fold more reactive than its partner toward iodoacetamide at pH 7.6. This selectivity was demonstrated by reaction of the 2-electron reduced enzyme with a low concentration of iodo[1-14C]acetamide under anaerobic conditions. The formation of a monolabeled derivative is accompanied by the reappearance of a spectrum of oxidized bound flavin, clearly different from that of the native enzyme. Alkylation of the remaining cysteine residues with iodo[12C]acetamide enabled the isolation of a tryptic version of the active center disulfide peptide. A single chymotryptic cleavage between the 2 alkylated cysteine residues generated a cationic and an anionic fragment containing 7% and 93% of the radioactivity of the purified tryptic peptide, respectively. The monolabeled derivative is catalytically inactive toward reduced or oxidized lipoamide, but is approximately 2-fold better as a transhydrogenase than the native protein using NADH and acetylpyridine adenine dinucleotide as substrates. Anaerobic titration with NADH leads to reduction of the flavin with concomitant formation of long wavelength absorption of low intensity. No intermediate reduced states were detected in this titration analogous to the red 2-electron form observed with the native enzyme. Similarly, intermediates during reduction of the enzyme by 1 eq of dithionite have not been detected.


Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin. The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.


Purification and characterization of an endo-beta-galactosidase produced by Diplococcus pneumoniae. An endo-beta-galactosidase acting on blood group A and B substances was found in the culture fluid of Diplococcus pneumoniae. The enzyme was purified 1000-fold, and its properties were studied in detail. The enzyme preparation, thus obtained, was practically free from various exoglycosidases, endo-beta-N-acetylglucosaminidase and proteases. The enzyme releases trisaccharides from blood group A and B active mucins purified from ovarian cyst fluid. The structures of the trisaccharides liberated from A and B active mucins were elucidated to be GalNAcalpha1 leads to 3(Fucalpha1 leads to 2)Gal and Galalpha1 leads to 3(Fucalpha1 leads to 2)Gal, respectively. The enzyme also hydrolyzes blood group A and B active oligosaccharides composed of type 2 chains, yielding the same products as in the case of ovarian cyst blood group substances. An H active mucin from ovarian cyst fluid, H active oligosaccharides, and A and B active oligosaccharides with type 1 chains were not hydrolyzed by the enzyme. Consequently, the enzyme catalyzes the following reaction, resulting in the degradation of blood type A and B determinants. (see article).


Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy. The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.


The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria. Protoporphyrinogen oxidase, an enzyme which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells, has been found in several mammalian tissues. It has been extracted from rat liver mitochondria by sonication in the presence of salt and detergent and partially purified. The enzyme is similar in many respects to yeast protoporphyrinogen oxidase. Based on its behavior on Sephadex G-200 the molecular weight of the enzyme is approximately 35,000. Catalysis by protoporphyrinogen oxidase was specific for proteoporphyrinogen IX (apparent Km of 11 muM) and proceeded maximally at pH 8.6 to 8.7. The effect of temperature on enzyme activity plotted according to Arrhenius gave a value of E of 9,100 calories per mol. Enzyme activity was inhibited in the presence of high salt concentrations and temperatures above 45 degrees. Oxygen was essential for protoporphyrinogen oxidase activity and an alternative elevtron acceptor has not yet been found. No requirement for a metal or other cofactor could be demonstrated. The presence of monothiol groups was indicated; however, it is not known whether the thiol groups are involved directly in the binding of substrate to the enzyme.


Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins. Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.


Ketopantoate hydroxymethyltransferase. II. Physical, catalytic, and regulatory properties. Some physical, catalytic, and regulatory properties of ketopantoate hydroxymethyltransferase (5,10-methylenetetrahydrofolate: alpha-ketoisovalerate hydroxymethyltranferase) from Escherichia coli are described. This enzyme catalyzes the reversible synthesis of ketopantoate (Reaction 1), an essential precursor of pantothenic acid. (1) HC(CH3)2COCOO- + 5,10-methylene tetrahydrofolate f in equilibrium r HOCH2C(CH3)2COCOO- + tetrahydrofolate  It has a molecular weight by sedimentation equilibrium of 255,000, a sedimentation coefficient (S20,w) of 11 S, a partial specific volume of 0.74 ml/g, an isoelectric point of 4.4, and an absorbance, (see article), of 0.85. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid analyses give a subunit molecular weight of 27,000 and 25,700, respectively; both procedures indicate the presence of 10 identical subunits. The NH2-terminal sequence is Met-Tyr---. The enzyme is stable and active over a broad pH range, with an optimum from 7.0 to 7.6. It requires Mg2+ for activity; Mn2+, Co2+, Zn2+ are progressively less active. The enzyme is not inactivated by borohydride reduction in the presence of excess substrates, i.e. it is a Class II aldolase. Reaction 1f is partially inhibited by concentrations of formaldehyde (0.8 mM) and tetrahydrofolate (0.38 mM) below or near the Km values, apparent Km values are 0.18, 1.1 and 5.9 mM for tetrahydrofolate, alpha-ketoisovalerate, and formaldehyde, respectively. For Reaction 1r, apparent Km values are 0.16 and 0.18 mM, respectively, for ketopantoate and tetrahydrofolate, and the saturation curves for both substrates show positive cooperativity. Forward and reverse reactions occur at similar maximum velocities (Vmax approximately equal to 8 mumol of ketopantoate formed or decomposed per min per mg of enzyme at 37 degrees). Only 1-tetrahydrofolate is active in Reaction 1; d-tetrahydrofolate, folate, and methotrexate were neither active nor inhibitory. However, 1-tetrahydrofolate was effectively replaced with conjugates containing 1 to 6 additional glutamate residues; of these, tetrahydropterolpenta-, tetra-, and triglutamate were effective at lower concentrations than tetrahydrofolate itself; they were also the predominant conjugates of tetrahydrofolate present in E. coli. Alpha-Ketobutyrate, alpha-ketovalerate, and alpha-keto-beta-methylvalerate replaced alpha-ketoisovalerate as substrates; pyruvate was inactive as a substrate, but like isovalerate, 3-methyl-2-butanone and D- or L-valine, inhibited Reaction 1. the transferase has regulatory properties expected of an enzyme catalyzing the first committed step in a biosynthetic pathway. Pantoate (greater than or equal to 500 muM) and coenzyme A (above 1 mM) all inhibit; the Vmax is decreased, Km is increased, and the cooperativity for substrate (ketopantoate) is enhanced. Catalytic activity of the transferase is thus regulated by the products of the reaction path of which it is one component; transferase synthesis is not repressed by growth in the presence of pantothenate.


Re-evaluation of the kinetics of lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by superoxide radicals in the presence of ethylenediaminetetraacetate. The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O2- is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O2- anion generated in the pulse). It is (1.0 +/- 0.2) X 10(5) M-1 S-1, consistent within a 13-fold variation in lactate dehydrogenase. NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO2 is estimated to be 2.0 X 10(6) M-1 S-1.


Purification and characterization of the alpha-D-mannosidase of rat liver cytosol. Three forms of alpha-D-mannosidase have previously been identified in rat liver, and each is localized in a different subcellular fraction: lysosomes, Golgi membranes, and cytosol. This communication reports the purification and characterization the cytosolic form. The enzyme was purified 12,000-fold in good yield to approximately 90% purity with the aid of the competitive inhibitor mannosylamine and dithioerythritol as stabilizers. The molecular weight of the enzyme is in the range of 372,000 to 490,000 depending on the method used. Since the subunit molecular weight is 110,000 by sodium dodecyl sulfate polyacrylamide electrophoresis, the enzyme is probably a tetramer. The pH optimum was shown to be between 5.5 and 5.9 (in the presence of 1 mM CoCl2) with the substrate p-nitrophenyl-alpha-D-mannoside. Normal Michaelis-Menten kinetics were observed with a Km of 0.14 mM. Mannosylamine was a competitive inhibitor with a Ki of 0.007 mM. The purified enzyme, stabilized by Co2+, Mn2+, and Fe2+ under some conditions, was unstable at low protein concentrations. Since an electrophoresed sample showed a positive periodic acid-Schiff stain, the enzyme may contain carbohydrate. The availability of purified cytosolic alpha-D-mannosidase should now make it possible to carry out substrate specificity, immunological, and structural studies which may shed light on the biological role of this enzyme.


Effects of two ionizing groups on the active site of human carbonic anhydrase B. Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B.


Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart. The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.


Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells. Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.


Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity. The Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.


Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation. DNA polymerase I has been purified to greater than 90% homogeneity from a strain of Escherichia coli K12 that bears the temperature-sensitive DNA polymerase I mutatation, polA12. The mutant enzyme has a reduced electrophoretic mobility and sedimentation rate. It is abnormally thermolabile and is rapidly inactivated at low salt concentrations. Its polymerase and 5' leads to 3' exonuclease activities are not grossly defective at 30 degrees, yet its capacity to promote the concerted 5' leads to 3' polymerization and the 5' leads to 3' exonucleolytic hydrolysis of nucleotides at a nick ("nick translation") is decreased 10-fold. These effects are probably the result of a significant alteration in the tertiary structure of the enzyme.


Purification of plasma membrane penicillinase from Bacillus licheniformis 749/C and comparison with exoenzyme. The membrane penicillinase of Bacillus licheniformis 749/C has been demonstrated to be a phospholipoprotein. The homogeneous enzyme gives a positive reaction for phosphorous and for unsaturated fatty acids, has a molecular weight of 33,000 in contrast to 29,000 for the exoenzyme, and contains 8 to 9 additional residues of aspartate or asparagine, 4 to 5 of serine, 7 of glutamate or glutamine, and 4 to 5 of glycine per mole. The COOH-terminal sequence of both membrane and exoenzymes is -Met-Asn-Gln-Lys-COOH; hence the extra peptide portion present in the membrane enzyme is not attached to the COOH-terminus of the exoenzyme. Procedures which readily detected the lysine residue at the NH2 terminus of the exoenzyme did not yield a positive test with the membrane form. The NH2 terminus of the membrane enzyme may be blocked by or linked to the phospholipid. A procedure for the preparation of membrane penicillinase on a large scale and an improved method for purification of the exoenzyme have been developed.


Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Temperature dependence of oxygen binding and pH dependence of subunit dissociation. The temperature dependence of the oxygen equilibrium of tadpole hemoglobin has been determined between 0 degrees and 32 degrees for the unfractionated but phosphate-free lysate and between 12 degrees and 32 degrees for each of the four isolated components between pH 6 and 10 in 0.05 M cacodylate, Tris, or glycine buffers containing 0.1 M NaCl and 1 mM EDTA. Under these conditions the Bohr effect (defined as deltalog p50/deltapH) of the unfractionated lysate is positive at low temperatures between pH 6 and 8.5 and is negative above pH 8.5 to 8.8 at any temperature. As the temperature rises the Bohr effect below pH 8.5 changes greatly. In the interval pH 7.0 to 7.5, the magnitude of the Bohr effect decreases from + 0.28 at 0 degrees to zero at about 24 degrees and becomes negative, as in mammalian hemoglobins, above this temperature. Measurements with the isolated components show that the temperature dependence of oxygen binding for Components I and II and for Components III and IV is very similar. For both sets of components the apparent overall enthalpy of oxygenation at pH 7.5 is about -16.4 kcal/mol and -12.6 kcal/mol at pH 9.5. The measured enthalpies include contributions from the active Bohr groups, the buffer ions themselves, the hemoglobin groups contributing buffering, and any pH-dependent, oxygenation-dependent binding of ions such as chloride by the hemoglobin. The apportioning of the total enthalpy among these various processes remains to be determined. Between pH 8 and 10.5 tadpole oxyhemoglobin undergoes a pH-dependent dissociation from tetramer to dimer. The pH dependence of the apparent tetramer-dimer dissociation constant indicates that at pH 9.5 the dissociation of each tetramer is accompanied by the release of approximately 2 protons. In this pH range the oxygen equilibrium measurements indicate that about 0.5 proton is released for each oxygen molecule bound. The results are consistent with the conclusion that one acid group per alphabeta dimer changes its pK from about 10 to 8 or below upon dissociation of the tetramer.


Dissociation of CO from carboxyhemoglobin. The reaction between carboxyhemoglobin and reduced microperoxidase (MP): Hb4(CO)4 + 4MP=Hb4 + 4MPCO, recently reported by us, has been further studied. By generating species Hb4(CO), Hb4(CO)2, and Hb(CO)3 in the stopped flow cuvette by the reaction of dithionite with the species of the general formula Hb4(O2)x(CO)y(x + y=4) in the presence of microperoxidase it has been possible to determine the stepwise CO dissociation rate constants l4, l3, l2, and l1. The overall CO dissociation rate constant l, which is the same in this system as l4, is not affected by 2,3-diphosphoglyceric acid. The activation energy of the reaction is 21,400 cal in 15-25 degrees range. The ratio deltal/deltapH is approximately 3 in 6.5 to 7.5 pH range. The kinetic data indicate that, compared to HbO2, the contribution to the cooperativity of the dissociation rate constants of carboxyhemoglobin is greatly reduced. The ligand-dependent differences in the reactions of Hb with CO, O2, and NO suggest that in the combination reactions the ligand plays an active role in the rate-limiting step.


Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH. Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...


A transition state analog of lysozyme catalysis prepared from the bacterial cell wall tetrasaccharide. Treatment of the cell wall tetrasaccharide GlcNAcbeta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc with alkali resulted in the formation of the unsaturated tetrasaccharide GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen. The same compound was also formed by transglycosylation upon incubation of the unmodified tetrasaccharide with the unsaturated disaccharide GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen (Tipper, D. J. (1968) Biochemistry 7, 1441-1449) and hen egg white lysozyme. The unsaturated tetrasaccharide was further characterized by paper electrophoresis, amino sugar analysis, and NMR. From NMR analysis it is concluded that the delta2,3-2-acetamido-2-deoxy-D-glucoseen at the reducing end of the unsaturated tetrasaccharide has a half-chair conformation. This conformation is similar to the one proposed for the sugar at subsite D in the lysozyme-substrate complex in the transition state. Addition of the unsaturated tetrasaccharide to a solution of hen egg white lysozyme quenched the fluorescence of the enzyme and shifted the fluorescence maximum to the blue, similar to the effect produced by the parent compound. The association constant of the unsaturated tetrasaccharide and lysozyme was measured at pH 6.0 and 24 degrees by spectrofluorimetry and microcalorimetry and found to be 1.45 X 10(5) M-1 and 2.5 X 10(5) M-1, respectively. The average value is 100 times higher than that found for the binding of unmodified tetrasaccharide to the enzyme under the same conditions. The unsaturated tetrasaccharide proved to be a better inhibitor of the lysis of Micrococcus luteus cells than the parent compound by a factor of 35. These results support the hypothesis that the active site of the enzyme is constructed so as to bind the transition state for the reaction it catalyzes more firmly than the substrate itself.


The study of 1-electron equivalent oxidation-reduction reactions by fast pulse generation of reagents. Cytochrome c/ferri-ferrocyanide system. The method of pulse radiolysis was used to generate reagents in situ in times (500 ns to 1.5 mus) short compared with the rates of the observed biochemical processes. This "instant" mixing technique is compared with rapid stopped flow measurements (limited in rates and concentrations) and T-jump measurements (limited to relaxation in the neighborhood of equilibrium) for the ferro-ferricytochrome c (C(II)-C(III))/ferro-ferricyanide (FCN(II)-FCN(III)) system. The reagents generated in situ were C(II) or FCN(III). Kinetically indistinguishable binding sites exist on C(II) and C(III) for hexacyanide anions. Reductive electron transfer to the protein proceeds within the FCN(II)-C(III) complex, with a rate of 400 s-1. The binding of FCN(II) on C(II) slows down the oxidation of C(II) by FCN(III). The sites of interaction on C(II) or C(III) with FCN(III) show effective charges of approximately +2. The association constant per binding site derived from the kinetics of electron transfer is greater than or equal to 10(4) M-1 for FCN(II)-C(II) and less than or equal to 10(4) M-1 for FCN(III)-C(III). Specific clusters of amino acids in the model of cytochrome C are suggested as binding sites. The oxidation-reduction reactions of FCN appear to involve electron equivalent transfer to and from such somewhat remote binding sites on the protein. Anions such as phosphate or sulphate also bind to these, less strongly than hexacyanides. In the presence of perchlorate the kinetics show the resolution of the pK=9.3 of C(III) into two parts: (a) optical changes at 695 nm due to ligand interchange on the heme-iron, unaffected by perchlorate and (b), a kinetic change leading to biphasic oxidation of C(II), with pK=7.4. This is attributed to the effect of perchlorate on water structure in the close environment of the binding sites. The high rate of oxidation of relaxed C(II) by FCN(III), (2 X 10(8) M-1 S-1 at mu=0) is not in agreement with an outer sphere Marcus mechanism. Nonrelaxed C(II) having a structure closer to C(III) transfers electron to FCN(III) even faster (k=3 X 10(9) M-1 S-1 at mu=0).


beta-Mannosidase from the mushroom Polyporus sulfureus. beta-D-Mannosidase (EC 3.2.1.25), a useful tool for the structural studies of heterosaccharide chains, has been isolated in a highly purified form from the fruiting bodies of the mushroom Polyporus sulfureus. This mushroom is unique among reported sources of this enzyme in that it has the advantage of being almost free of alpha-mannosidase activity. The purification procedure involves ammonium sulfate fractionation followed by Sephadex G-100 filtration and chromatography on columns of DEAE-cellulose and hydroxylapatite. The final enzyme preparation gives essentially a single band on disc gel electrophoresis. The purified enzyme liberates the beta-D-mannopyranosyl unit from various natural substrates such as the core glycopeptide, Man(GlcNAc)2-Asn isolated from ovalbumin, from Taka-amylase A, and from human alpha1-acid glycoprotein. It also hydrolyzes (Man)2-GlcNAc from the urine of an alpha-mannosidosis patient, 1,4-D-mannobiose and mannotriose isolated from ivory nut mannan, 4-O-beta-D-mannopyranosyl-L-rhamnose, 6-O-beta-D-mannopyranosyl-D-galactose and 4-O-beta-D-mannopyranosyl-N-acetylglucosamine. The molecular weight of this enzyme is estimated to be about 64,000 by gel filtration. For p-nitrophenyl-beta-D-mannopyranoside, the pH optimum is between 2.4 and 3.4 and the Km is 1.6 mM.


The carbamate reaction of glycylglycine, plasma, and tissue extracts evaluated by a pH stopped flow apparatus. We have used a stopped flow rapid reaction pH apparatus to investigate the carbamate equilibrium in glycylglycine solutions and in three biological tissues, human plasma, sheep muscle, and sheep brain, as well as to investigate the kinetics of carbamate formation in glyclyglycine solution and in human plasma. The rapid reaction apparatus was equipped with a pH sensitive glass electrode in order to follow the time course of pH from 0.005 to 100 s after rapid mixing of a solution of amine or protein and CO2. Two phases of the pH curve were observed: a fast phase representing carbamate formation, and a slow phase due to the hydration of CO2 which was uncatalyzed since a carbonic anhydrase inhibitor was added to the biological solutions. From the time course of pH change during the fast phase K2, the R-NH2 ionization constant, and Kc, the carbamate equilibrium constant as well as the velocity constant for the formation of carbamate, ka could be calculated from data at different pH and pCO2. The carbamate formed in glycylglycine solutions over a wide range of pH and pCO2 was found consistent with the theory of carbamate formation and with published data. At ionic strength 0.16 and 37 degrees pK is 7.67. pKc 4.58. The heat of the carbamate reaction (deltaH) was calculated to be -3.2 kcal/mol between 20 degrees and 37 degrees. Kt of glycylglycine depends quantitatively on ionic strength as predicted by the Debye-Huckel theory. With ionic strength 0.16 ku was found to be 2,500 M1 S1 at 37 degrees. The activation energy of carbamate formation is 6.7 kcal/mol. Carbamate measurements in human plasma at pCO2 from 38 to 359 Torr. pH from 6.9 to 8.3, temperature 37 degrees, and ionic strength 0.15 provided evidence that two kinds of amino groups participate in carbamate formation. From the equilibrium constants computed for the two species they could be identified as alpha- and  epsilon-amino groups. On the basis of a protein molecular weight of 69.000. 0.6 alpha-amino groups/molecule with pKz=7.0 and pKc=4.2, and 5.9 epsilon-amino groups/molecule with pKz=9.0 and pKc=4.3 contribute to carbamate formation. The velocity constant ka was estimated to be 4,950 M1 S1 for the alpha-amino groups and 13,800 M1 S1 for the epsilon-amino groups. Under physiological conditions (pCO2=40 Torr. pH=7.4). The concentration of carbamate in plasma is 0.6 mM and the half-time of carbamate formation is 0.05 s. In extracts prepared from sheep brain at 37 degrees pH=7 and pCO2=35 Torr. the carbamate formation was estimated to be 0.8 mM. With pCO2=70 Torr and the same pH and temperature the carbamate concentration in muscle approximates 0.3 mM and increases to 7 mM as pH rises to 8. It is concluded that, as in plasma, a considerable number of epsilon-amino groups appear to be available for carbamate formation in these tissues.


The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts. The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.


Variation in aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity in heteroploid and predominantly diploid rat liver cells in culture. Aryl hydrocarbon (benzo(a)pyrene) hydroxylase is present and inducible in Buffalo rat liver cells in culture. There is substantial variation in both basal and inducible hydroxylase activities among heteroploid subclones isolated from a heteroploid parent population, and among diploid subclones isolated from a diploid parent population. This variation is not related to differences in the growth characteristics of the subclones, or to differences in their chromosome number. The results indicate that substantial heterogeneity in both basal and induced hydroxylase activity develops during the growth of both heteroploid and diploid cell strains in culture. These findings indicate that diploid cell populations are not necessarily homogeneous with respect to aryl hydrocarbon hydroxylas activity. This observation may complicate the interpretation of experiments involving somatic cell hybridization or polycyclic hydrocarbon-induced transformation and/or cytotoxicity. This heterogeneity in hydroxylase activity develops rather rapidly (2-3 mo of culture), in the absence of any apparent mutational stress.


Isocratic separation of some purine nucleotide, nucleoside, and base metabolites from biological extracts by high-performance liquid chromatography. Separation of ATP, ADP, AMP, adenine, adenosine, cAMP, ITP, IDP, IMP, hypoxanthine, inosine, cIMP, the guanine series, NAD, NADPH, xanthine, 3-methylxanthine, theobromine, theophylline, and caffeine was accomplished using high-performance liquid chromatography with a microparticulate reversed-phase column. Under isocratic conditions all compounds could be eluted with reasonable resolution and retention time. Quantitation by peak height for several of the compounds was used to the 10-ng level.


Synthetic MIF has no effect on beta-MSH and ACTH hypersecretion in Nelson's syndrome. The effect of synthetic MIF (H-Pro-Leu-Gly-NH2) on beta-MSH secretion was studied in five patients with Nelson's syndrome and in one patient with Addison's disease. Two milligrams of the tripetide were injected intravenously (1 mg in an acute injection, followed by a 30-minute-infusion of 1 mg in 20 ml of saline solution). No consistent effect could be observed during the 90-minute period after the beginning of the infusion. In the same patients, LVP stimulation and dexamethasone suppression tests brought about significant changes in the plasma beta-MSH and ACTH levels.


Description of a polyvalent conjugate and a new serogroup of Bacteroides melaninogenicus by fluorescent antibody staining. A polyvalent conjugate (fluorescein isothiocyanate-labeled antibody reagent) containing serogroups A, B, and C conjugates was prepared. This polyvalent conjugate gave a positive fluorescent antibody (FA) stain with 49 stains of Bacteroides melaninogenicus representing serogroups A, B, and C. When additional strains (92 strains) of the three subspecies of B. melaninogenicus were examined by the FA stain, with A, B, and C, and polyvalent conjugates, nine strains of B. melaninogenicus subsp. intermedius failed to give a positive stain with any conjugate. Therefore, an FA conjugate was prepared with the antiserum to one of these strains (532-70A); all nine strains stained positively with this conjugate. These nine strains were biochemically characteristic of B. melaninogenicus subsp. intermedius; thus, these strains were designated as a new serogroup, serogroup C-1. A new polyvalent conjugate containing serogroups A, B, C, and C-1 was prepared. This polyvalent conjugate stained positively with 23 representative strains from serogroups A, B, C, and C-1. The new conjugates failed to stain positively with other anaerobes and aerobes tested. The four individual conjugates, as well as the polyvalent conjugate, may be used for a more rapid identification of B. melaninogenicus than is possible by biochemical testing.


Regulation of acid-base equilibrium in chronic hypocapnia. Evidence that the response of the kidney is not geared to the defense of extracellular (H+). It is generally believed that the reduction in plasma [HCO3] characteristic of chronic hypocapnia results from renal homeostatic mechanisms designed to minimize the alkalemia produced by.the hypocapneic state. To test this hypothesis, we have induced chronic hypocapnia in dogs in which plasma [HCO3] had previously been markedly reduced (from 21 to 15 meq/liter) by the prolonged feeding of HCl. The PaCO2 of chronically acid-fed animals was reduced from 32 to 15 mm Hg by placing the animials in a large environmental chamber containing 9% oxygen. In response to this reduction in PaCO2, mean plasma [HCO3] fell by 8.6 meq/liter, reaching a new steady-state level of 6.4 meq/liter. This decrement in plasma [HCO3] is almost identical to the 8.1 meq/liter decrement previously observed in normal (nonacid-fed) animals in which the same degree of chronic hypocapnia had been induced. Thus, in both normal and HCl-fed animals, the renal response to chronic hypocapnia causes plasma [HCO3] to fall by approximately 0.5 meq/liter for each millimeter of Hg reduction in CO2 tension. By contrast, the response of plasma [H+] in the two groups was markedly different. Instead of the fall in [H+] which is seen during chronic hypocapnia in normal animals, [H+] in HCl-fed animals rose significantly from 53 to 59 neq/liter (pH 7.28-7.23). This seemingly paradoxical response is, of course, an expression of the constraints imposed by the Henderson equation and reflects the fact that the percent fall in [HCO3] in the HCl-fed animals was greater than the percent fall in PaCO2. These findings clearly indicate that in chronic hypocapnia the kidney cannot be regarded as the effector limb in a homeostatic feedback system geared to the defense of systemic acidity.


Characterization of the protease activity in the chemotactic factor inactivator. The chemotactic factor inactivator (CFI) isolated from human serum contains a kininase activity that causes extensive hydrolysis of bradykinin. The highly chemotactic synthetic peptide Met-Leu-Phe was completely hydrolyzed by CFI preparations. The release of the constituent amino acids from this peptide coincided with a loss of its chemotactic activity. The N-formyl, but not the amide derivative, of the leukotactic peptide Met-Leu-Phe was resistant to the action of CFI, as evidenced by chemotactic and biochemical assays. Examination of the specificity of CFI proteolysis revealed that short polypeptide substrates are degraded sequentially from the amino terminus. Larger peptides are less extensively hydrolyzed, and the patterns of hydrolysis are more complex. Inactivation of the bacterial chemotactic factors by CFI was overcome by the addition of high concentrations of peptides which were substrated for CFI. CFI preparations readily hydrolyzed the peptide Arg-Phe-Ala. The constituent amino acids were conveniently identified by thin-layer chromatography method. This procedure afforded a rapid assay for measuring CFI activity in the whole human serum as well as in fractions throughout the purification steps. Moreover, CFI also hydrolyzed L-leucyl-beta-napthylamide at rates comparable to peptides. Thus, L-leucyl-beta-napthylamide served as a useful substrate for estimating  CFI activity in preparations at various stages of purification. This substrate was also useful in kinetic studies. The results from these studies indicate an aminopeptidase activity is the mechanism whereby CFI inhibits the activity of chemotactic substrates.


Antibody-dependent cell-mediated cytotoxicity in selected autoimmune diseases. Antibody-dependent cell-mediated cytotoxicity mediated by peripheral blood lymphocytes was studied in patients with systemic lupus erythematosus, polyarteritis nodosa. Sjogren's syndrome, and rheumatoid arthritis. The target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody. Antibody-dependent cell-mediated cytotoxic activity was normal in Sjogren's syndrome and rheumatoid arthritis but significantly decreased (P is less than 0.001) in active systemic lupus erythematosus and in two patients with polyarteritis nodosa. A partial regeneration of antibody-dependent cell-mediated cytotoxic activity was obtained by treatment with pronase and DNase followed by overnight incubation. Sera from patients with systemic lupus erythematosus inhibited antibody-dependent cell-mediated cytotoxic activity of normal lymphocytes. The inhibitory activity was studied by specific immunoadsorption and sucrose density geadient ultracentrifugation. Removal of IgG but not IgM greatly reduced inhibition. Inhibitory factors were present in 7S and heavier fractions containing IgG. Five systemic lupus erythematosus patients were studied serially to determine if improvement in clinical status could be correlated with a decrease in serum inhibitory factors as studied by inhibition of normal antibody-dependent cell-mediated cytotoxicity. Indeed, a greater serum inhibitory capacity was found in each patient during periods of greater disease activity.


The role of adrenergic mechanisms in the substrate and hormonal response to insulin-induced hypoglycemia in man. Sequential determinations of glucose outflow and inflow, and rates of gluconeogenesis from alanine, before, during and after insulin-induced hypoglycemia were obtained in relation to alterations in circulating epinephrine, norepinephrine, glucagon, cortisol, and growth hormone in six normal subjects. Insulin decreased the mean (+/-SEM) plasma glucose from 89+/-3 to 39+/-2 mg/dl 25 min after injection, but this decline ceased despite serum insulin levels of 153+/-22 mul/ml. Before insulin, glucose inflow and outflow were constant averaging 125.3+/-7.1 mg/kg per h. 15 min after insulin, mean glucose outflow increased threefold, but then decreased at 25 min, reaching a rate 15% less than the preinsulin rate. Glucose inflow decreased 80% 15 min after insulin, but increased at 25 min, reaching a maximum of twice the basal rate. Gluconeogenesis from alanine decreased 68% 15 min after insulin, but returned to preinsulin rates at 25 min, and remained constant for the next 25 min, after which it increased linearly. A fourfold increase in mean plasma epinephrine was found 20 min after insulin, with maximal levels 50 times basal. Plasma norepinephrine concentrations first increased significantly at 25 min after insulin, whereas significantly increased levels of cortisol and glucagon occurred at 30 min, and growth hormone at 40 min after insulin. Thus, insulin-induced hypoglycemia in man results from both a decrease in glucose production and an increase in glucose utilization. Accelerated glycogenolysis produced much of the initial, posthypoglycemic increment in glucose production. The contribution of glycogenolysis decreased with time, while that of gluconeogenesis from alanine increased. Of the hormones studied, only the increments in plasma catecholamines preceded or coincided with the measured increase in glucose production after hypoglycemia. It therefore seems probable that adrenergic mechanisms play a major role in the initiation of counter-regulatory responses to insulin-induced hypoglycemia in man.


The effect of hyperventilation on distal nephron hydrogen ion secretion. This study was designed to determine the effect of acute hyperventilation on distal nephron hydrogen ion secretion. The blood PCO2 declined and stabilized rapidly when bicarbonate loaded rats were hyperventilated. In contrast, the urine PCO2 declined slowly, resulting in an early increase in the urine minus blood (U-B) PCO2 which could not be obliterated by carbonic anhydrase infusion. Within approximately 50 min, the U-B PCO2 in the hyperventilated and carbonic anhydrase infused rats approached zero. Consequently, equilibrium between collecting duct urine and arterial blood PCO2 was then presumed to exist. This provided the basis for the subsequent studies on a series of rats. The U-B PCO2 decreased from a control of 22+/-1 mm Hg (mean+/-SEM) to 11+/-2 mm Hg (mean+/-SEM) with hypocapnia, and rose again to its control value when the blood PCO2 returned to prehyperventilation values. This decline in U-B PCO2 with acute hyperventilation could not be attributed to changes in urine flow, phosphate, or bicarbonate excretion, suggesting, therefore, a decrease in distal nephron (probably collecting duct) hydrogen ion secretion with acute hyperventilation. Possible pitfalls in the interpretation of the UB PCO2 are illustrated.


Enzymic basis for cyclic GMP accumulation in degenerative photoreceptor cells of mouse retina. The activities of guanylate cyclase, guanosine 3', 5'-monophosphate (cyclic GMP) phosphodiesterase and 5'-nucleotidase were measured during postnatal development in retinas of control and C3H/HeJ mice. In control retina, each of these enzyme activities increases in conjunction with photoreceptor cell differentiation and maturation. In C3H retina, guanylate cyclase and 5-nucleotidase activities increase with photoreceptor cell development and decrease with photoreceptor cell death. However, the activity of a class of cyclic GMP phosphodiesterase which distinguishes the photoreceptor cells of control mice and those of several other species is not demonstrable in retina of C3H mice at any age. It is suggested that the deficiency in cyclic GMP phosphodiesterase activity may account for the accumulation of cyclic GMP which has been shown to occur in the C3H photoreceptor cells before they degenerate.


beta-Adrenergic receptors in rat brain.

beta-Adrenergic receptors in rat brain. 125I-Iodohydroxybenzylpindolol ([125I] IHYP), a potent beta-adrenergic receptor antagonist, has been used to study beta-adrenergic receptors in rat brain. Binding of [125I] IHYP (30 pM) to a membrane fraction min and dissociation took place with a half time of about 16 min. Phentolamine (10(-4) M) decreased non-receptor binding but it had no effect on the binding of [125I] IHYP to beta-adrenergic receptors in cortex, cerebellum or caudate. In the presence of phentolamine specific binding (defined as binding which was blocked by 0.3 muM dl-propranolol) represented 70-85% of total binding. The binding of [125I] IHYP was inhibited by beta-adrenergic agonists and antagonists. d-Stereoisomers were 2-3 orders of magnitude less potent than the corresponding 1-isomers. The denstiy of [125I] IHYP binding sites was studied in membrane fractions from cerebral cortex, cerebellum, and caudate nucleus by means of Scatchard analysis. The K(D) of [125I] IHYP was similar in the three regions studied, and the density of [125I] IHYP binding sites was approximately 50% greater in the cortex and caudate than in the cerebellum. The Hill coefficient for the binding of [125I] IHYP to membranes from cerebral cortex was 1.02. The properties of the binding of [125I] IHYP are similar to those which would be expected of binding to beta-adrenergic receptors in vitro.


Effects of citrus pulp in high urea rations for steers. Effects of pelleted and conventional citrus pulp as a replacement for corn, with soybean meal added to keep protein comparable, were tested in rations with 5% urea and 33.33% sugarcane bagasse for fistulated steers. Thus, all rations were low in readily fermented carbohydrates other than those of corn or citrus pulp. Evaluation criteria were concentrations of urea in blood and of pH, ammonia, and volatile fatty acids of rumen fluid. Citrus pulp for diets 1, 2, 3, and 4 was 0, 19, 38, or 55%. Rumen fluid and blood were sampled 1 h before and 2, 4, 7, and 12 h after feed was placed directly into the rumen. No differences between pelleted and conventional pulp or among time trends were significant except that for both forms rumen ammonia was lower with the two highest percents of citrus pulp. Addition of citrus pulp at 0, 19, 38, or 55% of the ration reduced rumen pH (6.85, 6.65, 6.61, 6.51). Blood urea and rumen ammonia decreased in steers fed 19, 38, or 55% pulp; thus, the acetic to propionic ratio was higher. Butyric acid changed only in the time trend. Total volatile fatty acid concentrations were higher at 19, 38, and 55% than at 0% pulp. They were higher at 38 and 55 than at 19%.


Aspirin-induced gastritis and gastrointestinal bleeding. Aspirin-induced gastritis and gastrointestinal hemorrhage were reviewed and discussed on the basis of currently available literature. Acute hemorrhagic gastritis occurs in from 50% to 70% of all patients taking aspirin, is not directly related to dose size, and can be severe enough to cause death in a few cases. No tolerance appears to ever develop. The mechanism that causes this bleeding is not definite, but the back diffusion of H+ ions accross the gastric barrier seems to bear primary responsibility, with physical erosion, prolonged platelet bleeding, and the effect of low pH values also being possible explanations. There appears to be less acid present in the stomach when bleeding occurs, but this is a masking effect of the aspirin that causes increased absorption of the H+ ions. Factors important in determining pharmaceutical formulation are method of administration, particle size of the aspirin, duration of contact between the drug and the mucosa, presence of buffers in the drug to raise the gastric pH, dissolution rate of the drug in the stomach, and ionization characteristics of the drug itself. Gastrointestinal blood loss caused by aspirin can be minimized by administering the drug in one of these forms:--a dilute solution of acetylsalicylate;--an intravenously injected solution;--a very rapidly dissolving and rapidly absorbed tablet;--a solution with sufficiently large amounts of antacid added;--a fine-grain, highly buffered aspirin tablet;--an enteric-coated tablet that does not dissolve in the stomach; or--an aspirin substitute such as acetaminophen.


Urologic sepsis/shock.

Urologic sepsis/shock. At Columbia-Presbyterian Medical Center during the six-year period 1968-1973, there were 1236 cases of sepsis from Gram-negative pathogens; 124 of these originated in the urinary tract. Of these 124 patients, 19 died-a mortality rate of 15.3 percent. There were 205 deaths among the 1236 patients with sepsis from Gramnegative organisms-a mortality rate of 16.6 percent. Previously, in the 1959-1964 and 1965-19067 periods, the mortality rates had been 56.3 percent and 19.6 percent respectively. The lowered mortality rate during 1968-1973 for urologic sepsis/shock was associated with improved management procedures: a) preventive measures such as postponement of urologic instrumentation and surgical intervention in patients infected with drug-resistant urea splitters, until the infection is under control, with emergency surgical patients being treated by susceptibility-tested drugs to control possible postoperative complications; b) early diagnosis and treatment of sepsis and immediate administration of bactericidal antibiotics parenterally; c) immediate restoration of fluid/electrolyte balance, with monitoring of renal and pulmonary functions and metabolic acidosis; and d) early administration of large pharmacologic doses of glucocorticoids, with monitoring of the microcirculation and use of beta-adrenergic isoproterenol.


[Study of fetal heart rate in deliveries complicated by fetal acidosis]. A study has been carried out on the parameters of the graph of the fetal heart rate (the basal rate and dips) in 44 cases of acute fetal distress with a pH lower than 7.2 in the blood and in 30 normal deliveries. The statistical analysis confirms that there is a significant rise in the number of heart rate abnormalities such as persistent bradycardia or persistent tachycardia and with dips during deliveries with fetal acidosis. The frequency of these abnormalities increases with the degree of acidosis. Sometimes the abnormalities in the fetal heart rate precede the appearance of the acidosis. All the same the discovery of these abnormalities does not by itself make a precise diagnosis of fetal distress because we do find these abnormalities in a certain number of cases even in normal deliveries. Only measuring fetal pH at a definite time can establish the diagnosis of fetal distress and the severity of the condition.


The cleaning and disinfection by heat of bedpans in automatic and semi-automatic machines. This work is concerned with the cleaning and disinfection by heat of stainless-steel and polypropylene bedpans, which had been soiled with either a biological contaminant, human serum albumin (HSA) labelled with technetium-99m 99m(Tc), or a bacteriological contaminant, streptococcus faecalis mixed with Tc-labelled HSA. Results of cleaning and disinfection achieved with a Test Machine and those achieved by procedures adopted in eight different wards of a general hospital are reported. Bedpan washers installed in wards were found to be less efficient than the Test Machine, at least partly because of inadequate maintenance. Stainless-steel and polypropylene bedpans gave essentially the same results.


Factors affecting the sensitivity of replicating McCoy cells in the isolation and growth of chlamydia A (TRIC agents). Normal non-irradiated McCoy cell cultures provide a sensitive and reproducible method for the isolation of oculo-genital strains of chlamydia A directly from human secretions and for laboratory studies with these agents. Since September 1973, chlamydia have been isolated from 175 of 562 women (32-1%) attending venereal disease clinics. Freshly isolated and low passage strains have been used to determine the importance of centrifugation, constitution and pH of the tissue culture medium, and the temperature of incubation in controlling the efficiency of plating in the method.


Effect of six weekly transfusions on canine marrow grafts: tests for sensitization and abrogation of sensitization by procarbazine and antithymocyte serum. We have previously shown that blood transfusions can immunize a dog and lead to rejection of a subsequent marrow graft despite lethal total body irradiation (TBI). Sensitization to histocompatibility antigens induced by two prior transfusions of whole blood could be overcome by a regimen of procarbazine and anti-thymocyte serum (ATS) preceding TBI. The current study investigated a) whether this regimen could abrogate sensitization induced by six weekly transfusions given from days --50 to --15 preceding a marrow graft, and b) whether platelet survival studies and two in vitro tests of immunity could predict marrow graft rejection. All donor-recipient pairs were histoincompatible, unrelated, and of different breed. Twenty-two recipients received platelet concentrate transfusions and eight received whole blood transfusions. Recipients were given 1200 R TBI and a graft of marrow and peripheral blood leukocytes from the transfusion donor on day 0. Three of 15 recipients (20%) given procarbazine, 12.5 mg/kg i.v. on days --8, --6, and --4, and ATS, 0.6 ml/kg subcutaneously on days --7, --5 and --3, Rejected their grafts, whereas 11 of 15 dogs (73%) not given procarbazine and ATS rejected their grafts (p less than 0.01). Serum lymphocytotoxic antibodies, peripheral leukocyte migration inhibition, and in vivo donor platelet recovery and survival were studied in those recipients receiving six weekly transfusions and in 18 other recipients receiving a single donor transfusion 3 months before marrow grafting. No significant correlation was found among these in vitro and in vivo tests of sensitization. Sensitization to marrow grafts was not reliably detected by the presence of cytotoxic antibodies or leukocyte migration inhibition. Platelet survival, however, was positively correlated with the results of marrow grafting in 12 of 15 (80%) evaluable recipients (p approximately 0.15).


Fine structure of three different anti-fluorescein combining sites: induced circular dichroism of hapten bound to autologous and heterologous recombinants. We have previously reported the sequential appearance in hyperimmunized rabbits of three distinct types of antifluorescein-combining sites that can be distinguished by the characteristic induced circular dichroism (CD) of the bound hapten, fluorescein. Such induced CD depends on the configuration of the surrounding residues, and, in the case of anti-fluorescein, can be localized to the configuration of the sub-site which binds the hydroxyxanthenone moiety of fluorescein. In the present investigation, we have studied the fine structure of both autologous and heterologous recombinant sites and of the free chains by measuring the induced CD of bound hapten. Heavy chain dimers at pH 5.4 bound fluorescein that displayed a weak negative CD band which is different from that observed in any of the native antibodies. No induced CD was observed with light chains. Thus, the hydroxyxanthenone subsite does not exist intact in any of the isolated chains. Fluorescein bound to purified autologous recombinants, when studied at pH 7.5, exhibited CD spectra very similar to those observed for the original antibodies. Most significantly, fluorescein bound to purified heterologous recombinants, prepared in all possible combinations, showed CD spectra most similar to those exhibited by sites from which the heavy chain was derived. Thus, the microenvironment of the subsite appears to be due primarily to the heavy chain.


Biological expressions of lymphocyte activation. V. Characterization of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells. Supernatant fluids from murine spleen cell cultures incubated with concanavalin A for 48 hr contain a factor(s), soluble immune response suppressor (SIRS), which suppresses plaque-forming cell responses to sheep erythrocytes by murine spleen cells in vitro. In the present studies, some of the biochemical and biophysical properties of SIRS were investigated. SIRS was non-dialysable; the suppressive activity was stable at 56 degrees C for 30 min, but was destroyed by treatment at 70 degrees C for 30 min, 80 degrees C for 10 min, or at pH 2. The suppressive activity was not absorbed by the stimulating antigen, SRBC, or antisera against murine IgG or mu-chain, suggesting that SIRS does not contain immunoglobulin determinants. Murine spleen and thymus, but not kidney cells, however, absorbed SIRS activity. Enzyme treatments revealed that SIRS was resistant to DNase and RNase, but was destroyed by trypsin and chymotrypsin. In gel filtration with Sephadex G-100, SIRS activity eluted in the fraction corresponding to m.w. in the range between 48,000 and 67,000. With polyacrylamide gel electrophoresis, SIRS activity migrated in the region cathodal to albumin. Isopycnic centrifugation in a cesium chloride gradient suggested that SIRS is a glycoprotein. These supernatant fluids with SIRS activity were also found to contain macrophage migration inhibitory factor (MIF). In the experiments using gel filtration, polyacrylamide gel electrophoresis, and isopycnic centrifugation to fractionate supernatant fluids, SIRS and MIF activity were found in the same fractions, and to date we have been unable to dissociate definitively SIRS activity from MIF activity.


Two distinct cellular patterns in cutaneous necrotizing angiitis. Two distinct cellular patterns of necrotizing angiitis involving venules in skin of patients with clinically identical cutaneous lesions were appreciated by the 1 -mum-thick section technique. In those individuals with serum hypocomplementemia, there was a perivenular inflitrate composed predominantly of neutrophils with fibrin deposition and nuclear debris. In patients with normal serum complement levels, in addition to an infiltrate of neutrophils and fibrin deposition, perivenular lymphocytes in various stages of activation were prominent. In both patterns the venules and not the arterioles were affected, mast cells exhibited various degrees of hypogranulation, and basophils and eosinophils were recognized only rarely. Lesions of different clinical age obtained from one hypocomplelmentemic patient and one normocomplementemic patient exhibited consistent cellular patterns, as did a single crop of lesions biopsied twice, 24 hr apart, in a patient with hypocomplementemia. No patient with hypocomplementemia became normocomplementemic or vice versa with persistence of lesions.


Effect of 9-beta-D-arabinofuranosylhypoxanthine 5'-monophosphate on genital lesions and encephalitis induced by Herpesvirus hominis type 2 in female mice. 9-beta-D-Arabinofuranosylhypoxanthine 5'-monophosphate, administered orally or topically, increased the number of survivors and mean day of death in mice inoculated intravaginally with Herpesvirus hominis type 2. Topical application of the drug in ointment significantly reduced the severity of genital lesions. Orally administered drug did not appreciably reduce genital lesions, but it did cause significant increases in the number of survivors and mean day of death, even when administration was started as late as three days after inoculation of virus. Drug applied both topically and orally was more effective than drug applied by either route along. For topical application an ointment was found to be a better vehicle than polyvinyl alcohol or dimethysulfoxide. In some circumstances the vehicle of drug administration itself had some efficacy.


Histamine metabolism. I. Thin-layer radiochromatographic assays for histaminase and histidine decarboxylase enzyme activities. Thin-layer radiochromatographic methods for the measurement of histaminase and histidine decarboxylase activities have been developed. The assays are specific for the respective enzymes, are sensitive and reproducible, and can be performed using commercially available substrates. The histaminase assay permits determination of enzyme activity from 2.5 mul of pregnancy sera, 1-2 X 10(6) human granulocytes, and microgram quantities of partially purified human placenta histaminase with an error of less than 5 per cent. The histidine decarboxylase assay permits measurement of nanogram quantities of newly formed histamine from as few as 2 X 10(4) rat peritoneal mast cells or rat basophilic leukemia cells with an error of less than 5 per cent.


Effect of insulin on mitochondrial oxidative phosphorylation and energy charge of the perfused guinea pig liver. The effect of insulin was investigated in the isolated guinea pig liver perfused with Krebs-Ringer bicarbonate buffer containing red blood cells and albumin. In the mitochondria isolated from livers perfused with 10 units of insulin per hour, the phosphorylative activity with glutamate as a substrate increased to about 160 per cent of control 60 minutes after the beginning of perfusion (p less than 0.01). Such an enhanced phosphorylative activity was accompanied by increases in the respiratory control ratio, state 3 respiration, and P/O ratio. On the other hand, in the liver perfused with insulin, the levels of the energy charge and adenine nucleotide quotient increased to a significant degree as compared to the liver without insulin (p less than 0.01 and p less than 0.05, respectively). It is suggested that insulin plays an important role as a portal factor in regulating mitochondrial oxidative phosphorylation and the levels of the phosphorylated adenine nucleotides.


The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis. Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.


The metabolism of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid into cholic: an enzyme assay using homogenates of human liver. An enzyme assay was developed to measure the conversion of the bile acid precursor, 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid (THCA), into cholic acid using homogenates of human liver biopsies. The average rate of metabolism of THCA into cholic acid was found to be 3.9 +/- 0.5 (+/- 1 SD) pmoles of cholic acid formed/mg liver/minute in twelve normal liver biopsies. This assay system can be used to determine if the syndrome of neonatal cholestasis associated with a metabolic block in the conversion of THCA into cholic acid is transmitted as a genetic trait.


Improved methods for the study of hepatic HMG CoA reductase: one-step isolation of mevalonolactone and rapid preparation of endoplasmic reticulum. Two new methods are described for the study of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. (1) Endoplasmic reticulum was rapidly prepared by diluting a 10,000 g supernatant with buffer containing 8 mM calcium chloride. The yield of protein and the specific activity of HMG CoA reductase in the pellet subsequently obtained by low speed centrifugation were nearly identical to those in the microsomal pellet prepared by ultracentrifugation. This technique may be particularly useful in studies of the rapid, in vitro modulation of the enzyme. (2) Mevalonolactone was extracted into benzene from the HMG CoA reductase assay mixture with an efficiency of 58%. There was less than 1% extraction of HMG CoA, acetoacetate, or beta-hydroxybutyrate. The extracted mevalonolactone was at least 98% pure as judged by thin-layer chromatography with four different solvent systems. These improved methods should significantly aid studies of the physiological importance of HMG CoA reductase.