Effect of the mouse mutants testicular feminization and sex reversal on hormone-mediated induction and repression of enzymes. The mouse mutants testicular feminization and sex reversal have been used to investigate hormone-mediated induction and repression of enzymes. Tfm/Y animals were already known to be androgen insensitive, rendering the androgen-inducible enzymes ADH and beta-glucuronidase noninducible because of an inherited deficiency of a cytosol androgen-receptor complex. The animals display female secondary sexual characteristics. Sxr/+,XX animals display male primary and secondary sexual characteristics with small testes. We demonstrate (1) that the Tfm mutation is pleiotropic, preventing repression of an androgen-repressible enzyme (ornithine aminotransferase) as well as induction of androgen-inducible enzymes, (2) that an estrogen-inducible enzyme (histidine decarboxylase) is not affected by the Tfm mutation, and (3) that Sxr/+,XX animals produce enough androgen for malelike activities of androgen-sensitive enzymes. It was also discovered that histidine decarboxylase repressed by androgen in normal animals, rather than being unaffected by it in Tfm/Y animals, is in fact induced. This unexpected phenomenon is discussed and an explanation is suggested for it.


Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide. The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.


The effect of beta adrenergic blockade on pulmonary hypertension, right ventricular hypertrophy and polycythaemia, induced in rats by intermittent high altitude hypoxia. Adult male rats were used to study the effect of a beta blocking agent on pulmonary hypertension and right ventricular hypertrophy induced by intermittent high altitude (IHA) hypoxia (8 hr daily, 5 days a week, stepwise up to the simulated altitude of 7000 m). Trimepranol was injected subcutaneously in a single dose of 10 mg/kg/b.w. one hour before each IHA exposure. Administration of the beta blocking drug caused significant changes of haematocrit values even in animals kept under normoxic conditions. The initial deep decrease was followed by a slow return to control values; prolongation of treatment led to a further significant decrease of the haematocrit curve. The polycythaemic response of IHA-exposed and Trimepranol-treated animals was, therefore, significantly less pronounced as compared with the hypoxic non-treated group. Administration of Trimepranol to IHA-exposed rats significantly decreased the values of right ventricular systolic and mean pressure, right ventricular hypertrophy as well as the degree of muscularization of pulmonary arteries. It may be assumed that the protective effect of Trimepranol is due to a) changes in pulmonary vascularization, b) reduction of polycythaemia, and c) lower cardiac output, induced by the negative inotropic and chronotropic effect of this drug.


Reconstitution of chlorophyllide formation by isolated etioplast membranes. 1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the additon of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation. 6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35 micron respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.


Effects of glucagon and insulin on fatty acid synthesis and glycogen degradation in the perfused liver of normal and genetically obese (ob/ob) mice. 1. Rapid effects of hormones on glycogen metabolism and fatty acid synthesis in the perfused liver of the mouse were studied. 2. In perfusions lasting 2h, of livers from normal mice, glucagon in successive doses, each producing concentrations of 10(-10) or 10(-9)M, inhibited fatty acid and cholesterol synthesis. In perfusions lasting 40--50 min, in which medium was not recycled, inhibition of fatty acid synthesis was only observed with glucagon at concentrations greater than 10(-9)M. This concentration was about two orders of magnitude higher than that required for the stimulation of glycogen breakdown. Glucagon did not inhibit the activity of acetyl-CoA carboxylase, assayed 10 or 20 min after addition of glucagon (10(-9) or 10(-10)M). It is proposed that the action of glucagon on hepatic fatty acid biosynthesis could be secondary in time to depletion of glycogen. Insulin prevented the effect of glucagon (10(-10)M) on glycogenolysis, but not that of vasopressin. 3. Livers of genetically obese (ob/ob) mice did not show significant inhibition of lipid biosynthesis in response to glucagon, although there was normal acceleration of glycogen breakdown. This resistance to glucagon action was not reversed by food deprivation. Livers of obese mice exhibited resistance to the counteraction by insulin of glucagon-stimulated glycogenolysis, which was reversible by partial food deprivation.


Cerebral oxidized and reduced nicotinamide-adenine dinucleotide phosphate and glucose 6-phosphate dehydrogenase in mice during exposure to high oxygen pressure. NADP+, NADPH and glucose 6-phosphate dehydrogenase were determined in the cerebral cortex of mice exposed to high O2 pressure for 0, 8 and 16 min. These time intervals corresponded to 0, 50 and 100% of the CT50 (the time taken for 50% of the mice to convulse). Cerebral NADP+, NADPH and glucose 6-phosphate dehydrogenase also were determined in O2-exposed mice exhibiting hyperactivity, convulsions, and in mice killed 10s after convulsions. Similar increases in cortical NADP+ and decreases in NADPH were found in mice exposed to 610kPa (6 atm.) of 100% O2 for 0, 50 and 100% of the CT50, during hyperactivity, onset of seizure and 10s after convulsions. The NADP+/NADPH ratio increased approx. 25% at 0% of the CT50, and remained at this increased value at all O2-exposure periods including the hyperactive state, onset of seizure and 10s after convulsions. Identical changes in cerebral NADP+ , NADPH and the NADP+/NADPH ratio were found in mice exposed for 16min to 100% O2 at 100, 350 or 610kPa. No change in cerebral glucose 6-phosphate dehydrogenase was found in mice exposed to 610kPa of 100% O2 during the various stages of O2 toxicity. Only in the 10s post-convulsive group was a statistically significant decrease in glucose 6-phosphate dehydrogenase observed. Disulfiram [bis(diethylthiocarbamoyl) disulphide], an effective O2-protective agent, did not prevent the O2-induced increase in cerebral NADP+ and the NADP+/NADPH ratio, or decrease in NADPH.


Catalase model systems. Decomposition of hydrogen peroxide catalysed by mesoferrihaem, deuteroferrihaem, coproferrihaem and haematoferrihaem. The catalytic decomposition of H2O2 by deuteroferrihaem, mesoferrihaem, coproferrihaem and haematoferrihaem was studied as a model for the mechanism of action of catalase. For haematoferrihaem, anomalous but reproducible results were obtained, which could not be adequately explained. For each of the other ferrihaems studied, both monomeric and dimeric species catalysed decomposition, although the activity of monomer (aM) was much greater than that of dimer (aD). The pH variation of aD in the range 6.5--11 was consistent with an inverse dependence on [H+]1/2. The molecular mechanism whereby such a dependence could be achieved is not apparent. A study of the pH-dependence of aM in the range 6.5--11 revealed a linear inverse relationship with [H+]. This is interpreted in terms of attack by HO2- on ferrihaem monomer. The specific pH-independent rate constants for this reaction were in the order coproferrihaem greater than protoferrihaem greater than or equal to mesoferrihaem congruent to deuteroferrihaem. The order of magnitude of these rate constants is the same as that for catalysis by Fe(H2O)63+ and the second-order rate constant for decomposition of H2O2 by catalase. The implications on the mechanism of action of catalase are discussed.


The role of peroxide in haem degradation. A study of the oxidation of ferrihaems by hydrogen peroxide. The oxidation of ferrihaems by H2O2 was studied as a model for haem catabolism. Rates of ferrihaem oxidation were evaluated by using a new computer-based method that measures the loss in catalytic activity of the ferrihaem during oxidation. For protoferrihaem, deuteroferrihaem, coproferrihaem and mesoferrihaem, oxidation proceeded via the monomeric species and no dimer contribution was detectable. The pH-dependence of oxidation was studied in the range 6.5--11. Within experimental error, the data were compatible with an inverse linear dependence on [H+]. This was interpreted in terms of attack by HO2- on monomeric ferrihaem. The specific second-order rate constants for oxidation of monomeric species by HO2- were of the same order of magnitude for all the ferrihaems, and were in the sequence coproferrihaem greater than protoferrihaem greater than mesoferrihaem congruent to deuteroferrihaem. A model is suggested involving formation of a ferrihaem monomerperoxide complex, which may either dissociate with the formation of a peroxidatic intermediate or be involved in an intramolecular oxidation of the ferrihaem. Haem catabolism may occur via the same or a similar intermediate.


Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates. 1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.


Regulation of synthesis of guanosine 3':5'-cyclic monophosphate in neuroblastoma cells. The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.


The mitochondrial localization of coproporphyrinogen III oxidase. The location of coproporphyrinogen III oxidase in mitochondria was studied in rat liver by using the digitonin method or hypo-osmotic media for fractionation. The enzyme was found in the intermembrane space with a fraction loosely bound to the inner membrane. This fraction was released by washing the inner-membrane-matrix complex with alkaline solutions or solutions of high ionic strength. The enzyme in both fractions had the same Km (0.16 micrometer) for coproporphyrinogen III. When incubation was performed in a medium that avoided destruction of enzyme membrane binding, a dramatic increase in activity was observed after sonication of whole mitochondria or of the inner-membrane-matrix complex.


IgA-related defective neutrophil chemotaxis in ulcerative colitis. A case of ulcerative colitis with defective neutrophil chemotaxis, improved after short sulfasalazine therapy, is reported. This is the first case of this disease in which the chemotactic defect was characterized as a serum inhibitor, directed toward autologous and homologous neutrophils and associated with circulating IgA. Preincubation of normal neutrophils with increasing concentrations of patient's serum resulted in a dose-related inhibition, suggesting a stoichiometric relationship between humoral inhibitory power and neutrophils.


Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha. Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.


Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups. Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).


DNA bifunctional intercalators. I. Synthesis and conformational properties of an ethidium homodimer and of an acridine ethidium heterodimer. An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.


Subunit dissociation in the allosteric regulation of Glycerol kinase from Escherichia coli. 3. Role in desensitization. The mechanism of desensitization of glycerol kinase to allosteric inhibition by fructose 1,6-bisphosphate caused by salt, urea, and high pH has been examined in the light of the model proposed in an earlier paper [de Riel, J. K., and Paulus H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding. KCl (0.4 M) causes a tenfold decrease in the affinity of tetrameric glycerol kinase for fructose, 1,6-bisphosphate but has no significant effect on the dissociation process itself. Urea (2 M) causes a large increase in the equilibrium constant for the dissociation of the glycerol kinase tetramer to dimer but has no effect on the affinity of the tetramer for the allosteric inhibitor. High pH (9--10) has only a small effect on the subunit dissociation constant but greatly reduces the rates of subunit association and dissociation. Desensitization of glycerol kinase to allosteric inhibition can thus occur by three different mechanisms, two of which are directly related to the polysteric nature of the enzyme.


Acyl carrier protein from Escherichia coli: characterization by proton and fluorine-19 nuclear magnetic resonance and evidence for restricted mobility of the fatty acid chain in tetradecanoyl-acyl-carrier protein. The acyl-carrier protein (ACP) of Escherichia coli is a protein of molecular weight 8847 with a 4'-phosphopanthetheine prosthetic group. ACP functions (via the SH of the prosthetic group) as a coenzyme in the synthesis of fatty acids and complex lipids. We report proton nuclear magnetic resonance (NMR) studies of the structure of ACP under various experimental conditions. The motion of the fatty acyl chain of acyl-ACP has been investigated by 19FNMR studies of difluorotetradecanoyl-ACP. 31PNMR studies of the prosthetic group phosphorus of ACP and acyl-ACP are also reported. We make the following conclusions: (1) the structure of ACP is stabilized by surface charge, and (2) the fatty acid residue of acyl-ACP does not move freely and seems immobilized by an interaction with the protein moiety.


Sequence of reactions which follows enzymatic oxidation of propargylglycine. The nonenzymatic reactions which follow enzymatic oxidation of the gamma-delta acetylenic amino acid propargylglycine (2-amino-4-pentynoate) have been studied. The product which accumulates in solution has been identified as 2-amino-4-hydroxy-2,4-pentadienoate gamma-lactone, formed by intramolecular attack of the carboxylate anion on the electrophilic fourth carbon of 2-iminium-3,4-pentadienoate. This previously unknown substance was characterized by its reactions in acid and base and by its nuclear magnetic resonance spectrum. The lactone is preceded in the pathway by 2-amino-2-penten-4-ynoate, a transient electron-rich species which binds tightly to D-amino-acid oxidase and induces a charge-transfer complex with the electron-deficient bound flavin coenzyme. The aminediene lactone is converted by base treatment to 2-amino-4-keto-2-pentenoate, which is also a strong inhibitor of D-amino-acid oxidase and induces a charge-transfer complex.


Sequence of reactions which follows enzymatic oxidation of allylglycine. The pathway following flavoprotein-catalyzed oxidation of allylglycine (2-amino-4-pentenoate) has been studied and found to be dependent on the incubation conditions. In N-2-hydroxyethyl-N'-2-ethanesulfonic acid (Hepes) buffer, the oxidation product 2-iminium-4-pentenoate predominantly reacts to form 2-amino-2,4-pentadienoate, a strong noncovalent inhibitor of D-amino-acid oxidase. However, in pyrophosphate buffer, the more rapid reaction is hydrolysis to form 2-keto-4-pentenoate, which has been found to be a substrate for L-lactic dehydrogenase. 2-Keto-4-pentenoate is in rapid equilibrium with 2-hydroxy-2,4-pentadienoate, which is also a strong noncovalent inhibitor of D-amino-acid oxidase. In both systems, these metastable intermediates react in subsequent slower steps to yield trans-2-keto-3-pentenoate, which accumulates in the incubation. Syntheses of trans-2-amino- and trans-2-keto-3-pentenoate are described. Comparisons between the reactivities of acetylenic and olefinic species have been made based on the differences between this pathway and that following oxidation of propargylglycine [Marcotte, P., and Walsh, C. (1978), Biochemistry 17 (preceding paper in this issue)].


Characterization of human platelet glutathione reductase. Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.


Characterization of glutathione reductase from porcine erythrocytes. Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) was purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2',5'-ADP-Sepharose 4-B. Analytical ultracentrifugation experiments were analysed to give the following physical parameters for the enzyme: s20,w = 5.7 S, D20,w = 50 microgram2/s, and Mw = 103 000 (protein concentration, 0.5 mg/ml). The frictional ratio was 1.37 and the Stokes radius was 4.3 nm. The enzyme molecule is a dimer composed of subunits of equal size each containing a FAD molecule. The amino acid compositions and circular dichroism spectra of the porcine and human enzymes indicated extensive structural similarities. The isoelectric point was at pH 6.85 (at 4 degrees C). The absorption spectrum of the oxidized enzyme had maxima at 377 and 462 nm. In vivo the enzyme appears to be partially reduced. At a physiological concentration of reduced glutathione the apparent Michaelis constants for glutathione disulfide and NADPH were higher than in the absence of reduced glutathione. At 0.15 M ionic strength the catalytic activity obtained with NADPH as reductant was optimal at pH 7 and more than 200 times higher than that obtained with NADH. S-sulfoglutathione and some mixed disulfides of glutathione were poor substrates with the exception of the mixed disulfide of coenzyme A and reduced glutathione. The purified enzyme displayed low transhydrogenase activity with oxidized pyridine nucleotide analogs and diaphorase activity with 2,6-dichlorophenolindophenol as acceptor substrates; both NADPH and NADH served as donors.


Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH. The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.


Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms. Bovine liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been obtained in three forms designated transferase I, A and B on the basis of their elution positions from chromatography on phosphocellulose. All forms have been shown to have a molecular weight of about 152 000, each being composed of four similar subunits. Amino acid analysis of transferase A and B, the two major forms, revealed a close relationship between both forms with almost identical amino acid composition and arginine as N-terminal residue. The three transferases differ with respect to their redox state and their multiplicity of forms with isoelectric points of 6.9, 7.5 and 8.8, into which the transferases I and A were spontaneously transformed upon isoelectric focusing or rechromatography on phosphocellulose. Transferase B represents a stable enzyme form with an isoelectric point of 8.8. Although the redox state of transferase B can be adjusted to that of transferase A still a difference in charge and in the multiplicity of forms exists, thus indicating different protein states.


The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues. Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.


Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease. The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.


Effects of amino acids, adenine nucleotides and inorganic pyrophosphate on glutamine synthetase from Anabaena cylindrica. Glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from Anabaena cylindrica was inhibited by alanine, glycine, serine and aspartate. The effects of alanine and serine were uncompetitive with respect to glutamate, while those of glycine and asparatate were uncompetitive with respect to glutamate, while those of glycine and aspartate were non-competitive and mixed type respectively. Different pairs of amino acids and their various combinations caused a cumulative inhibition of the enzyme activity. Glutamine synthetase was also inhibited by ADP and AMP and both nucleotides affected the enzyme competitively with respect to ATP and non-competitively for glutamate. Inorganic pyrophosphate, between 2 and 3 mM, produced a very pronounced inhibiton of enzyme activity. The inhibition by PPi was uncompetitive for ATP. Various combinations of the adenine nucleotides, PPi and Pi exerted a cumulative inhibitory effect on the enzyme activity, as did the amino acids, in different combinations with either adenine nucleotides, PPi or Pi. The effects of the adenine nucleotides and the amino acids were more pronounced at higher concentrations of ammonia. Except for serine similar responses of these effectors were obtained with increasing concentrations of Mg2+. It is proposed that changes in the free concentrations of Mg2+ are important in energy-dependent regulation of the enzyme activity in this alga.


A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. A rapid and sensitive method has been developed to determine lysopine dehydrogenase (EC 1.5.1-) and nopaline dehydrogenase activities in crown gall tumour tissues. By this method, enzyme activities as low as 0.2  micrometerol octopine or nopaline per h per g fresh weight tumour tissue can still be detected. In non-infected young pea seedlings, no lysopine dehydrogenase activity was detected.


Spectrophotometric titration of phenolic groups of pepsin. The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.


Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies. Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.


Laser Raman studies on cobrotoxin.

Laser Raman studies on cobrotoxin. Laser Raman spectra of cobrotoxin under various conditions have been obtained. Comparison of the spectra of native cobrotoxin in lyophilized form and in aqueous solution indicates that the secondary structures of cobrotoxin are not significantly affected by the removal of the aqueous solvent. On going from the native to the partially reduced and the completely reduced, carboxy-methylated forms, characteristic peaks of the C-S-S-C and tyrosine ring in the region of 500--900 cm-1 showed definite changes in structure. The partially reduced form gave two peaks at 502 and 524 cm-1, suggesting difference in the conformation of the remaining disulfide bonds. As indicated by the present work, the conformation of the main chain of cobrotoxin in the native unperturbed state, in the partially reduced and in the completely reduced forms are the coexistence of beta-pleated sheet with random-coil structure, predominantly random coil, and predominantly random coil with the existence of an alpha-helix type structure, respectively. The effect of pH on the conformation of cobrotoxin in solution appeared to give rise to the change of the local structure of two aromatic residues common to all snake neurotoxins.


Reaction of horseradish peroxidase with azide and some implications for the heme environmental structure. NMR and kinetic studies. The azide complex of horseradish peroxidase was studied by high resolution 1H and 15N NMR spectroscopy and by the temperature-jump method. The heme peripheral methyl proton peaks and the ligand 15N resonance were resolved to show that binding of azide by horseradish peroxidase occurs only in acidic solution below pH 6.5. It was also found that the chemical exchange rate of azide with the ferric enzyme was much faster on the 1H and 15N NMR time scale. This was further substantiated by kinetics of azide binding by horseradish peroxidase where the chemical exchange rate was confirmed to be in the microseconds range at pH 5.0 and 23 degrees C. This rate is salient in usual ligand exchange reactions in hemoproteins so far reported. pH dependences of the first order association and dissociation rate constants were also studied by the temperature-jump method to suggest a strong linkage of the azide binding with a proton uptake of an amino acid residue on the enzyme. These results were compared with the case of horse metmyoglobin and were interpreted to indicate that a heme-linked ionizable group on the enzyme facilitates the fast entry of the ligand to the coordination site. A histidyl residue is a possible candidate for the ionizable group of the enzyme.


13C NMR investigations on Npi-[13C1]carboxymethyl-histidine-119 ribonuclease. The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12.


pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase. A pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase was carried out at 25 degrees C and 0.1 M ionic strength. The net charge on cytochrome c peroxidase due to proton association and dissociation varies from +32 at pH 2 to --50.2 at pH 12, while that of apocytochrome c peroxidase varies between +24.5 at pH 3 to --48 at pH 12. The apoprotein tented to aggregate below pH 3. Between pH 4 and 8, the titration behavior of both the native enzyme and the apoenzyme are consistent with the semi-empirical Linderstrøm-Lang theory. Between pH 9 and 12, the titration behavior of both the holo- and apoproteins suggest they assume a more extended conformation which reduces the electrostatic interaction charged groups on the surface. In the acid region, between pH 4 and 3, a similar transition occurs in which the protein expands 40% based on the electrostatic factor of the Linderstrøm-Lang theory.


Isoelectric focusing of ferredoxins, flavordoxins and a rubredoxin. Isoelectric points of ferredoxins, flavodoxins and a rubredoxin from a range of sources were measured by electrofocusing over the pH range between 2.5 and 5.0 on thin layers of polyacrylamide gel. The pH gradient along the gel was measured directly by a surface electrode. The isoelectric points of the plant-type ferredoxins were between approx. 3.15 and 3.35, and those of the flavodoxins close to 3.5. Ferredoxin, rubredoxin and flavodoxin from Clostridium pasteurianum had isolectric points of the of 2.75, 2.9, and 3.1, respectively. The values for the isoelectric points ferredoxins are significantly lower than previous results in the literature suggest.


Tyrosine aminotransferase converting factor: kinetic properties, cellular localization, and tissue distribution. Utilizing a more rapid procedure for determining tyrosine aminotransferase-converting factor activity, the kinetic properties of this factor were characterized further. Tyrosine aminotransferase-converting factor is a heat-labile substance present in the particulate fraction of rat liver that converts tyrosine aminotransferase form III to I at 4 degrees C. Analysis of the distribution of marker enzymes for mitochondria and lysosomes, and of converting factor, following differential and discontinuous sucrose gradient centrifugation indicated that this factor was associated with light lysosomes. The activity of converting factor was not altered following administration of cortisol. Converting factor activity, equivalent to that in liver, was also observed in particulate fractions from kidney and spleen, and to a lesser extent, in pancreas and salivary gland. No detectable activity was observed in brain, heart, small intestine, skeletal muscle, red blood cells, serum, or plasma. The presence of converting factor activity in kidney and spleen suggests that other proteins are substrates for this factor since tyrosine aminotransferase is virtually absent from these tissues. Alternatively, the absence of converting factor from other tissues need not indicate they are devoid of converting factor-like activity merely, that such activity in these tissues has different specificities and does not utilize tyrosine aminotransferase as a substrate.


Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes. An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.


Collagen-induced platelet aggregation and release. I Effects of side-chain modifications and role of arginyl residues. To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen. These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.


Optical characteristics of carboxyl group in relation to the circular dichroic properties and dissociation constants of glycosaminoglycans. Circular dichroism studies of glycosaminoglycans including chemically transformed heparins at various pH values reveal that carboxyl chromophore plays an important role in the dichroic behavior of the polymers. With decreasing pH, iduronic acid-containing glycosaminoglycans show increased negative ellipticity near 220 nm whereas the polymers containing glucuronic acid display enhanced negative dichroism near 230 nm and decreased negative dichroism around 210 nm. The pH-dependent optical properties have been utilized to determine the pKa values of uronic acid moieties. The acid strengths of the iduronic acid-containing glycosaminoglycans are inherently smaller than those of corresponding glucuronic acid-containing polymers. Glycosaminoglycans in which the amino sugars are linked with iduronic acid display a very weak n leads to pi* amide transition, or none. The rotational strength at 210 nm of these polymers is largely due to iduronic acid moieties. The CD variations above 200 nm with change in pH do not indicate any major conformational transition of the molecules but the difference between dermatan sulfate and heparin can be attributed to difference either in iduronic acid conformation or in intersaccharide linkages.


Effect of pH on chloroplast photosynthesis. Inhibition of O2 evolution by inorganic phosphate and magnesium. 1. The pH optimum of CO2-dependent O2 evolution by barley (Hordeum vulgare L.) chloroplasts was found to be between 7.8 and 8.2. The addition of 1 mM MgCl2 in the dark inhibited O2 evolution over the entire pH range tested and resulted in a much sharper pH profile centered around pH 8.2. 2. The pH optimum for O2 evolution, in the presence and absence of 1 mM MgCl2, was acid-shifted 0.3--0.4 pH units by 2 mM NH4Cl. The pH optimum of O2 evolution, with and without 1 mM MgCl2, was base-shifted by 2 mM sodium acetate, approx. 0.5 pH units relative to the controls. 3. O2 evolution in the presence of bicarbonate plus 3-phosphoglycerate or ribose-5-phosphate was considerably less sensitive to pH than CO2-dependent O2 evolution in the absence of substrate. With these substrates, both in the presence and absence of 1 mM MgCl2, the pH optimum was broad and was centered around pH 7.8. 4. Inhibition of CO2-dependent O2 evolution by inorganic phosphate and magnesium increased as the pH of the reaction mixture was decreased below the optimum. Decreasing the pH from 8.2 to 7.6, reduced over 3-fold the concentration of inorganic phosphate required to inhibit O2 evolution completely. For magnesium, a similar change in pH reduced the concentration required to inhibit O2 evolution 50% approx. 5-fold. At pH 8.2, magnesium inhibition required inorganic phosphate. Magnesium was not required for inhibition of O2 evolution by inorganic phosphate, but incresaed the relative inhibition observed. 5. Illumination of intact barley chloroplasts increased the activity of NADP-glyceraldehyde-3-P dehydrogenase, phosphoribulokinase and fructose-1,6-diphosphatase. MgCl2 and inorganic phosphate prevented this increase in enzyme activity at concentrations that completely inhibited CO2-dependent O2 evolution. 6. The results obtained suggest that magnesium inhibition of O2 evolution may be caused by enhanced phosphate exchange across the chloroplast envelope.


Studies on the succinate dehydrogenating system. I. Kinetics of the succinate dehydrogenase interaction with a semiquindiimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine. 1. The activities of the soluble reconstitutively active succinate dehydrogenase (EC 1.3.99.1) measured with three artificial electron acceptors, e.g. ferricyanide, phenazine methosulfate and free radical of N,N,N',N'-tetramethyl-p-phenylenediamine (WB), have been compared. The values estimated by extrapolation to infinite acceptor concentration using double reciprocal plots 1/v versus 1/[acceptor] are nearly the same for ferricyanide and phenazine methosulfate and about twice as high for the WB. 2. The double reciprocal plots 1/v versus 1/[succinate] in the presence of malonate at various concentrations of WB give a series of straight lines intercepting in the third quadrant. The data support the mechanism of the overall reaction, in which the reduced enzyme is oxidized by WB before dissociation of the enzyme-product complex. 3. The dependence of the rate of the overall reaction on WB concentration shows that only one kinetically significant redox site of the soluble succinate dehydrogenase is involved in the reduction of WB. 4. Studies of the change of V and Km values during aerobic inactivation of the soluble enzyme suggest that only 'the low Km ferricyanide reactive site' (Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys, Res. Commun. 65, 1264--1269) is involved in reoxidation of the reduced enzyme by WB. 5. The pH dependence of V for the succinate-WB reductase reaction shows that the group of the enzyme with the pKa value of 6.7 at 22 degrees C is responsible for the reduction of dehydrogenase in the enzyme-substrate complex. 6. When WB interacts with the succinate-ubiquinone region of the respiratory chain, the double reciprocal plot 1/v versus 1/[WB] gives a straight line. The thenoyltrifluoroacetone inhibition of succinate-ubiquinone reductase or extraction of ubiquinone alter the 1/v versus 1/[WB] plots for the curves with a positive initial slope intercepting the ordinate at the same V as in the native particles. The data support the mechanism of succinate-ubiquinone reduction, in which no positive modulation of succinate dehydrogenase by ubiquinone exist in the membrane.


Conditions limiting the use of ionophore A23187 as a probe of divalent cation involvement in biological reactions. Evidence from the slow fluorescence quenching of type A spinach chloroplasts. The conditions under which ionophore A23187 can be used as a probe of Mg2+ involvement in the reactions of intact (Type A) spinach chloroplasts have been investigated by monitoring ionophore-induced reversal of slow fluorescence quenching. The following observations were made: (1) A23187-dependent reversal of quenching is a strong function of pH. This is consistent with competition between protons and divalent cations for the carboxylic acid moiety of the ionophore. (2) In the presence of exogenous Mg2+, quenching reversal by A23187 is significantly slowed. It is suggested that formation of the dimeric A23187 . Mg2+ complex delays action of the ionophore at the thylakoid membrane by slowing equilibration of the ionophore among chloroplast membrane phases. (3) In the absence of Mg2+, significant interaction of A23187 with certain monovalent cations--Li+ and Na+, but not K+--is observed. Evaluations of the interaction of ionophore A23187 with specific biological systems and inferences of divalent cation involvement, or lack thereof, must take these limitations into account.


Characterization of two quenchers of chlorophyll fluorescence with different midpoint oxidation-reduction potentials in chloroplasts. The properties of two redox quenchers of chlorophyll fluorescence in chloroplasts at room temperature have been investigated. (1) Redox titration of the fluorescence yield reveals two n = 1 components with Em7.8 at--45 and --247 mV, accounting for approx. 70 and 30% of the total yield, respectively. (2) Neutral red, a redox mediator often used at redox potentials below --300 mV, preferentially quenches the fluorescence controlled by the --247 mV component. Titrations using neutral red artifactually create an n = 2 quenching component with Em7.8 = --375 mV. (3) Analysis of fluorescence induction curves recorded at different redox potentials indicates that both the --45 and --247 mV components can be photochemically reduced. The reduction of the --247 mV component corresponds to a fast phase of the induction curve whilst the slower reduction of the 45 mV component accounts for the tail phase. (4) The excitation spectra for the fluorescence controlled by the two quenchers show small differences in the ratio of chlorophyll a and b. (5) Whereas the --247 mV component readily shows a 60 mV per pH unit dependency on solution pH, the ability of the --45 mV component to respond to pH change is restricted. (6) Triton Photosystem II particles contain both quenchers but the --247 mV component accounts for approx. 70% of the fluorescence and the high component has an Em7.8 of +48 mV. The relative merits of sequential and parallel models in explaining the presence of the two quenchers are considered.


Purification and properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from a methanol-utilizing yeast, Candida boidinii. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase, EC 1.1.1.44) were purified approx. 1700 fold and 330 fold, respectively, from Candida boidinii grown on methanol. The final enzyme preparations were homogeneous as judged by polyacrylamide gel electrophoresis. The molecular weights of the enzymes were estimated to be 118 000 and 110 000, respectively. Both enzymes are composed of two probably identical subunits and the molecular weights of the polypeptide chains were calculated to be 61 000 and 58 000, respectively. From a consideration of enzyme activities and types of inhibition by different metabolites the role of these two enzymes in glucose- and methanol-metabolism is discussed.


Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers. A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0--12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0--40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40--55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.


Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity. The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.


Effect of salts on D-glycerate dehydrogenase kinetic behavior. Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.


Multiple acid proteinases in the cellular slime mould Dictyostelium discoideum. Proteinase activity in the cellular slime mould Dictyostelium discoideum has been analyzed by electrophoresis on polyacrylamide gels containing denatured hemoglobin. At least eight bands due to acid proteinases have been defined using extracts of myxamoebae, four bands A-D which move faster than the fifth and major band E, a minor band E' which moves just behind E and two slow bands G and H. Fruiting body formation was accompanied by the appearance of one new proteinase band F. The proteinases were present in extracts of both axenically-grown and bacterially-grown cells. Differences between the pH dependence and stability of the individual proteinases were detected. Inhibitor studies suggested that the faster proteinases A-D may be cathepsin B-like, whilst the slower enzymes E, E' and F do not fit readily into any known group of proteinases since they were sensitive to HgCl2 but not to other inhibitors of cathepsin B and not to inhibitors of cathepsin D-like proteinases under standard conditions. None of the proteinases was apparently formed during or after preparation of extracts and the proteinases could be re-run on polyacrylamide gels to give only the band expected from the first run. The bands are believed to reflect multiple proteinase activities within the cell.


Nuclear phosphoprotein phosphatase from calf liver. Calf liver nuclear phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphophosphorylase, phosphohistones f1 and f2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase greater than phosphohistone f1 greater than phosphohistone f2b greater than phosphoprotamine. The Km values toward phosphophospharylase and phosphohistone f1 are 17 and 28 micron phosphate, respectively. Dephosphorylated histone f1 and orthophosphate are competitive inhibitors of the enzyme with respective Ki values of 11 micron and 4.1 mM. NaCl and divalent metal ions inhibit the enzyme but CaCl2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.


Alpha-mannosidase in human red cells.

Alpha-mannosidase in human red cells. 1. A search for lysosomal hydrolases and related enzymes has been made in hemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for alpha-mannosidase, neutral alpha-glucosidase and beta-hexosaminidase. 2. alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5--6.0. Electrophoresis on cellulose acetate showed three bands. Hemolysates from four patients with mannosidosis were not deficient in alpha-mannosidase. pH activity curves and elctrophoretic pattern were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell mannosidase differs from the lysosomal acid mannosidase.


Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands. A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.


Response waveforms of vertebrate photoreceptors: what are the underlying mechanisms? A class of models is investigated using computer simulation in which the inner and outer segments of the vertebrate photoreceptor are coupled through a pump. The outer segment membrane conductance is controlled by an internal transmitter, activated by photolysis of the photosensitive molecules in the cell. Several possibilities for the coupling dynamics are investigated. The analysis favors the conclusion that the hyperpolarizing transient at high intensity stimuli arises from the coupling dynamics, (unless there is an extracellular current shunt path). It predicts, moreover, that the transient should be observed intracellularly, but not extracellularly to the outer segment. This is, in fact, the case. It also predicts that the transient should become more marked, as the steady state ratio of inner to outer segment currents decreases. The computer simulations are concerned with the intracellularly recorded responses; the long term adaptation parallel to pigment bleaching and regeneration is not considered explicitly here. In conclusion, it is shown that the state conditions as well as the response waveforms can be related to physiologically significant variables.


Characteristics of the contingent negative variation in patients suffering from specific phobias. The essential clinical feature of phobic neurosis is the anticipatory fear of certain objects and situations. Since an important factor in the generation of the contingent negative variation (CNV) is the anticipation of the imperative stimulus, records of CNVs were used as an indicator of electrocortical activity in a group of 14 patients suffering from specific phobias. After clinical evaluation, a CNV to a picture of a nondisturbing object was obtained. The amplitude of the CNV and duration of the PINV (postimperative negative variation) were taken as reference values against which response to the phobic objects were evaluated. Reaction time was automatically measured between the period of S2 and the button press. Significant differences, viz., larger CNV amplitude, longer PINV duration, and shorter reaction time, were found with phobogenic than with nondisturbing stimuli. After behavioral recovery with behavior therapy, no differences were noted in the CNVs obtained with the presentation of non-disturbing and the ones that were phobogenic stimuli. The significance of the results for an understanding of phobic neurosis and behavior therapy are discussed.


Response to lithium carbonate.

Response to lithium carbonate. We examined the clinical and research records of 29 acutely ill hospitalized patients with affective disorder who received only lithium carbonate during their first week of treatment. Nineteen patients (Group I) could be continued on lithium ion alone, while 10 patients (Group 2) needed additional somatic treatment. Compared with Group 1, Group 2 patients were significantly younger at illness onset, more severely ill on admission, clinically more "colorful" in dress and behavior, stayed more than twice as long in the hospital, and (although not statistically significant) had more than twice the morbidity risk for affective disorder in first-degree relatives. At discharge, both groups were equally improved, and 70% of Group 2 patients were receiving lithium alone. We did not confirm previous reports that nonresponders to lithium alone (Group 2) were more overactive or paranoid--destructive or less euphoric--grandiose than responders to lithium alone (Group 1). Our Group 2 patients had a more severe or penetrant form of illness than our Group 1 patients, requiring neuroleptic drugs or ECT in addition to lithium therapy. Eventually, however, they had a satisfactory outcome, suggesting therapeutic optimism and tenacity even in those patients who initially fail lithium alone and require polytreatment.


Base stacking in the dinucleoside phosphate rapa. The pH-induced unstacking of rRpA has been investigated by batch calorimetry and uv spectroscopy. Equilibrium uv melting curves confirmed that the adenine bases in rApA are stacked at pH7 but unstacked at pH 1.5. The enthalpy change accompanying this pH-induced unstacking is +2.65 kcal (mole of A-A stack)-1 as measured by batch calorimetry. This represents the first direct determination of this important parameter for a dinucleoside phosphate. It is noted that the calorimetrically determined value reported here is considerably lower than published van't Hoff enthalpies but is consistent with values that can be derived from calorimetric data on polymers.


[Age and the course of nephrotoxic nephritis in rats]. In experiments on two groups of mongrel rats (4 weeks old and 4 months old) with induced nephrotoxic nephritis it was revealed that in comparison with adult rats the course of nephritis in ratlings was characterized by lesser proteinuria, selective in nature, by lesser reducticn of endogenous creatinine clearance and diuresis. The acido- and ammo-niogenesis decreased in ratlings and adult rats to the same extent. Morphological changes in the kidneys of ratlings were less pronounced than in adult animals, and were mostly localized in the convoluted tubules. The level of DNA-synthetic activity of the epithelial nuclei of the glomeruli prevailed over this index of the convoluted tubules epithelium. The weight index of the kidneys increased less in ratlings with nephritis than in adult rats. beta-lipoproteinemia in ratlings increased 8 times. Normalization of the urine and blood indices occurred more rapidly in ratlings than in adult rats.


Factors influencing the release of acetylcholine from the myenteric plexus of the ileum of the guinea-pig and rabbit. 1 The effects of electrical stimulation, changes in external ion concentrations and various drugs on acetylcholine release from the myenteric plexus were measured by bioassay in the presence of physostigmine and by recording the responses of the longitudinal muscle. In preparations from the guinea-pig, the acetylcholine output per pulse increased with decreasing frequency of stimulation and reached its maximum at a frequency of 0.017 Hz (1/min) and thus ensured that the output per unit of time was constant at frequencies below 0.5 Hz. Spontaneous release was suppressed during stimulation at 0.017 Hz. 2 In the rabbit, the fractional acetylcholine release was lower than in the guinea-pig. The output per pulse increased with decreasing frequency of stimulation but at a lesser rate, with the effect that the output per unit decreased between 0.5 and 0.017 Hz. 3 In the guinea-pig, reduction of the Ca2+ concentration, addition to the bath fluid of Mn2+, ganglion-blocking drugs, morphine and catecholamines reduced output more at low than at high frequencies of stimulation. In the rabbit, acetylcholine output was less sensitive to changes in Ca2+ concentration and insensitive to Mn2+ and morphine. 4 In the guinea-pig, morphine and catecholamines depressed both the contractile response and acetylcholine output whereas Mn2+ in concentrations up to 125 muM, bretylium and ganglion-blocking drugs depressed only acetylcholine output. 5 In preparations from the guinea-pig, drugs blocking noradrenergic neurons or alpha-adrenoceptors, e.g. bretylium, phenoxybenzamine, thymoxamine and phentolamine, increased acetylcholine output during stimulation at high (1.5 to 10 Hz) but not at low frequencies. 6 The implications of these findings for the release of acetylcholine from different pools in the heterogeneous myenteric plexus are considered. The possible errors, introduced by the effects of physostigmine, on the size of the acetylcholine pools and on the transmission of impulses within the myenteric plexus are discussed.


4-Aminopyridine and evoked transmitter release from motor nerve endings. 1 In the presence of tetrodotoxin, electrotonic depolarization of frog motor nerve terminals causes the appearance of stimulus-graded endplate potentials. When 4-aminopyridine is added, the graded endplate potential is converted into a triggered all-or-none response resulting in giant endplate potentials of about 70 mV amplitude and 50 ms duration. The triggered endplate potentials are abolished in Ca(2+)-free saline and are blocked by Mn(2+) ions. Sr(2+) but not Ba(2+) can replace Ca(2+) in supporting transmitter release. Mg(2+) fails, even in concentrations as high as 32 mM, to affect the amplitude and the shape of the endplate potential but abolishes it when the Ca(2+) concentration is reduced to 0.2 mM.2 Despite the large amplitude of the triggered endplate potential in the presence of 4-aminopyridine and tetrodotoxin, repetitive stimulation up to 10 Hz causes only a small decline in amplitude of successive endplate potentials. However, in the presence of (+)-tubocurarine or gallamine, repetitive nerve stimulation produces a marked decline in successive endplate potential amplitude. The fall is counteracted when evoked transmitter release is reduced in the presence of 0.2 mM Ca(2+). The results suggest that in the presence of 4-aminopyridine such large amounts of transmitter are released that even during repetitive stimulation (5 to 10 Hz) endplate potentials are of maximal amplitude.3 4-Aminopyridine causes a prallel shift to the right of the dose-response curve to Mg(2+) for blockade of nerve impulse-evoked transmitter release (in the absence of tetrodotoxin). A similar parallel shift occurs in the presence of tetraethylammonium and guanidine.4 It is concluded that 4-aminopyridine increases transmitter release by enhancing the transport efficacy for Ca(2+) across the nerve terminal membrane during nerve terminal depolarization.


Rehearsal relief of a nightmare.

Rehearsal relief of a nightmare. A recurrent nightmare of 14 years' duration rapidly disappeared after it was rehearsed repeatedly in treatment. The nightmare was independent of co-existing compulsive rituals ans sexual problems, but was related to co-existing depression.


Brain alpha-adrenergic receptors: comparison of [3H]WB 4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex. The ability of a series of adrenergic agents to displace the binding to brain membranes of [3H]WB 4101, a potent alpha-adrenergic antagonist (WB 4101 = 2-[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the alpha-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [3H]WB 4101 rapidly bound to a high affinity site (KD = 2.7 nM) in membranes from cerebral cortex. Binding came to equilibrium by 2 min at 37 degrees C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 greater than phentolamine greater than naphazoline) in displacing binding of [3H]WB 4101 were comparable to the potencies of these agents as inhibitors of the alpha-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10--30 times more potent in inhibiting cyclic AMP accumulation than in displacing [3H]WB 4101 binding. The potency of classical alpha-adrenergic agonists in displacing WB 4101 (epinephrine greater than norepinephrine greater than methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation (r = 0.87, P less than 0.005) was found between WB 4101 binding and alpha-adrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with alpha-adrenergic receptor properties. The identification of these binding sites as receptors depends on a correlation of binding with a known alpha-adrenergic receptor-mediated response in brain. These data demonstrating that WB 4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the alpha-adrenergic accumulation of cyclic AMP in rat cerebral cortex.


Early increase in phenylethanolamine-N-methyltransferase activity in a new strain of spontaneously hypertensive rats. By repeated inbreeding, 2 strains of spontaneously hypertensive and normotensive rats have been simultaneously selected. The activities of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase were determined in various central catecholaminergic nuclei (C1, C2, A6 and A9) and in two peripheral tissues (adrenal glands and superior cervical ganglion). These assays were performed on rats belonging to the normotensive or the hypertensive strain at 3 ages which characterize the development of hypertension (5, 9 and 21 weeks). Except for a decrease in the C1 region of 9-week-old rats, no significant change in tyrosine hydroxylase activity occurred in central or peripheral structures of the spontaneously hypertensive rats when compared to the normotensive rats. In contrast, the activity of the phenylethanolamine-N-methyltransferase (PNMT), was increased in the C2 adrenergic group of the medulla oblongata in young spontaneously hypertensive rats: +43% (P less than 0.001) at 5 weeks of age and +32% (P less than 0.001) in 9-week-old rats. However, there was no significant difference between the 21-week-old rats. No modification of the PNMT activity was found in the C1 adrenergic group of the medulla oblongata. PNMT activity was increased significantly in the adrenal glands of 5-week-old hypertensive rats (+22%, P less than 0.001). By 9 weeks, the difference in PNMT activity in the adrenals was no longer significant. Thus, in young rats of the hypertensive strain, there was an increase in the capacity to synthetize adrenaline in the C2 area of the medulla oblongata and in the adrenal glands. While the enzymatic change present in the adrenals seems to be specific to this new strain of hypertensive rats, the elevation of PNMT activity in a specific region of the medulla oblongata (C2 group) is a characteristic common to at least two independently derived strains of genetically hypertensive rats.


Fine structural localization of glutamine synthetase in astrocytes of rat brain. The distribution of glutamine synthetase was determined in rat brain by ultrastructural immunocytochemistry. Except for trace amounts in a rare indeterminate glial cell, all the reaction product was located in astrocytes. Specifically, none was observed in neurons, synaptic endings, oligodendrocytes, microglial cells, pericytes, endothelial cells and other mesenchymal vascular elements. The results of this study clearly indicate that the astrocyte forms the compartment in brain concerned with glutamine synthesis, thereby assigning a key role to the astrocyte in the metabolism of ammonia and the putative neurotransmitters, glutamic acid and gamma-aminobutyric acid. The localization of glutamine synthetase in astrocytes additionally provides a valuable marker for a number of neurobiological studies.


Dynamics of serum potassium change during acute respiratory alkalosis. In summary, one should be aware that hyperventilation and hypokalaemia occur frequently in the operating room under ordinary clinical conditions. An awareness of the rapidity with which these changes occur, and their extent as well as their potential hazards, should cause consideration of more aggressive attempts to maintain normocarbia and to use supplemental potassium during anaesthesia, particularly in patients with cardiac disease.


Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa. Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate-Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate alpha-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360,000 and as having a sedimetation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate alpha-helix content of 23-24%. The subunit generated by treatment with urea was found to be 45,000 daltons by gel filtration methods and a molecular weight of 46,000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45,000 daltons.


3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in beef adrenal cortex. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity (mevalonate:NADP+ oxidoreductase )CoA-acylating) EC 1.1.1.34) was demonstrated in beef adrenal cortex. Most of the HMG-CoA reductase activity is in the microsomal fraction while a small percentage of the activity is associated with the mitochondria, Mitochondria purified on a linear sucrose gradient are enriched in HMG-CoA reductase and cytochrome c oxidase activities. The reductase present in microsomal preparations from the whole adrenal cortex has an apparent Km of 5.6 X 10(-5) M for (R,S)-HMG-CoA. Reductase activities found in the microsomal fractions from the zona glomerulosa, the zona fasciculata, and the zona reticularis were 1.32, 7.37, and 9.74 nmol mevalonate formed per milligram protein in 30 min respectively.


Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography. The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.


Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine. The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.


Production and properties of polygalacturonate lyase by an alkalophilic microorganism Bacillus sp. RK9. Bacillus sp. RK9 was isolated from soil and produced a constitutive polygalacturonate lyase. Production of the enzyme required the presence of complex nitrogen (peptone and yeast extract). Highest activity was obtained with an initial pH of 9.7. The organism was alkalophilic. No growth occurred below pH 7.5. The enzyme was purified by salt precipitation and diethylaminoethyl (DEAE) cellulose ion-exchange chromatography. The pH optimum for activity was 10.0 in 0.01 M glycine-NaOH buffer. Calcium alone, of divalent cations, activated the enzyme by 2.9-fold. Complete inhibition of enzyme activity was achieved by 1 mM ethylenediaminetetraacetic acid (EDTA). Hydrolysis of substrate occurred in a random fashion and the enzyme was 50% more active towards acid soluble pectic acid (ASPA) than towards sodium polypectate.


DNA-dependent RNA and polyadenylic acid polymerase from phototrophically grown Rhodospirillum rubrum. DNA-dependent RNA and polyadenylic acid polymerases have been purified from phototrophic Rhodospirillum rubrum. Their properties have been found to be very similar to those of the previously reported heterotrophic R.  rubrum enzymes. However, several important differences do exist between the enzymes from the phototrophic and the heterotrophic cells, such as the lack of response to added polyadenylic acid for poly A synthesis and the presence of the sigma subunit in the phototrophic enzymes. Furthermore, additional purification steps were necessary for preparation of phototrophic enzyme fractions with high DNA-dependence.


Cellulase and beta-glucosidase production by a basidiomycete species. The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.


alpha,alpha-Trehalase of Trichoderma reesei. A simple adsorption and elution of the trehalase of Trichoderma reesei on bentonite increased the specific activity 70-80 times, with a recovery of 90%. This alpha,alpha-trehalase has an optimum pH of 4.4, a pl of 5.7, a Km of 3.1 X 10(-3) M, and a specific activity of 50 mumol/mg. min-1.


Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously. Hybridization of cells of defined and different histotypes has been carried out to investigate whether the expression (or reexpression) of parental functions is mutually exclusive, as is expected if the generally assumed rule of discreteness of differentiation applies to hybrid cells. A cross of pigmented mouse melanoma cells and albumin-producing rat hepatoma cells gave rise to hybrids containing essentially one set of chromosomes from each parent and producing neither melanin nor albumin. Cells of one hybrid clone are shown to retain the potential to reexpress both parental differentiations. Successive subclonings of this hybrid have shown that cells which reexpress one function may retain the potential to reexpress the other, and that freshly isolated, morphologically homogeneous subclones may produce pigment or albumin, but not both; there successive and exclusive shifts of phenotype are documented, and in these cases, chromosome loss is very slight. The use of immunoadsorbed antisera has revealed that most (if not all) of the albumin produced by the hybrid cells is of the mouse type. We conclude that both parental determinations are retained by the hybrid cells, and that the parental differentiations are reexpressed only in a mutually exclusive fashion.


Mobility of histones on the chromosome of simian virus 40. Linear simian virus 40 (SV40) chromosomes were prepared by Eco R1 nuclease cleavage of the circular SV40 chromosomes released from virions with dithiothreitol at pH 9,8. Chromatin-DNA hybrids were constructed with segments of 3H-labeled, naked SV40 DNA covalently joined via the Eco R1-generated cohesive ends to segments of linear SV40 chromosome. Upon incubation of chromatin-DNA hybrids at 37 degrees C and moderate ionic strength, histones migrated onto the labeled DNA while retaining the nucleosome structure. This was shown first, by the pattern of micrococcal nuclease digestion of labeled DNA; second by nitrocellulose filter binding of labeled DNA after redigestion of the chromatin-DNA hybrids with Eco R1; and third, by examination of chromatin-DNA hybrids in the electron microscope. Migration was slow, being apparent after several hours. Parallel experiments in which naked DNA and chromosomes were mixed without joining showed no transfer of nucleosomal histones between DNA molecules. The kinetics of Eco R1 cleavage of the DNA in virion-derived SV40 chromosomes are also consistent with the notion that nucleosomal histones, in the absence of other proteins, can move on DNA.


Prospects for the prevention of bacterial meningitis with polysaccharide vaccines. MOST SUPPURATIVE INFECTIONS OF THE MENINGES ARE CAUSED BY FIVE BACTERIAL SPECIES: Escherichia coli, Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and group B streptococcus. The immune response of adults to pneumococcal capsular polysaccharides has been studied in great detail and their responses to meningococcal and H. influenzae type b capsular polysaccharides are quite similar. Immune responses of adults to E. coli and group B streptococcal antigens are disappointing. The responses of children below the age of 7 years differ both quantitatively and in duration. Early experience shows that useful antibody titres can be achieved with certain antigens but further studies are required. In order to prevent bacterial meningitis by immunization, three vaccine formulations will need to be developed. When epidemic meningococcal disease occurs in a population, the vaccine containing only components of the meningococcus would be applied to a large segment of the population to terminate the epidemic. The second vaccine would contain components of H. influenzae type b, pneumococcus, and the meningococcus and would be administered in the first year of life, and repeated at suitable intervals to maintain life-long immunity. The third vaccine, designed to prevent neonatal meningitis caused by E. coli K1 and group B streptococci, would be administered to women preferably during the third trimester of pregnancy, so that their offspring would inherit sufficient antibodies to protect them during the first 3 months of life.The vaccine against the meningococcus is a reality and has been used extensively during major epidemics, with excellent results. The two vaccines for control of endemic bacterial meningitides do not exist as yet, but the prospects are good.


The American Cleft Palate Association: its first 36 years. History involves memory, and sometimes memory is faulty. History requires documentation, and sometimes documents cannot be found. History demands winnowing, and sometimes the wheat and the chaff cannot be completely separated. History is an assembly of details, and details may become burdensome. History is a challenge, to which the historian tries to respond. Surely, earlier Historians of the American Cleft Palate Association must have encountered this challenge. The present Historian acknowledges the work of her predecessors: George H. Foster, W. J. Robinson, and William Harkins from dentistry and Gretchen M. Phair and Asa J. Berlin from speech pathology. In preparing this review of events that took place from 1943 to 1978, in the United States and in other countries, during times of war and times of relative peace, through difficulties and accomplishments, I have attempted to be as accurate as possible. If I have distorted facts and if I have omitted people and happenings that should have been included, I have not done so intentionally. As Historian of the American Cleft Palate Association, I have appreciated the opportunity to learn about the organization and the people, past and present, who have built and maintained it. Thank you for giving me this assignment.


An improved method for preparation of samples for the simultaneous assay of some antiepileptic drugs by gas-liquid chromatography. A new simple extraction method, e.g., for subsequent quantitative determination of some antiepileptic drugs by GLC is described. HCl and an internal standard are added to 100 microliter of plasma or any biological fluid. This mixture is applied directly to an Extrelut column. Lipophilic substances (i.e. the antiepileptic drugs diphenylhydantoin, primidone and phenobarbital) are then extracted into 20 ml of ether and the solvent is evaporated to dryness. Direct GLC analysis of the on-column methylated drugs is performed using a P-N-detector.


A new procedure for the visualization of multiple forms of gamma-glutamyl transferase (GGT). Results in normals, patients receiving enzyme-inducing drugs and patients having liver parenchymal lesions. The activity of gamma-glutamyl transferase (GGT, EC 2.3.2.2) in sera of 68 healthy individuals, of 38 patients receiving anti-epileptic drugs, and of 27 patients having liver parenchymal lesions, was visualized after electrophoresis on cellulose-acetate gel using the substrate gamma-L-glutamyl-p-nitroanilide as a new colour reagent. The liberated p-nitroaniline was converted in situ into a lilac-coloured product using the Bratton-Marshall reaction. In most samples two bands were demonstrated, one located between albumin and alpha1-globulin, called GGT 1, the other located in the alpha2-globulin region, GGT 2. In a few samples a third band, GGT 3, with far less activity than the other bands, occurred in the beta-globulin region. When it occurred, an increase in total GGT activity was mainly due to an increase of GGT-1 activity. The possible role of the determination of GGT-1 activity as a monitor of microsomal enzyme induction is discussed.


Urinary albumin determination by the immediate bromcresol green method. It has been demonstrated recently that the reaction of serum samples with bromcresol green (BCG) reagent proceeds in two steps. Albumin is responsible for the immediate reaction while other serum proteins produce the slow reaction. In this paper the immediate BCG reaction has been used for the determination of urinary albumin concentration in patients with proteinuria by a slightly modified method with a primary pH adjustment of the urine and the use of a urine blank. Comparison of the immediate BCG method (y) with Laurell "rocket" technique (x) gave the following equation: y = 17.2 + 1.006x (n = 98; r = 0.99) mg/l. The coefficient of variation (within-day), C.V. (%), ranged between 0.9 and 2.7% depending on the albumin concentration. It is thus possible to carry out rapid, accurate and precise albumin determinations in urine samples using this simple method.


Diagnosis of Niemann-Pick disease using a simple and sensitive fluorimetric assay of sphingomyelinase activity. Phosphodiesterase activity of cultured cells was determined with bis-(4-methylumbelliferyl) phosphate as substrate. In the presence of Triton X-100 an acid component was evident and results indicated that this enzyme was identical with sphingomyelinase. Acid phosphodiesterase activity was specifically inhibited by sphingomyelin. In fibroblasts from patients with Niemann-Pick diseases types A, B and C, acid phosphodiesterase activity was deficient whereas neutral activity was normal. Neutral activity could, however, be removed by acid precipitation or by binding to DEAE-cellulose. Hence a simple and sensitive fluorimetric method is described for the assay of sphingomyelinase activity in the diagnosis of Niemann-Pick disease.


Brain catecholamines and catecholamine-synthesizing enzymes in renovascular hypertension in the rat. 1. Noradrenaline content of several rat brain stem and hypothalamic nuclei falls transiently at 72 h after initiation of renovascular hypertension (one-kidney Goldblatt model). 2. Tyrosine hydroxylase activity is significantly reduced in posterior, paraventricular and periventricular nuclei of hypothalamus at this time but returns to control value by 7 days. 3. Treatment with hydrallazine, 5 mg/kg intraperitoneally, twice daily or methaoxamine, 5 mg/kg, three times daily for 3 days respectively raises and lowers the noradrenaline content of brain nuclei, suggesting that short-term changes in noradrenaline may be secondary to afferent baroreceptor input. 4. At later times after the development of renovascular hypertension (7 and 28 days) activity of phenylethanolamine-N-methyl transferase is increased in the nucleus of the solitary tract and the locus coeruleus. 5. Brain catecholamines may participate both early in the development and later in the maintenance of renovascular hypertension.