Effects of iodination of tyrosyl residues on the binding and action of glucagon at its receptor. The binding and action of glucagon at its receptor in hepatic plasma membranes have been compared, as a function of pH, with that of glucagon containing iodotyrosyl residues. Iodinated glucagon, at pH 7.0 and below, binds to the receptor and activates adenylate cyclase with an affinity about threefold higher than that of native glucagon. At pH 8.5, the affinity of the receptor for native glucagon is the same as that seen at pH 7.0. However, iodinated glucagon binds with a lowered affinity with increasing pH. The decreased affinity of the iodinated hormone correlates with ionization of the iodotyrosyl phenoxy groups, which has a pKa of 8.2. It is suggested that the decreased affinity is actually due to the inability of the ionized iodoglucagon to bind to the receptor. The relative potency of native and iodoglucagon will depend, therefore, on the concentrations of ionized and un-ionized species of iodoglucagon, which in turn depend on the pH of the medium. We conclude that incorporation of iodine atoms in the tyrosyl residues of glucagon has two major effects: (i) the iodine atom increases hydrophobic interaction of the hormone with the receptor and (ii) ionization of the phenoxy groups results in the loss of biological activity possibly as the result of loss of hydrogen bonding capability. Thus, the tyrosyl residues in glucagon are critically involved in the function of the hormone.


Steady state kinetic analysis of the mechanism of guanosine triphosphate hydrolysis catalyzed by Escherichia coli elongation factor G and the ribosome. The mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor G and the ribosome in the absence of other participants in protein synthesis was examined by steady-state kinetic analysis. Optimal hydrolytic conditions were determined to be approximately pH 8.0, 20 mM Mg2+, and 80 mM NH4+. Kinetic analyses were performed under these conditions at constant elongation factor G concentrations and variable ribosome and GTP concentrations. The resulting double-reciprocal plots in conjunction with the inhibition patterns obtained with GDP indicated that the reaction occurs by an ordered mechanism in which GTP is the leading obligatory substrate. Dissociation constants for GTP and guanosine diphosphate (GDP), as well as limiting Michaelis constants for GTP and ribosomes, were calculated from the double-reciprocal plots. These values are: KSGTP = 37.0 muM, KSGDP = 16.5 muKMGTP = 8.0 muM, KMR = 0.22 muM. Inhibition was also observed at high ribosomal concentrations and suggests that inhibition was due both to the decreased breakdown of the tertiary elongation factor G-GDP-ribosome posthydrolytic complex and to the formation of a nonproductive elongation factor G-ribosome complex. A sequential mechanism with a dead-end elongation factor G-ribosome complex has been constructed to describe the hydrolysis of GTP catalyzed by elongation factor G and the ribosome.


Nuclear magnetic resonance determination of intramolecular distances in bovine pancreatic trypsin inhibitor using nitrotyrosine chelation of lanthanides. Nitration of tyrosine has been investigated as a means for chemically introducing lanthanide chelating sites at known positions in proteins. The low-field portions of the 250-MHZ and 270-MHZ 1H nuclear magnetic resonance spectra of native and chemically modified bovine pancreatic trypsin inhibitor have been studied in the presence of lanthanide ions. Comparisons of spectral changes observed with native, mononitro (tryosine 10) and dinitro (tyrosines 10 and 21) derivatives enable these changes to be separately attributed to metal bound at nitrotyrosine 21, nitrotyrosine 10, or the set of five carboxyl groups. The pH dependence of Pr(III) and Eu(III) induced chemical shifts yields stability constants of 50 and 159 M-1 for the association between lanthanides and nitrotyrosines 10 and 21, respectively. Correlation times for the interactions with Gd(III) bound to specific nitrotyrosines are estimated from the induced line broadening of resonances of the nitrotyrosine ring protons. These stability constants and correlation times are used to determine the distances from the different metal binding sites to buried backbone NH protons having resolved resonances. Comparisons with distances in the x-ray crystal structure give assignments of the NH resonances to a small set of buried backbone NH's.


Existence of electrogenic hydrogen ion/sodium ion antiport in Halobacterium halobium cell envelope vesicles. Illumination causes the extrusion of protons from Halobacterium halobium cell envelope vesicles, as a result of the action of light on bacteriorhodopsin. The protonmotive force developed is coupled to the active transport of Na+ out of the vesicles. The light-dependent ion fluxes in these vesicles were studied by following changes in the external pH, in the fluorescence of the dye, 3,3'-dipentyloxadicarbocyanine, in the 22Na content of the vesicles, and in [3H]dibenzyldimethylammonium (DDA+) accumulation. During Na+ efflux, and dependent on the presence of Na+ inside the vesicles, the initial light-induced H+ extrusion is followed by H+ influx, which results in net alkalinization of the medium at pH greater than 6.5. When the Na+ content of the vesicles is depleted, the original net of the medium is restored and large deltapH develops, accompanied by a decrease in the electrical potential. Data reported elsewhere suggest that the driving force for the transport of some amino acids consists mainly of the electrical potential, while for others it comprises the Na+ gradient as well. Glutamate transport appears to be energized only by the Na+ gradient. The development of the Na+ gradient during illumination thus plays an important role in energy coupling. The results obtained are consistent with the existence of an electrogenic H+/Na+ antiport mechanism (H+/Na+ greater than 1) in H halobium which facilitates the uphill Na+ efflux. The light-induced protonmotive force thereby becomes the driving force in forming a Na+ gradient. The presence of the proposed H+/Na+ antiporter explains many of the light-induced pH effects in intact H. halobium cells.


Fractionation of DNA from mammalian cells by alkaline elution. The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.


Polymerization pattern of insulin at pH 7.0. Sedimentation equilibrium results, obtained with bovine zinc-free insulin (with and without a component of proinsulin) at pH 7.0, I o.2, 25 degrees C, and up to a total concentration of 0.8 g/l., are shown to be consistent with three different polymerization patterns, all involving an isodesmic indefinite self-association of specified oligomeric species. The analysis procedure, based on closed solutions formed by summing infinite series, yields for each pattern a set of equilibrium constants, It is shown that a distinction between the possible patterns can be made by analyzing sedimentation equilibrium results obtained in a higher total concentration range (up to 4 g/1.) with insulin freed of zinc and proinsulin, account being taken of the composition dependence of activity coefficients. The favored pattern, which differs from that previously reported in the literature, involves the dimerization of monomeric insulin (mol wt 5734), governed by a dimerization constant of 11 X 10(4) M-1 and the isodesmic indefinite self-association of the dimer, described by an association constant of 1.7 X 10(4) M-1. This polymerization pattern is also shown to be consistent with the reaction boundary observed in sedimentation velocity experiments.


Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases. A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.


Human liver alcohol dehydrogenase: purification, composition, and catalytic features. Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.


Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Kinetic studies and fluorescence titration. Mechanisms involved in the action of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1) have been investigated by means of kinetic studies and fluorescence titration. Our results, as well as those from previous investigations, suggest that the allosteric MWC model (Monod, J., Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88-118) may be used as a first step for the explanation of the properties of the transhydrogenase. The basic reaction of the enzyme is the oxidation of reduced triphosphopyridine nucleotide (TPNH) by diphosphopyridine nucleotide (DPN+). In terms of the model, the functional R state is favored by TPNH, whereas the product triphosphopyridine nucleotide (TPN+) behaves as an allosteric inhibitor, and is therefore assumed to favor the nonfunctional T state. To a slight extent, the T state is also favored by inorganic phosphate. On the other hand, adenosine 2'-monophosphate and several other 2'-phosphate nucleotides function as activators, and hence are presumed to shift the allosteric equilibrium toward the R state. The studies in this paper suggest a specific regulatory site for the transhydrogenase.


Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Active enzyme ultracentrifugation studies. Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an S20,W value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme (S20,W = 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan, N. O. (1970), J. Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 651-664), appears, therefore, to be an inactive species. The physiological implications of the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.


Coenzyme binding by triphosphopyridine nucleotide dependent isocitrate dehydrogenase from beef liver. Equilibrium and kinetics studies. The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 muM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 muM. On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 muM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was decreased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (k1 = 20 muM-1 s-1). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzyme-NADPH complex. Then interaction between the conenzymes and the involvement of ternary complexes in the catalytic mechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry, 15, 1761-1766).


Oxidation-reduction potential measurements on chloroperoxidase and its complexes. The oxidation-reduction potential of chloroperoxidase, an enzyme which catalyzes peroxidative chlorination, bromination, and iodination reactions, has been investigated. In addition to catalyzing biological halogenation reactions, chloroperoxidase is unusual in that the carbon monoxide complex of ferrous chloroperoxidase shows the typical long wavelength Soret absorption associated with P-450 hemoproteins. The pH dependence of the chloroperoxidase oxidation-reduction potential shows a discontinuity around pH 4.7. Similarly, measurements of the affinity of ferrous chloroperoxidase for carbon monoxide monitored both by spectroscopic and potentiometric titration exhibit a discontinuity in the pH 4.7 region. Oxidation-reduction potential measurements on chloroperoxidase in a CO atmosphere also show a discontinuous pH profile. These results suggest that ferrous chloroperoxidase undergoes reversible modification at low pH and that these changes are reflected in the oxidation-reduction potential. The oxidation-reduction potential of chloroperoxidase at pH 6.9 is - 140 mV, close to that measured for cytochrome P-450cam in the presence of substrate. The oxidation-reduction potential of chloroperoxidase at pH 2.7, the pH optimum for enzymatic chlorination, is +150 mV. The oxidation-reduction potentials of the halide complexes of chloroperoxidase (chloride, bromide, and iodide) are essentially identical with the potential measurements on the native enzyme. These observations suggest that, although halide anions bind to the enzyme, they probably do not bind as an axial ligand to the heme ferric iron.


The influence of postnatal nutritional deprivation on the phospholipid content of developing rat lung. It has been previously reported that fasting may result in decreased lung surfactant production. In order to investigate this relationship and the role of nutrition in lung phospholipid synthesis, 21-day-old rats were exposed for 60 h to one of five dietary regimens: standard rat chow (controls), fasting, pure glucose, pure fat, or pure protein. After the period of fasting there was a 33% decrease in lung protein content, but there was no change in DNA content. Exposure to any of the experimental diets resulted in a decrease in tissue total phospholipid and phosphatidylcholine content per lung, but not per unit lung protein. Similarly lung lavage phospholipid and phosphatidylcholine content was decreased by 25% after fasting when expressed per lung or per unit DNA, but not per unit protein. Pulmonary cholinephosphotransferase (EC 2.7.8.2) activity was decreased in the fasted animals and those fed the protein diet, but not in the glucose or fat-fed animals. The activities of acetyl-CoA carboxylase (EC 6.4.1.2) and microsomal fatty acid elongation were decreased in all the experimental groups except for the  glucose-fed group. It is concluded that fasting results in a decrease in lung cell size but not in lung cell number. Total phospholipid and phosphatidylcholine content in lung tissue and lung lavage is decreased per cell but not per unit cell mass.


Regulatory control and function of alanine dehydrogenase from a thermophilic bacillus. L-alanine dehydrogenase, (L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.1) synthesis in a thermophilic bacillus was found to be subjected to regulatory control. Addition of L- and D-alanine and L-serine to cultures growing in the presence of either succinate or pyruvate, induced an accelerated synthesis of the alanine dehydrogenase enzyme. Synthesis of the enzyme was dependent on the presence of inducer during growth and was arrested by addition of glucose. Catabolite repression by glucose was abolished by limiting the ammonium concentration during growth. The apparent Km values of the substrates involved in alanine dehydrogenase activity are as follows (M): NH4+, 4-10(-2); pyruvate, 5-10(-4); NADH, 6-10(-5); L-alanine, 3.1-10(-3) and NAD, 2-10(-4). Alanine dehydrogenase activity was measurable at temperatures below the minimal growth temperature (at 25 degrees C) and the highest activity was found at 65 degrees C; heat denaturation occurred at 80 degrees C.


Studies on phenylalanine and tyrosine hydroxylation by rat brain tyrosine hydroxylase. Tyrosine hydroxylase (EC1.14.16.2), presumably the rate-limiting enzyme in the biosynthesis of catecholamines, is known to catalyze the hydroxylation of both phenylalanine and tyrosine. Using both an isolated enzyme preparation and a synaptosomal preparation, where some architectural integrity of the tissue has been preserved, we have attempted to evaluate the manner in which these two substrates are hydroxylated by rat brain tyrosine hydroxylase. In the presence of tetrahydrobiopterin the isolated enzyme catalyzes the hydroxylation of phenylalanine to 3,4-dihydroxyphenylalanine with the release of free tyrosine as an obligatory intermediate. In contrast, the rat brain striatal synaptosomal preparation in the presence of endogenous cofactor converts phenylalanine to 3,4-dihydroxyphenylalanine without the release of free tyrosine.


Kinetic studies of Rhus vernicifera laccase. Role of the metal centers in electron transfer. The reactions of Rhus vernicifera (monophenol,dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) with the reducing substrates hydroquinone and ascorbic acid have been investigated with the stopped-flow technique. Rhus laccase appears to be present in two molecular forms with a pH-sensitive equilibrium constant regulating the relative concentrations of each species. A model for the reaction of Rhus laccase with reducing substrates has been formulated. The model is similar to one formulated earlier for the anaerobic reduction of laccase from Polyporus versicolor (Andréasson, L.-E., Malström, B.G., Strömberg, C. and Vänngård, T. (1973) Eur. J. Biochem. 34, 434-439) and accounts for the reduction also of this enzyme. The essentials of the model are as follows: Electrons are taken up from reductants one at a time. The type 1 Cu2+ has a central role in mediating the transfer of at least one of the electrons needed for the reduction of the co-operative two-electron acceptor. Intramolecular reactions determine the concentrations of two molecular forms of the enzyme and influence the rate of reduction of the two-electron acceptor. The model, which has been used for successful simulations of the anaerobic reduction of Rhus laccase, is capable of explaining the reduction of laccases also in the presence of the inhibitor F-. In addition, the model gives an explanation of the behaviour of the laccases when reducing substrates and O2 are simultaneously present and is consistent with earlier observations of the post-steady-state reduction of the type 1 Cu2+ and the two-electron accetor (Holwerda, R.A. and Gray, H.B. (1974) J. Am. Chem. Soc. 96, 6008-6022).


Evidence for sulfhydryl groups at the active site of catechol-O-methyltransferase. Earlier studies using affinity labeling reagents have suggested the existence of two nucleophilic groups at the active site of catechol-O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6). Both nucleophilic residues are critical for catalytic activity. In an effort to elucidate the nature of these residues and to further characterize the relationship between the chemical structure and the catalytic function of this enzyme, inactivation studies using N-ethylmaleimide were undertaken. Inactivation of the enzyme by N-ethylmaleimide under pseudo first-order conditions exhibited a non-linear relationship between the log of the fraction of enzyme activity remaining and preincubation time. Kinetic analysis of this inactivation process suggested the modification by N-ethylmaleimide of two residues at the active site of the enzyme, both crucial for catalytic activity. Detailed analysis of the inactivation process including substrate protection studies, pH profiles of inactivation, and incorporation studies using N-ethyl[2,3-14C2]maleimide provided additional evidence to support this conclusion.


Microheterogeneity of arylsulfatase A from human tissues. Human arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase, EC 3.1.6.8) exhibited microheterogeneity on isoelectric focusing in polyacrylamide gels. Pure urinary enzyme gave 3 bands of activity with pI values of 4.7, 4.8 and 4.9, whereas purified liver enzyme yielded six equally spaced bands from pI 4.4 to 4.9. Detection of enzyme in the gel was made by either methylumbelliferyl sulfate or nitrocatechol sulfate. Crude enzyme preparations from human liver, kindey, placenta, brain and testis showed the six-banded pattern with varying amounts of activity in the different bands. The banding pattern of cultured human fibroblast extracts was distinctive: in addition to activity in the area of Bands 1-6 a sharp band at pI 5.1 was observed with both enzyme stains. This latter band was also present in metachromatic leukodystrophy fibroblast extracts. However, in this case the band did not appear when the specific aryl-sulfatase A stain was used. Enzyme Bands 1, 2 and 3 from urine were isolated by extraction of the gel. The three bands refocused in their initial positions; showed nearly identical enzymatic activities toward methylumbelliferyl sulfate, mitrocatechol sulfate, cerebroside sulfate and ascorbic acid 2-sulfate; and demonstrated equivalent immunological competence by antibody titration. The banding pattern of urinary arylsulfatase A was unchanged with neuraminidase treatment, whereas Bands 4-6 of the liver enzyme were converted to Bands 1-3 by this treatment. It appears that Bands 4-6 are due to sialylation of aryl-sulfatase A but that Bands 1-3 are probably due to some other type of post-ribosomal protein modification.


Coupling of the Penicillium duponti acid protease to ethylene-maleic acid (1 : 1) linear copolymer. Preparation and properties of the water-soluble derivative. The coupling of the thermostable acid protease (EC 3.4.23.-) of Penicillium duponti K 1014 to ethylene-maleic acid (1 : 1) linear copolymer in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide at pH 3.0, afforded a soluble enzyme derivative with a protein incorporation yield of 67% under optimal conditions. The protein content of the enzyme-polymer complex, the molecular weights of the reactants, and the mean value of 2.2 lysine residues per mol of enzyme found in amide linkage to the matrix, support a structure consisting of two polymer chains per mol of protease, each chain acylating a single lysine residue of the enzyme. The isoelectric point of the coupled enzyme was found to be 3,47, a value lower than that measured on the free protease (3.81). The specific activity of the bound protease against casein, at pH 3.7 and 30 degrees C, was 34% of that of the free enzyme, and at 75 degrees C increased to 70%. The increased size of the coupled enzyme resulted in an improved retention of activity by ultrafiltration membranes over that observed with free protease, alone or in admixture with ethylene-maleic acid copolymer. A water-soluble, coupled pepsin was prepared in 43% yield on protein basis by using the aminoethylmonoamide of ethylene-maleic acid copolymer and the same water-soluble carbodiimide.


Neutral elastolytic proteinase from canine leucocytes. Purification and characterization. 1. A neutral proteinase (EC 3.4.-.-) with elastolytic activity was isolated from canine bloodstream leucocytes, and purified to apparent homogeneity by a two-step procedure consisting of DEAE-Sephadex chromatography and molecular sieving on Sephadex G-75. 2. The molecular weight of the enzyme was 23 500, and the absorbance (A1%1cm) at 282 nm was 6.1. Amino acid analysis showed high content of glycine, aspartic acid, and valine, and low proportion of methionine, lysine and histidine as well as the absence of tyrosine in the enzyme molecule. 3. The proteinase was active against several protein substrates as well as towards N-t-butyloxycarbonyl-L-alanine p-nitrophenyl ester, N-acetyl-L-alanyl-tyrosine ethyl ester. 4. The enzyme was inactivated by diisopropylfluorophosphate, N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone, and N-p-tosyl-L-phenylalanine chloromethyl ketone. Inhibition by some natural proteinase inhibitors was also noted.


Purification and properties of the extracellular metallo-proteinases of Chromobacterium lividum (NCIB 10926). Four extracellular proteolytic enzymes (I-IV) (EC 3.4.22.-) were identified in static cultures of Chromobacterium lividum (NCIB 10926) by agar gel electrophoresis and isoelectric focusing. Proteinases I-III were freed of non-enzymic protein by chromatography on TEAE-cellulose and CM-cellulose. The enzyme mixture was then fractionated in a pH gradient by isoelectric focusing. All three enzymes were shown to be heat-labile metallo-enzymes. Optimal activity occurred at pH 5.6 for enzyme I and at pH 6.2 for enzymes II and III. Remazolbrilliant Blue-hide powder was a sensitive substrate for these enzymes. Proteinase I was also shown to degrade haemoglobin and casein effectively, but not myoglobin, ovalbumin or bovine serum albumin. Proteinases I-III exhibited molecular weight values of 75 000, 72 000 and 67 000 by exclusion chromatography and 71 000 and 66 000 by sodium dodecyl sulphate-poly-acrylamide-gel electrophoresis for enzyme I and II, respectively. The amino acid compositions of enzymes I and II were somewhat similar. Proteinase I was inhibited by  EDTA, 1,2-di(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic activity. Mg2+ could substitute for Ca2+ or Mn2+ for Co2+. The interrelationship of proteinases I-III is discussed.


Photocontrol of urease activity in spiropyran collagen membrane. 1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.


Magnetic studies of Chromatium flavocytochrome C552. A mechanism for heme-flavin interaction. Electron paramagnetic resonance and magnetic susceptibility studies of Chromatium flavocytochrome C552 and its diheme flavin-free subunit at temperatures below 45 degrees K are reported. The results show that in the intact protein and the subunit the two low-spin (S = 1/2) heme irons are distinguishable, giving rise to separate EPR signals. In the intact protein only, one of the heme irons exists in two different low spin environments in the pH range 5.5 to 10.5, while the other remains in a constant environment. Factors influencing the variable heme iron environment also influence flavin reactivity, indicating the existence of a mechanism for heme-flavin interaction.


Conformational transitions of monellin, an intensely sweet protein. Conformational transitions of monellin, an intensely sweet protein from the berries of Dioscoreophyllum cumminsii, were studied by the circular dichroism (CD) probe. According to the CD spectra, monellin has a low content of the helical structure and a significant amount of the pleated sheet (beta) conformation. The native conformation was found to be sensitive to alkali, sodium dodecyl sulfate, and guanidine-HC1, but it was stable in acid (e.g. pH 2.4) as shown by CD and persistence or the disappearance of sweet taste. The main chain conformation of the alkali-denatured monellin (pH 10.9) was restored upon acidification (pH 3.3) of the alkaline solutions. The tertiary structure, however, was not completely restroed, as indicated by CD in the 230-300 nm spectral zone, although the sweet taste reappeared. If the pH of a neutral solution was raised to 9.6, the CD in the near ultraviolet was significantly altered, though the sweet taste persisted. This indicates that a slight conformational change did not interfere with the effects on the taste buds. While sodium dodecyl sulfate readily disorganized the tertiary structure, the main chain was reconstructed by this reagent into a new form of higher helix content than in the native macromolecule. Reconstruction into a modified conformation of higher helix content was achieved also with 50% ethanol. The main chain conformation was not affected by 25% ethanol which produced slight changes in the CD at 230-260 nm zone and did not abolish the sweet taste.


In vitro activation of glycoprotein hormones. Hybridization of subunits from thyrotropin, lutropin and human choriogonadotropin. In vitro assembly of thyrotropin alpha and beta subunits led to an increase in content of alpha helix and beta sheet very similar to that found for gonadotropins. This association-dependent active folding involved the burying of three tyrosine residues tentatively assigned to Tyr alpha 41, Tyr beta 37 and Tyr beta 59 and common to all studied glycoprotein hormones. In vitro hybridizations between alpha and beta subunits of various hormones (thyrotropin, lutropin and choriogonadotropin) from different species (ovine, bovine and human) triggered the same molecular events as assembly of homologous subunits: the burying of three tyrosine residues and the increase of periodic structure of the folding. These changes are slow, time-dependent processes. Rates and yields of hybrid formation measured by sedimentation analysis and difference spectroscopy of tyrosines are identical, within experimental error, with the rates and yields measured by the recovery of the biological activity either the stimulation of chick thyroids for thyrotropin-beta hybrids or binding to porcine testis receptors for gonadotropin-beta hybrids. Whatever the origin of the alpha subunit, the thyrotropin-beta hybrids were not able to bind to testis receptors although active on chick thyroids. Rates and yields of hybrid formation essentially depended on the origin of the beta subunit. All the hybrids could be dissociated at acid pH with rates similar to those of native hormone. The extension to thyrotropin and various hybrids of the structural features of the in vitro assembly already recognized for gonadotropins strengthens the hypothesis that one deals with a basic activation process which also occurs in vivo after the synthesis of the subunits.


A new method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes. Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.


Study of the biological significance of cytochrome methylation. I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c. The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.


Physical studies on the size and structure of the covalently closed circular chloroplast DNA from higher plants. The size and structure of the covalently closed circular chloroplast DNAs (ctDNA) from pea, lettuce, and spinach plants, have been studied by analytical ultracentrifugation. The values of so20,w,Na+ of the native and denatured forms of the open and closed circular DNAs from these plants have been determined. The absolute molecular weight of purified closed circular pea ctDNA monomers has been determined by buoyant equilibrium sedimentation to be 89.1 (S.D. +/- 0.7)-10(6). The value of the so20,w,Na+ of open circular pea ctDNA and its molecular weight, in conjunction with corresponding values for other sizes of circular DNA, has been used to derive an empirical relationship between so20,w,Na+ and molecular weight for open circular DNAs. Using this relationship, the molecular weights of lettuce and spinach ctDNAs have been determined to be 98.2 (S.D. +/- 1.5)-10(6) and 97.2 (S.D. +/- 1.5)-10(6), respectively. At pH values 12.7 and 13, closed circular lettuce and pea ctDNAs have been found to exist as mixtures of reversibly and irreversibly denatured closed circular DNAs.


Further characterization of a DNA polymerase activity in mouse sperm nuclei. The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.


The rye embryo system as an alternative to the wheat system for protein synthesis in vitro. Isolated rye embryos are a readily available source for the preparation of very active, cell-free, protein-synthesizing systems. Incorporation levels up to 2000 pmol leucine per 50 mul assay are routinely obtained at saturating TMV (Tobacco mosaic virus) RNA concentrations; at limiting messenger RNA concentrations the incorporation exceeds 1000 leucine molecules per TMV RNA molecule. The characteristics of this cell-free system for the translation of TMV RNA are identical with those of a similarly prepared wheat germ system. The major advantage of the rye embryo system is its high reliability as compared to the unpredictable wheat germ system. Sucrose gradient analysis of the reaction mixture during the incubation shows an extensive polysome formation with TMV RNA and demonstrates efficient polypeptide chain release.


Raman pH profiles for nucleic acid constituents I. Cytidine and uridine ribonucleosides. Raman spectra of aqueous solutions of uridine and cytidine have been recorded as a function of pH with the band intensities and vibrational frequencies monitored to determine bands which may be considered as diagnostic of the concentration of the various species. Quantitative band intensity measurements indicate that not all pH-sensitive bands can be considered as diagnostic of the pK value for the acid form of the nucleoside, and for the percent species in solution. Although the accuracy of the Raman band intensity method is inherently less than that of the titrimetric or visible-ultraviolet spectrophotometric methods, the pK values and percent species agree well with those obtained from these methods. The utility of the results obtained from the pH profiles for cytidine is discussed in terms of the effect of acidification on the structural and conformational characteristics of polycytidylic acid in solution.


Raman ph profiles for nucleic acid constituents. II. 5'-AMP and 5'-GMP ribonucleotides. Raman spectra of aqueous solutions of 5'-AMP and 5'-GMP have been recorded as a function of pH. Band intensities and frequencies have been monitored to determine bands which may be considered as diagnostic for the concentration and the pK of the solution species. Quantitative band intensity measurements indicate only a selected number of bands can be considered as diagnostic of the base or the secondary phosphate proton dissociation. The utility of the pH profiles derived from specific band intensity variations for 5'-AMP is discussed in terms of the effect of acidification on solution characteristics of polyadenylic acid.


Solubility of phospholipid polar group model compounds in water. The aqueous solubilities of the Na+ and Ca2+ salts and the free acid forms of phosphorylcholine, phosphorylethanolamine and D,L-phospho-serine, respectively, were found to be below the polar group concentrations calculated for bilayers of the corresponding phospholipids.


Photophosphorylation as a function of illumination time. II. Effects of permeant buffers. (1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.


Effect of ionophores A23187 and nigericin on the light-induced redistribution of Mg2+, K+ and H+ across the thylakoid membrane. Passive redistributions of Mg2+ and K+ ions across the thylakoid membranes, occurring in association with the light-driven electrogenic influx of hydrogen ions have been examined in suspensions of broken spinach chloroplasts under a variety of conditions. (i) In accord with results of Hind el al. (Proc. Natl. Acad. Sci. U.S. (1974) 71, 1484), it was found that at a low K/Mg concentration ratio in the medium, the K-efflux is negligibly small, whereas a substantial Mg-efflux is observed. The converse is true when the K/Mg concentration ratio in the medium is high. (ii) In the presence of A23187, which was found to cause approximately a 60% inhibition of the light-induced pH-gradient, a significant influx of Mg2+ was observed in the light at a high K/Mg concentration ratio. Conversely the Mg-influx was small in the presence of A23187 when the K/Mg concentration ratio in the medium was low. Under these conditions, the Mg-influx was considerably increased upon the addition of valinomycin. A23187 was found not to affect the K-efflux in the light. (iii) The light-induced K-influx observed in the presence of nigericin also was found to be dependent on the concentration ratio of the monovalent and divalent cation. Its magnitude increased upon an increase in the K/Mg ratio. The results are interpreted in terms of a simplified model in which the total passive efflux of cations, driven by the potential set by the electrogenic proton pump, is considered to be a constant fraction of the proton influx. According to this, an increase in the flux of an ion species, induced either by raising its concentration, or by increasing its permeability through the membrane, will cause a decrease in the flux of the other cations. The relevance of the results is discussed with respect to conclusions about the involvement and relative magnitudes of the passive K and Mg effluxes across the thylakoid membrane during energization of intact chloroplasts and chloroplasts in situ.


Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal. The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculate mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with gx values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11beta-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spinal signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spinal parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and a high spin ferric gxy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.


Secretion of lecithin: cholesterol acyltransferase from isolated rat hepatocytes. 1. Lecithin:cholesterol acyltransferase is secreted from isolated rat heptocytes. 2. The secretion is stimulated when serum is added to the incubation medium. 3. Optimal conditions for secretion are: 5-10(6) hepatocytes per ml, 5 h incubation, pH 7.3-7.4 and 25% serum in the incubation medium. 4. Concomitantly with the secretion of lecithin:cholesterol acyltransferase there is a secretion of unesterified cholesterol and triacylglycerol. 5. Colchicine or cycloheximide in the incubation medium inhibits secretion of lecithin:cholesterol acyltransferase.


Distinct testicular 17-ketosteroid reductases, one in interstitial tissue and one in seminiferous tubules. Differential modulation by testosterone and metabolites of testosterone. The final step in the biosynthesis of testosterone is the reduction of androstenedione, which is catalyzed by the microsomal enzyme 17-ketosteroid reductase. Evidence is presented which suggests that there are two distinct 17-ketosteroid reductases in rat testes, one in interstitial tissue and one in seminiferous tubules. The two enzymes have different pH optima, 5.6 for the one from interstitial tissue and 6.5 for the one from seminiferous tubules. At the optimum pH, a 70-fold difference in Km values was observed, 17 muM for the interstitial tissue enzyme and 0.25 muM for the enzyme from seminiferous tubules. Testosterone and metabolites of testosterone have very different effects of each of these enzyme activities. The interstitial tissue enzyme activity is inhibited by testosterone and several 5alpha-reduced metabolites of testosterone and by estrogens. The most potent inhibitor of the steroids investigated was 5alpha-androstane-3alpha, 17beta-diol, followed by 17beta-estradiol approximately equal to dihydrotestosterone greater than testosterone greater than estrone greater than estriol. 5alpha-Androstane-3alpha, 17beta-diol and 17beta-estradiol were shown to act by competitive inhibition with apparent Ki values of 2.2 and 3.7 muM, respectively. In contrast, it was demonstrated that among the above steroids, only dihydrotestosterone inhibits the 17-ketosteroid reductase activity of seminiferous tubules and this inhibition was only observed at very high concentrations of inhibitor. Testosterone stimulated the 17-ketosteroid reductase activity of seminiferous tubules. 5alpha-Androstane-3alpha, 17beta-diol at low concentrations stimulated the enzyme activity from seminiferous tubules, while it had no effect at high concentrations. The remainder of the steroids tested had no effect on the 17-ketosteroid reductase activity of seminiferous tubules. The difference in response of the two enzyme activities suggests a mechanism for local regulation of testosterone synthesis in each testicular compartment that does not involve directly pituitary gonadotropins.


[Cathepsin D from horse spleen. II. Study of certain enzymatic properties]. This work reports some enzymatic properties of highly purified horse spleen cathepsin D. Hydrolysis rate of several proteins are compared. The Kinetic constants (Km = 4.95 10(-5) M and Vm = 1,76 delta DO/mn/mug) have been determined in the presence of a denatured haemoglobin substrate. Stability of the enzymatic preparation is discussed according to the pH, concentration and time of storage. Some investigations concerning the active site are described. Enzymatic and chemical results show that dicarboxylic and tryptophanyl residues seem to be involved in the hydrolytic process. Catalysis does not depend on sulfhydryl or seryl residues. Different salts, particularly nitrate, nitrite and polyphosphate are potent inhibitors of enzymatic activity.


The chemical ecology of Biomphalaria glabrata: the effects of ammonia on the growth rate of juvenile snails. When juvenile specimens of Biomphalaria glabrata were subjected to concentrations of ammonia ranging from 1-100 mug/ml in various media the following effects were observed: the addition of ammonia to borate buffered media caused mortality. Both borate and tris-buffered media caused a decrease in the growth rate of snails when compared with controls in SSW. The growth rates of the snails could be enhanced by increasing the concentration of ammonia to critical thresholds, but further increases beyond these thresholds resulted in growth inhibition. The toxicity of ammonia in ambient water was augmented by an an increase in pH. The possible causation and ecological significance of these effects are discussed. There are indications that the snails are physiologically well-adapted to utilize ammonia when required and also to control its excretion and uptake from the medium.


[Spectral effects of denaturation of B- and C-phycoerythrins]. B-phycoerythrin (B-PhE) from red alga Porphyridium cruentum and C-phycoerythrin (C-PhE) from blue-green alga Nostoc punctiforma were isolated. Their absorption and fluorescence spectra were measured at room and liquid nitrogen temperature. The drastic change of fluorescence and absorption maxima under dissociation of the proteins into subunits was observed. Dissociation of the C-PhE into two subunits (molecular weight 16 000 and 12 000) was revealed by SDS-acrylamide gel electrophoresis in 0.01% SDS solution at pH 7.0. The absorption spectra of subunits of both B-PhE and C-PhE were similar. The fluorescence quenching by oxidants and destructive photooxidation were negligible and increased after denaturation.


[Catalytic properties and stability of horseradish peroxidase immobilized in polyacrylamide gel]. Effect of polyacrylamide (PAA) gel on properties of horseradish peroxidase, immobilized by means of the incorporation into PAA gel is studied. Catalytic properties of immobilized enzyme are studied. Km value and pH-dependency of the enzyme activity are found to be close to those of soluble enzyme, kcat value is 3 times lower at pH 7.0. PH-stability of immobilized peroxidase at 20 degrees C and thermostability of soluble and immobilized peroxidases at pH 7.0 within the temperature range from 20 to 81 degrees C are studied. The stability of peroxidase in PAA gel is found to decrease (in 3 times at 20 degrees C, and in 17 times at 56 degrees C). A mechanism of the effect of PAA gel on catalytic properties and stability of peroxidase is discussed.


Acid-base changes of mature and premature neonates following exchange transfusion. The changes of acid-base values of 60 mature and premature neonates treated by exchange transfusion were followed. It could be shown that the blood conserves having acidic pH values caused no acidosis if the acid-base balance of the patients had been normal before transfusion. An existing acidosis frequently increased significantly in the first day of life of the mature but also in the later days in premature neonates. Thus, the determination of acid-base state prior and after transfusion of such patients seems to be important. In the case of pronounced acidosis its correction and also the control of the acid-base values after the exchange transfusion are necessary.


Active enzyme gel chromatography. I. Experimental aspects. Transport properties of active enzyme species can be studied effectively by layering a small band of enzyme-containing sample on a gel chromatographic column previously saturated with substrate. The column is optically scanned at successive time intervals to yield profiles representing the appearance of chromophoric product or disappearnce of chromophoric substrate. These profiles permit determination of the specific activity and rate of transport of the active species. Initial studies on mechanic of the technique establish the feasibility of accurately determining transport properties of active enzyme species chromatographed on gel columns. Illustrative results are presented for L-glutamate dehydrogenase and for homoserine dehydrogenase studied in both forward and reverse reactions. It is shown that the partititon cross sections derived from the rates of motion of catalytic activity are the same as those determined by equilibrium saturation experiments which directly measure the degree of partitioning by the protein. These results establish the validity of the technique for a variety of future studies. Active enzyme gel chromatography appears comparable in precision to the active enzyme sedimentation technique at current stages of development.


A calorimetric study of polyguanylic acid at neutral pH. The structure of polyguanylic acid (poly G) at neutral pH has been studied by optical and calorimetrical methods. It can be shown that diverging from earlier findings Poly G reversibly undergoes a cooperative thermal transition. Thermal denaturation curves are recorded at 253 nm as a function of the sodium ion concentration. The denaturation enthalpy of poly G in dilute aqueous solution is determined to 2.2 kcal/mole g. It is concluded, that the part of the ordered poly G structure, which gives rise to a temperature dependent cooperative transition, arises from stacking interactions of adjacent bases in the single strand.


Electron transport by C-type cytochromes. I. The reaction of horse heart cytochrome c with anionic reductants. The kinetics of reduction of horse heartcytochrome c have been investigated using the reductants sodium dithionite and potassium ferrocyanide. Sodium dithionite reduction at pH 7.0 yields rate constants of 2.8 X 10(8)M(-1)sec-1 for SO2 AND 6 X 10(5) M-1 sec-1 for S2O4 at infinite dilution. Moreover, the data presented demonstrates the participation of positively charged amino acid side chains at the site of electron transfer. The effect of pH on the reduction of ferricytochrome c requires a minimum of two pK values for description (pK1 = 7.0 +/- 0.4, pK2 = 9.3 +/- 0.3). Based on the pK values determined, one or more lysines and a residues(s) with a low pK are implicated as the positively charged residues participating in electron transfer. From a comparison of the rates of reduction of various denatured forms of cytochrome c we feel that the most viable conclusion is that electron transfer takes place at the exposed heme edge in the vicinity of the amino acid side chains indicated above. Ferrocyanide reduction of ferri-horse heart cytochrome c takes place in a kinetically complex manner. A mechanism is described which includes complexes of ferrocyanide and ferricytochrome c and ferricyanide and ferrocytochrome c. As was found for dithionite reduction a positively charged region of the cytochrome c participates in electron transfer. Combining our results with ferrocyanide and dithionite we conclude that avaible data is compatible with a single mechanism of electron transfer. It is suggested that the kinetic distinction between different reductants lies in the lifetime of the transient complex formed, with the order ferrocyanide greater than S2O4 greater than SO2.


Flash photometric experiments on the photochemical cycle of bacteriorhodopsin. The photochemical reaction cycle of bacteriorhodopsin was investigated by means of flash photometric methods. Three different intermediates with absorption maxima at about 630 nm, 411 nm, and 646 nm could be detected. Kinetic data of the occurrence of these intermediates were obtained from isolated purple membrane in different mediums and from intact halobacteria. An activation energy of 14.1 +/- 0.4 kcal-mol-1 and of about 19 kcal-mol-1 for formation of bacteriorhodopsin 411 and of bacteriorhodopsin 565, resp., was calculated. pH-changes in the medium caused by the reaction cycle of bacteriorhodopsin were detected by use of the pH-indicator bromocresol green.


A new alkali-resistant hemoglobin alpha2J Oxford gammaF2 in a Sicilian baby girl with homozygous beta0 thalassemia. A 10-mo-old baby girl with homozygous beta0 thalassemia and alphaJOxford, presenting the clinical picture of homozygous beta thalassemia is described. Hemoglobin electrophoresis showed three bands: the first two with the mobilities of hemoglobin Hb A2 (1%) and Hb F (69%), respectively, the third migrating a little faster than Hb A (30%). About 30% of her alpha chains were J Oxford which, bound to her gamma chains, produced a new alkali-resistant hemoglobin, alpha2 J Oxford gamma F2, which has not been described previously. Hemoglobin synthesis in vitro showed the absence of beta chain synthesis and an alpha/non-alpha ratio of 2. The patient's father was heterozygous for both the Hb J Oxford and beta0 thalassemia genes, the mother a carrier of beta0 thalassemia; four other relatives were carriers of Hb J Oxford, and one was a carrier of beta thalassemia.


Effect of osmotic pressure, ionic strength and dibutyryl cyclic adenosine monophosphate on the adhesion of hen erythrocytes. In the presence of lysolecithin at physiological pH it was found that the increase of ionic strength facilitates the adhesion of hen erythrocytes. In this medium, dibutyryl cyclic adenosine monophosphate (DBcAMP) increases the adhesion index of the cells. If the osmotic pressure is elevated without a proper increase of ionic strength, the lysolecithin induced hemolysis and adhesion are found to be lacking.


Report of the first hemispheric meeting on meningococcal disease. The first Hemispheric meeting on meningococcal meningitis was held in São Paulo and Brasília on 23-28 February 1976. Organized by the Pan American Health Organization in collaboration with the Government of Brazil and other PAHO Member States, the meeting had three principal aims: to review the general subject of cerebrospinal meningitis and the special situation in Brazil; to analyze experience gained in Brazil regarding laboratory diagnosis, treatment, and reduction of mortality among hospitalized patients; and to develop prevention and control strategies based on a review of available scientific knowledge and techniques for prevention and control of meningococcal disease.


The contribution of extraneuronal uptake to the trachea-blood vessel selectivity of beta-adrenoceptor stimulants in vitro in guinea-pigs. 1 The potencies relative to isoprenaline of isoetharine, tertiary butyl noradrenaline, salbutamol, orciprenaline, Me 506, rimiterol, fenoterol, carbuterol and terbutaline on isolated preparations of guinea-pig trachea and blood vessels (perfused hind limb) were determined. All the compounds were selective for trachea and selectivity values, i.e. relative potency on trachea divided by relative potency on hind limb, ranged from 2.3 to 21.4. 2 Responses to isoprenaline (the reference compound), tertiary butyl noradrenaline and isoetharine were potentiated on trachea by 50 muM phenoxybenzamine (PHB) and by other inhibitors of extraneuronal uptake (ENU). Under these conditions the selectivity values of all the compounds was close to unity. 3 Selectivity values were also close to unity if they were calculated from data obtained without ENU inhibition, provided that only those compounds not potentiated by PHB on trachea were used. 4 It is proposed that the trachea-blood vessel selectivity shown by beta-adrenoceptor stimulants can be caused by the influence of ENU upon them, rather than by their ability to distinguish between two beta2-adrenoceptors. 5 The suggestion that differences exist between beta2-adrenoceptors in respiratory and vascular smooth muscle is not supported by the in vitro experiments described.


Adrenoreceptors of the guinea-pig urinary bladder. 1 Adrenaline, noradrenaline and isoprenaline (5 mug/ml) did not affect the resting tone of the isolated urinary bladder of the guinea-pig. 2 The catecholamines (1-2 mug/ml) inhibited neuronally evoked contractions at various stimulation frequencies; the inhibition was maximum at 2 Hz and minimum at 50 Hz. Isoprenaline produced maximum inhibition. 3 Propranolol (0.5 mug/ml) completely blocked the catecholamine-induced inhibition at all the frequencies employed. The concentration-response curves of isoprenaline at 2, 10 and 50 Hz were characteristically shifted by propranolol (50 ng/ml). Phenoxybenzamine (0.2 mug/ml) was totally ineffective. 4 In some experiments adrenaline significantly raised the tone of the bladder exposed to propranolol; this effect could be blocked by phenoxybenzamine. 5 Acetylcholine-induced bladder contractions were inhibited by adrenaline (2 mug/ml); the inhibition was completely blocked by propranolol (0.5 mug/ml). 6 The results indicate the presence of an inhibitory beta-adrenoceptor and suggest the possibility of an excitatory alpha-adrenoceptor in guinea-pig urinary bladder.


The cat lung strip as an in vitro preparation of peripheral airways: a comparison of beta-adrenoceptor agonists, autacoids and anaphylactic challenge on the lung strip and trachea. 1 A new in vitro preparation, the isolated lung strip of the cat, is described for investigating the direct effect of drugs on the smooth muscle of the peripheral airways of the lung. The preparation comprises a thin strip of lung parenchyma which can be mounted in a conventional organ bath for isometric tension recording. Its pharmacological responses have been characterized and compared with the isolated tracheal preparation of the cat. 2 The lung strip exhibited an intrinsic tone which was relaxed by catecholamines, aminophylline and flufenamate. It was contracted strongly by histamine, prostaglandin F2alpha, acetylcholine, compound 48/80, potassium depolarizing solution and alternating current field stimulation. In contrast, the cat trachea was unresponsive to histamine and prostaglandin F2alpha and did not exhibit an intrinsic tone. 3 (-)-Isoprenaline and (-)-adrenaline were much more potent in relaxing the lung strip than the trachea. The potency order of relaxation responses to isoprenaline, adrenaline and (+/-)-noradrenaline in the lung strip was isoprenaline greater than adrenaline greater than noradrenaline but in the trachea was isoprenaline greater than noradrenaline greater than or equal to adrenaline. 4 beta2-Adrenoceptor selective agonists salbutamol and terbutaline were more potent in the lung strip than the trachea, suggesting beta2-adrenoceptors predominated in the lung strip. Propranolol was equipotent in inhibiting isoprenaline relexations of the lung strip and trachea, whereas practolol was much less effective in inhibiting lung strip than trachea, further supporting a predominance of beta2-adrenoceptors in lung strip and beta1-adrenoceptors in trachea. 5 Strong Schultz-Dale type contractions were elicited in both lung strips and trachea by Ascaris lumbricoides antigen in actively sensitized cats. The initial phase of the contractile response of the lung strip following challenge was shown to be due to histamine release and was absent in the trachea. The delayed phase of the contraction which took several minutes to develop in both the mepyramine-treated lung strip and trachea was not due to prostaglandins E1, F2alpha or bradykinin, the probable mediator being slow reacting substance of anaphylaxis (SRS-A). 6 It is concluded that the isolated lung strip of the cat is useful as an in vitro model for investigating the effect of drugs on the smooth muscle of the peripheral airways of the lungs.


Imaginal flooding and exposure to real phobic situations: treatment outcome with agoraphobic patients. Each of thirty-six female agoraphobic out-patients were treated by one of three methods: 8 sessions of imaginal flooding followed by 8 sessions of practice in the real situation; 16 sessions of combined flooding and practice; or 16 sessions of practice alone. Three therapists treated equal numbers of patients in each group, and there was some evidence that patients' response varied according to the therapist seen, irrespective of treatment group. There were no significant differences between treatment groups after 8 sessions, 16 sessions or on six-month follow-up. It is concluded that there are no long-term differences between the effects of treatments involving exposure to either imaginal or real phobic situations or to a combination of both, provided that patients are encouraged to practise between sessions.


Imaginal flooding and exposure to real phobic situations: changes during treatment. This paper reports the results of measures taken during treatment in the study of imaginal flooding and exposure to real phobic situations previously described by Mathews, Johnston, Lancashire, Munby, Shaw and Gelder (1976). On weekly measures of change of similar reduction in phobic behaviour in all treatments was found, confirming the previous findings. Differences in therapist effectiveness were also confirmed. On measures of the immediate effects of treatment, exposure to the phobic situation had consistent positive effects, imaginal flooding had little or no detectable effect. It is proposed that the treatments studied differ in their immediate effects on phobic behaviour but also have the common effect of facilitating counterphobic behaviour outside the treatment situation, and that this is the main agent of therapeutic change.


Fresh symptom emergence after intensive behaviour therapy. The outcome of a standard four-day intensive symptomatic treatment programme with 39 agoraphobics is examined in relation to the incidence of fresh symptom emergence. Twenty-six patients suffered fresh symptom emergence during follow-up, and there was a significant association of fresh symptom emergence with poorer outcome at one-year follow-up. About 18 percent of patients were adversely affected by the treatment programme, as judged on a wide range of symptoms and measures of inter- and intra-personal adjustment. Sixteen individually treated patients are then compared with the 39 group-treated patients and differences in drop-out rate are discussed.


Personality, expectancies and group psychotherapy. The paper reports an extension of work into the relevance of personality and pre-treatment expectancies for allocation and response of patients to group psychotherapy. The results show that subjects who are internally directed in interest and who have a liberal attitude to a variety of social issues and a "psychological" set to treatment are more responsive to group psychotherapy as seen by their therapists as well as by themselves. Those who are externally directed in interest and who have a conservative attitude to life and a more "medical-physical" set to treatment are more likely to be referred for behaviour therapy; if referred for group psychotherapy they are likely either to drop out or to show very limited response to treatment.


Further characterization of morphine-like peptides (endorphins) from pituitary. Pituitary peptides with opioid pharmacologic action (endorphins) can exist in several forms with different basicities and molecular weights from about 3000 to less than 1000 daltons. Partial purification yielded an endorphin at least 4 times more potent than normorphine and 16 times more potent than the endogenous brain pentapeptide methionine enkephalin. It is proposed that pituitary endorphin or a similar peptide in brain may be the precursor of brain enkephalin.


Separately developing axonal uptake of 5-hydroxytryptamine and norepinephrine in the fetal ileum of the rabbit. Uptake of 5-hydroxytryptamine (5-HT) by adult and fetal rabbit's ileum was studied. The adult myenteric plexus accumulated tritium when incubated with tritiated 5-HT. However, in addition to labeled 5-HT, tritiated 5-hydroxyindole acetic acid and, when monoamine oxidase (MAO) was inhibited, 5-HT-o-glucuronide were found in the tissue. Two uptake processes differing in affinity could be defined. Only the high affinity process was saturable. Fetal ileum took up tritiated 5-HT but glucuronidation did not occur when MAO was inhibited. The uptake of tritiated 5-HT by the fetal ileum was due to a single, saturable, temperature sensitive (Q10 at 27-37 degress C = 2.4) process inhibited by ouabain. It was identical to the high affinity uptake found in adult tissue. This specific high affinity uptake could be found as early as the 16th day of gestation, 5-8 days before uptake of norepinephrine (NE) begins. Light and electron microscope radioautography revealed that the uptake of 5-HT was primarily into axons and a characteristic structure called the expanded process, both in the myenteric plexus. Both contained dense-cored vesicles. Axons were not labeled by tritiated NE until after 24 days and the expanded process was never labeled by tritiated NE. This study shows that uptake of 5-HT is a property of distinct system of axons in the mammalian myenteric plexus which develops prior to adrenergic axons during ontogeny.


A comparison of the 12,000 dalton proteins synthesized by Aplysia neurons L11 and R15. The 12,000 dalton proteins of neurons L11 and R15 of the Aplysia abdominal ganglion were labeled by incubation of the ganglion in [3H]leucine and compared in terms of their subcellular localization, solubility in various media, and molecular charge. Both proteins are cytoplasmic constituents. Their solubility behavior is identical: both are insoluble in aqueous media of low and high ionic strength as well as chloroform-methanol, and both are solubilized by Triton X-100 + urea and by LIS. They are essentially identical in molecular weight as determined by SDS gel electrophoresis but differ by a single charge per molecule at low pH. The broad similarity between these proteins suggests that they could serve similar functions, while the observed charge difference might be important in terms of previously discovered differences in their processing.


[Consequences of the administration of alpha or beta drenolytic agents on the mobilization of plasma free fatty acids as induced by noradrenaline and isoprenaline in the dog]. Noradrénaline or isoprenaline (a beta sympathicomimetic drug) infusion in fasting awake dog provoque a rise of plasmatic FFA. This effect is abolished by administration of propranolol (beta blocking agent) but not by phentolamine (alpha blocking agent). Phentolamine potentiate the rise of FFA in response to noradrenaline but not to isoprenaline. This alpha blocking drug supress the fall of FFA induced by phenylephrine a sympathomimetic drug.


[Influence of variations in blood pH on the bronchodilator action of salbutamol and terbutaline]. In the cat, the broncho-dilatator effects of salbutamol and terbutaline are influenced by blood pH variations. Gazeous or metabolic alkalosis increases, acidosis decreases, the broncho-dilatation provoked by salbutamol or terbutaline.


Effect of structural analogs of butaclamol (a new antipsychotic drug) on striatal homovanillic acid and adenyl cyclase of olfactory tubercle in rats. The 3-isopropyl (I), 3-cyclohexyl (II) and 3-phenyl (III) analogs of the new antipsychotic drug butaclamol, which contains a 3-tertiary butyl group, and their respective (+)-enantiomers, but not (-)-enantiomers, caused a dose related elevation of rat striatal homovanillic acid concentration, indicative of an increased dopamine (DA) turnover; droperidol also exhibited this activity. The order of activity of the (+)-enantiomers was (butaclamol) approximately II greater than I greater than III. A decrease in striatal DA was observed with (+)-I and (+)-III at the highest dose used, but not at one-half the dose. Each analog antagonized the DA-induced increase in adenyl cyclase (EC 4.6.1.1) activity of olfactory tubercle homogenates, the order of activity of the racemates (except for II) AND (+)-ENANTIOMERS BEING (BUTACLAMOL) APPROXIMATELY I greater than III greater than II. The (+)-enantiomers of butaclamol and analogs were two to four times more potent than their respective racemates, with (+)-butaclamol and (+)-I displaying activity generally equivalent to fluphenazine. The respective (-)-enantiomers were ineffective indicating a stereochemical specificity for DA-receptor blockade. Such analogs presented should be of value in elucidating dopaminergic mechansims.


Catalepsy induced by morphine or haloperidol: effects of apomorphine and anticholinergic drugs. To investigate the extent of cholinergic involvement in opiate-induced catalepsy, the effects of three anticholinergic drugs were studied on morphine-induced catalepsy. Haloperidol-induced catalepsy was also examined. Maximum catalepsy in rats was obtained with 30 mg/kg morphine or 3 mg/kg haloperidol. The anticholinergic drugs atropine, benztropine, and scopolamine were unable to antagonize morphine-induced catalepsy, yet readily antagonized haloperidol-induced catalepsy. Low doses of apomorphine (7.5 mg/kg), on the other hand, readily antagonized morphine catalepsy, but 13-fold higher doses of apomorphine were needed to block haloperidol-induced catalepsy. The results are compatible with the idea that catalepsy can be mediated via the striatum or the amygdala; morphine-dopamine antagonism may occur in the amygdala, whereas morphine-dopamine-cholinergic interactions occur in the striatum.


Behavioral studies on the enantiomers of butaclamol demonstrating absolute optical specificity for neuroleptic activity. Butaclamol is a member of a new chemical class for which antipsychotic activity in humans has been demonstrated. Butaclamol, a racemate, has been resolved into its optical isomers and a separation of activities was found to occur between the (+) and (-) enantiomers. The present experiments show that at doses ranging from 0.1 to 0.3 mg/kg the (+) enantiomer abolished amphetamine-induced (a) stereotyped behavior and (b) rotational behavior in rats with unilateral lesions in the substantia nigra. It also inhibited the lever-pressing response in the continuous (Sidman) avoidance procedure, blocked discriminated avoidance behavior, and decreased ambulation and rearing in the open field. In contrast, the (-) enantiomer was devoid of behavioral activity at 100-500 times larger doses. At considerably higher doses (+)-butaclamol antagonized epinephrine-induced mortality. Again, the (-)-butaclamol was devoid of this activity as well. The significance of absolute optical specifity manifested by a neuroleptic drug is discussed in the light of dopaminergic and adrenergic mechanisms.


Effects of acidity, cations and alcoholic fractionation on absorption of heparin from gastrointestinal tract. Heparin was introduced into the stomach or duodenum of mice separately in doses of ca. 250 mg/kg. A slight anticoagulant effect in the systemic circulation was detected in whole blood clotting times and factor X inhibition. In contrast to most drugs, more heparin was absorbed from the stomach than from the intestine. Suppressing ionization of heparin by simultaneous administration of acid resulted in improved absorption of heparin from the small intestine. Heparin was separated with ethanol into five molecular weight fraction: I, 17 999; II, 13 i99; III, 10800, IV, 8 700; and V, 6 700. Each was introduced into the duodenum of mice with citric acid. The maximum hypocoagulability was produced with fraction IV. When administered in distilled water instead of in citric acid, this heparin fraction did not produce an anticoagulant effect. These studies demonstrated that improvement of heparin absorption from the gastrointestinal tract can be obtained by the combination of suppressing ionization and selecting molecular size.


Extracellular nuclease produced by a marine bacterium. II. Purification and properties of extracellular nuclease from a marine Vibrio sp. Extracellular nuclease produced by a marine Vibrio sp., strain No. 2, was purified by salting out with ammonium sulfate and by chromatography on a DEAE-cellulose column and twice on a Sephadex G-200 column. The nuclease was eluted as a single peak in which the deoxyribonuclease (DNase) activity and ribonuclease (RNase) activity appeared together. Polyacrylamide disc gel electrophoresis showed a single band of stained protein which had both DNase and RNase activity. The molecular weight of the enzyme was estimated to be 100 000 daltons. When using partially purified enzyme from the DEAE-cellulose column, the optimum pH for activity was 8.0, and the enzyme was activated strongly by 0.05 M Mg2+ ions and stabilized by 0.01 M Ca2+ ion. These concentrations of Mg2+ and Ca2+ ions are similar to those of the two cations in seawater. Indeed, the enzyme revealed high activity and strong stability when kept in seawater. The presence of particulate matter, such as cellulose powder, chitin powder. Hyflosupercel, Kaolin, and marine mud increased the stability of the enzyme. When the hydrostatic pressure was increased from 1 to 1000 atmospheres, the decrements of the enzyme activity were more pronounced at 30 and 40 degrees C than at 25 or 50 degrees C. The enzyme activity was restored after decompression to 1 atm at 30 degrees C.


Ecological distribution of Spirillum lipoferum Beijerinck. A survey in various countries revealed that the N2-fixing Spirillum lipoferum Beijerinck is a very common root and soil inhabitant in the tropics. More than half of the grass root and soil samples collected in tropical countries (four African countries and Brazil) contained abundant S. lipoferum populations, while less than 10% of the samples collected in temperate South Brazil, Kenya, and the U.S.A. contained the organism. There is a pronounced vegetation effect. Panicum maximum seems the most favorable among the forage grasses, while few positive samples were found under virgin tropical forest. Legume roots contained less S. lipoferum than adjacent soils. More than 80% of the samples from cereal roots (maize, sorghum, wheat, and rye) grown in fields fertilized with PK and Mo, in Rio de Janeiro State, were positive. Maize and sorghum grown under similar conditions in Wisconsin contained less than 10% of positive samples, but when maize fields were inoculated 90% of the root samples contained S. lipoferum. Alluvial soils were more favorable than eroded hill soils. Occurrence in soil was strongly pH-dependent with a pH around 7, being optimal (correlation coefficient r = 0.90). Sporadic occurrence was observed even in soils with pH 4.8. Surface-sterilized P. maximum roots collected from soils with pH ranging from 4.8 to 7.2 contained high S. lipoferum numbers which did not correlate with soil pH (r = 0.41). Amendment with malate of acid soils was not very effective in increasing nitrogenase (N2-ase) activity, but in two soils with pH above 6.4, high N2-ase activity was obtained after 16 to 48 h of incubation. In two soils from a temperate climate region, inoculation with S. lipoferum increased N2-ase activity produced through malate amendment.


Thermothrix thioparus gen. et sp. nov. a facultatively anaerobic facultative chemolithotroph living at neutral pH and high temperature. Thermothrix thioparus gen. et ep. nov. occurs naturally in a New Mexico hot spring at a temperature of 74 degrees C, a pH of 7.0, and a HS- concentration of 1 mg/litre. The organism is gram-negative, non-motile, 0.5-1.0 X 3-20 mum, and forms cell chains up to 1 cm in length. The resulting filaments do not possess a sheath. Sulfur is deposited extracellularly. The organism was isolated using an autotrophic medium with HS- as the energy source and NO3- as the terminal electron acceptor. Anaerobically either NO2- or NO3- is required, NO2- is formed from NO3-, and no observable gas is evolved. Oxygen can also be used as the terminal electron acceptor, but growth is poor because of the decreased solubility of O2 at temperatures required for growth. Alternate energy sources used aerobically and anaerobically include hexose, HS-, SO3-, and S2O3=. The temperature optimum is 70-73 degrees C and growth occurs from 62 to 77 degrees C. The organism's thermal and physiological characteristics are compared to those of Bacillus stearothermophilus, Methanobacterium thermoautotrophicum, Sulfolobus acidocalderius, Thermus aquaticus, Thermus flavus, as well as Thiobacillus denitrificans, the latter being the only other facultatively anaerobic chemolithotroph which has been isolated and described.


Comparison of talc-Celite and polyelectrolyte 60 in virus recovery from sewage: development of technique and experiments with poliovirus (type 1, Sabin)-contaminated multilitre samples. For virus recovery from sewage, a mixture of talc and Celite was tested as a possible inexpensive substitute for polyelectrolyte 60 (PE 60). After adjustment of pH to 6 and the addition of 45-60 plaque forming units (PFU)/ml of poliovirus type I (Sabin) to the sewage sample under test, 100 ml of it was passed through either a PE 60 (400 mg) or a talc (300 mg)-Celite (100 mg) layer; the layer-adsorbed virus was eluted with 10 ml of 10% fetal calf serum (FCS) in saline (pH 7.2). In these experiments, PE 60 layers recovered 73-80% (mean 76%) of the input virus. In comparison, virus recoveries with the talc-Celite layers were 65-70% (mean 68%). Passage of 5 litres of raw sewage (containing 50 to 1.26 X 10(5) PFU/100 ml of the poliovirus) through the talc (15 g)-Celite (5 g) layers and virus elution with 50 ml of 10% FCS in saline gave virus recoveries of 33-63% (mean 49%). Except for pH adjustment and prefiltration through two layers of gauze to remove large solids, no other sample pretreatment was found to be necessary. Application of this technique to recovery of indigenous viruses from field samples of raw sewage and effluents has been highly satisfactory.


The effects of pH and temperature on the assay of superoxide dismutase. A simple and reliable method for the measurement of superoxide dismutase (EC 1.15.1.1) activity is described. The method is based on a linear inhibition of the reduction of acetylated cytochrome c by superoxide dismutase.


Biosynthesis of lysine in Saccharomyces cervisiae: properties and spectrophotometric determination of homocitrate synthase activity. A rapid assay is described for homocitrate synthase (EC 4.1.3.21) of the lysine biosynthetic pathway of Saccharomyces cerevisiae. The alpha-ketoglutarate-dependent cleavage of acetyl-coA was measured spectrophotometrically as decrease in absorbance at 600 nm in the presence of 2,6-dichlorophenol-indophenol and enzyme from the wild type strain X2180. This activity was also present in citrate synthaseless glutamate auxotroph glu3, and the activity was inhibited by 5 mM L-lysine. Radioactive homocitric acid was obtained from a reaction mixture containing [1-14C]acetyl-coA. Homocitrate synthase activity was dependent upon time, both substrates, and enzyme. The activity exhibited a pH and temperature optimum of 7.5-8.0 and 32 degrees C, respectively, and was inhibited by metal-chelating and sulfhydryl-binding agents.


Dependence of the superficial layers of Spirillum putridiconchylium on Ca2+ or Sr2+. Chelating agents disrupted the superficial layers on Spirillum putridiconchylium and adsorption of cationized ferritin indicated that both upper and lower surfaces of superficial layer fragments, as well as the outer membrane surface, possessed areas which were negatively charged. Growth of the bacterium in 1% casamino acids (vitamin free) resulted in cells which were devoid of the superficial layers, and negative staining of these cells revealed in amorphous precipitate together with a vesicular outer membrane component extruding from their surfaces into the medium. Addition of either 1 mM Ca2+ or 1 mM Sr2+ to the growth medium produced the typical regularly structured cell surface, whereas addition of equal concentrations of Li+, Na+, K+, Mg2+, Ba2+, Mn2+, Fe3+, or three polyamines produced the structureless surface.


Some properties of p-coumarate decarboxylase from Cladosporium phlei. The optimal pH and temperature of p-coumarate decarboxylase were 6.0 and 23 degrees C respectively. The enzyme activity was reduced to three quarters by heat treatment at 35 degrees C for 5 min and by half at 25 degrees C in 24 h, but kept almost unchanged at -20 degrees C at least for 10 days. The activity was not inhibited by potassium cyanide, sodium diethyldithiocarbamate, ethylenediaminetetraacetic acid disodium salt, or sodium citrate at 10 mM concentration, but was inhibited by p-chloromercuribenzoate or iodoacetate at 0.1 mM, the inhibition by the former being prevented to a great extent by the presence of reduced glutathione or dithiothreitol. The activity was inhibited by maleic acid cinnamic acid, or p-methoxycinnamic acid, but not by fumaric acid, acrylic acid, p-hydroxystyrene, furcatin p-hydroxyphenylacetic acid, or phloretic acid. An unsubstituted p-hydroxy group on the benzene ring and an acrylic acid side chain were required for the enzyme activity. Km value for trans-p-coumaric acid was about 6.5 X 10(-4) M.


Production of volatiles from decomposing plant tissues and effect of these volatiles on Rhizoctonia solani in culture. Volatiles, of which NH3 is a major component, were evolved from decomposing immature corn tissue (c:n9) and affected R. solani in culture two ways: they supplied additional nitrogen to the growth medium so that fungal mycelial growth increased; and they raised substrate pH from 5.5 to 8.2 which induced melanization of mycelium. Volatiles increased fungus growth and pigmentation within 2 weeks of amendment addition to soil. Increases were concomitant with NH3 production from corn tissue. More NH3 evolved from decomposing corn tissues of C:N9 and 17 than from those of C-N 33 and 81. More growth and pigmentation occurred in flasks through which volatiles from decomposing corn (C:N9) were passed than in flasks through which volatiles from nonamended soil or decomposing corn (C:N81) were passed. Carbon dioxide from decomposing tissues did not affect growth or pigmentation. Twice as much NH3 evolved from corn tissue (C:N9) which decomposed in saturated soil than from tissue which decomposed in soil at 50% of its water-holding capacity. Pigment production doubled under saturated conditions.


Factors affecting rate of methane formation from acetic acid by enriched methanogenic cultures. A stable enrichment culture converting acetic acid to methane was successfully obtained from a pear waste digester, using a synthetic substrate solution with acetic acid as the main carbon source. This enrichment culture converted up to 10 mmol of acetic acid per litre per day at 35 degrees C and did not use hydrogen or formic acid in appreciable amounts as substrate for methane production instead of, or in addition to, acetic acid. The rate of conversion of acetic acid to methane was maximum at temperature of 40-45 degrees C, at a pH of 6.5 to 7.1, and was adversely affected by exposure to air, reducing agents, and high salt concentrations. The rate of conversion was independent of acetic acid concentration between 0.2 and 100 mM, but dropped markedly at concentrations below 0.2 mM.


Roles of low pH, carbon and inorganic nitrogen source use in chlamydospore formation by Fusarium solani. Citrate and malate were poorer sources of exogenous carbon than several hexose, pentose, or disaccharide sugars for supporting macroconidial germination by Fusarium solani at high conidial density (1 X 10(5) condia/ml). Only citrate, however, failed to block chlamydospore morphogenesis to a degree comparable to glucose or other readily used sugars. Mostly immature chlamydospores were formed in the presence of citrate. At low conidial density (5 X 10(3) conidia/ml), exogenous carbon-independent macroconidial germination and subsequent rapid chalmydospore formation on germ tubes was not inhibited by ammonium or nitrate nitrogen. The citrate-phosphate buffered, low pH (4.0) medium of Cochrane induced more immature chlamydospore formation by F. solani than a pH 6.0 medium, but few mature chlamydospores were formed in either medium. Condensation of hyphal cytoplasm into developing chlamydospores, a character typical of chlamydospore formation, did not occur extensively and macroconidia, hyphae, and immature chlamydospores stained deeply with Sudan III, suggesting lipid biosynthesis. This inhibition of chlamydospore maturation may be due partly to nitrogen deficiency imposed by the high C:N ratio of the medium and to the presence of citrate. Only vesiculate hyphal cells were formed by F. solani f. sp. phaseoli in both media. Field soils to which the clone of F. solani used is indigenous had mean pH values ranging from 5.2 to 6.0.


Transport of phenylalanine by conidia of Fusarium sulphureum. L-Phenylalanine was actively transported by conidia of Fusarium sulphurenum Schlect (isolate 1). Uptake was optimal at pH 7, 30 degrees C; respiration-dependent; and was unaffected by relatively high internal concentrations of phenylalanine. The Km for transport was 1-3 X 10(-5) M and the Vmax was 2.5-4 nmol/min per milligram dry weight. Phenylalanine is transported by a general transport system for basic and neutral amino acids. Sucrose repressed uptake of phenylalanine and this repression was largely negated by cycloheximide. Efflux of accumulated phenylalanine was influx-dependent; this transport system deteriorated slowly with aging of the conidial culture.


Characterization of a strain of Methanospirillum hungatti. The results of morphological, base ratio, nutritional, temperature, and pH studies on a strain of Methanospirillum hungatii, isolated from an anaerobic pear waste digester, are described. The isolate, designated as strain GP 1, was compared with some of the characteristics of type-strain M. hungatii JF 1. Strain GP 1 is Gram-negative, weakly motile, and a strict anaerobe with a guanine plus cytosine (G +C) content of 46.5 mol%. The preferred substrates for methane production are hydrogen, carbon dioxide, and formate. Acetate is used under certain conditions but its specific contribution to cell carbon and (or) methane formation was not established. The optimum temperature for both growth and methane production is 35 degrees C, but growth and methane production occur over the range 25-45 degrees C. Methane production is optimal at pH 7.0.


Psychotropic drug use among women.

Psychotropic drug use among women. The consistent 2:1 ratio of women to men in the receipt of prescriptions for psychotropic drugs is reflected in the higher rates for women of neurotic illness, symptoms of both physical and mental discomfort, and help-seeking and drug-taking behaviour. Physicians' perceptions of the problems presented by their male and female patients influence their prescribing of these drugs. Recent statistics in Ontario indicate that greater use of physicians' services by women is an inadequate explanation of the higher rate of prescribing of psychotropic drugs to women. A longitudinal study of a large insured population in Ontario showed that almost twice the proportion of females, compared with males, received a prescription for psychotropic drugs in 1970-71 and in 1973-74, a higher proportion of females received multiple prescriptions for each drug class, and males were more likely than females to have received only one prescription in a year.


Adoptive immunotherapy of a Gross virus producing lymphoma and a methylcholanthrene-induced fibrosarcoma in tolerant rats. Immunological tolerance to Gross virus-specific transplantation antigens in rats given neonatae transfer of donor lymphoid cells beneath the kidney capsule of syngeneic recipient rats. Immune or normal donor cells invariably developed a cell-mediated immune reaction in kidneys of GV-tolerant recipients, presumably against GV antigens present on the surface of recipient lymphoid cells in the kidney. Spleen and lymph node cells from tolerant rats failed to develop a reaction in tolerant recipients, but developed a strong reaction to histoincompatible antigens in the kidneys of semisyngeneic tolerant rats. The immunologically tolerant state in the rats could be broken by adoptive transfer of spleen and lymph node cells from syngeneic rats immunized with GV-induced lymphoma cells. Immunotherapy of a GV-induced and also a GV-infected methylcholanthrene-induced fibrosarcoma growing in tolerant rats was successful when immune spleen and lymph node cells were administered i.p. 3 days after s.c. inoculation of 2 X 10(7) tumor cells in the case of the lymphoma, and 1 day after inoculation of 5 X 10(6) tumor cells in the case of the fibrosarcoma.