bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide inhibitors of Streptomyces
dd-carboxypeptidases
Authors: Nieto Manuel , Perkins Harold R. , Leyh-Bouille Mélina , Frère Jean-Marie , Ghuysen Jean-Marie
Published: August 2015

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms.
Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties
Authors: Battelli M. G. , Lorenzoni E. , Stirpe F.
Published: August 2015

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD+-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD+. 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.
Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena
Authors: Reyes F. , Byrde R. J. W.
Published: August 2015

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a β-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN′-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The Km value for hydrolysis of p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside at 37°C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.
Short Communications. The binding of polyribosomes to smooth and rough endoplasmic-reticulum membranes (Short Communication)
Authors: Rolleston Francis S. , Mak Dennis
Published: August 2015

In vitro the binding of polyribosomes to smooth endoplasmic-reticulum membranes is more sensitive to ionic strength than is the binding to rough endoplasmic-reticulum membranes. Polyribosomes from the free and membrane-bound fractions bind with equal efficiency to endoplasmic-reticulum membranes.
Effect of steady illumination on the binding of carbon monoxide by carboxymethylated cytochrome c
Authors: Wilson M. T. , Brunori M. , Bonaventura J. , Bonaventura C.
Published: August 2015

The quantum yield, [unk], is reported for the photodissociation of CO from reduced carboxymethylated cytochrome c. The values of [unk] obtained are low relative to that for myoglobin and are pH-independent, being 0.23 at pH6.1 and 0.27 at pH9.7.
The control of the enzymes degrading histidine and related imidazolyl derivatives in Pseudomonas testosteroni
Authors: Coote J. G. , Hassall H.
Published: August 2015

1. The induction of the enzymes for the degradation of l-histidine, imidazolylpropionate and imidazolyl-l-lactate in Pseudomonas testosteroni was investigated. 2. The activities of histidine ammonia-lyase, histidine–2-oxoglutarate aminotransferase and urocanase are consistent with these enzymes being subject to co-ordinate control under most growth conditions. However, a further regulatory mechanism may be superimposed for histidase alone under conditions where degradation of histidine must take place for growth to occur. 3. Experiments with a urocanase− mutant show that urocanate is an inducer for the enzymes given above and also for N-formiminoglutamate hydrolyase and N-formylglutamate hydrolase. 4. N-Formiminoglutamate hydrolase and N-formylglutamate hydrolase are also induced by their substrates, and it is suggested that these two enzymes may be different gene products from those expressed in the presence of urocanate. 5. Induction of the enzyme system for the oxidation of imidazolylpropionate is dependent on exposure of cells to this compound.
The plasma transport and metabolism of retinoic acid in the rat
Authors: Smith John Edgar , Milch Peter O. , Muto Yasutoshi , Goodman DeWitt S.
Published: August 2015

The transport of retinoic acid in plasma was examined in vitamin A-deficient rats maintained on small doses of radioactively labelled retinoic acid. After ultracentrifugation of serum adjusted to density 1.21, most of the radioactivity (83%) was associated with the proteins of density greater than 1.21, and not with the serum lipoproteins. Gel filtration of the labelled serum on Sephadex G-200 showed that the radioactive label was associated with protein in the molecular-weight range of serum albumin. On polyacrylamide-gel electrophoresis almost all of the recovered radioactivity migrated with serum albumin. Similar esults were obtained with serum from a normal control rat given a single oral dose of [14C]retinoic acid. These findings indicate that retinoic acid is transported in rat serum bound to serum albumin, and not by retinol-binding protein (the specific transport protein for plasma retinol). Several tissues and the entire remaining carcase of each rat were extracted with ethanol–acetone to determine the tissue distribution of retinoic acid and some of its metabolites. The total recover of radioactive compounds in in the entire body of the rat was about 7–9μg, representing less than 5% or 10% respectively of the total administered label in the two dosage groups studied. The results confirm that retinoic acid is not stored in any tissue. Most of the radioactive material was found in the carcase, rather than in the specific tissues analysed. Two-thirds of the radioactivity in the carcase appeared to represent unchanged retinoic acid. Of the tissues examined, the liver, kidneys and intestine had relatively high concentrations of radioactive compounds, whereas the testes and fat-pads had the lowest concentrations.
The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vitro
Authors: Herzfeld Annemarie
Published: August 2015

Two common ways of measuring the potential for glutamine synthesis in a tissue are the rates of formation of γ-glutamylhydroxamate either by synthesis from glutamate (the glutamylhydroxamate synthetase reaction) or by transfer from glutamine (the glutamyltransferase reaction); it has not been established, however, that either reaction is a specific measure of glutamine synthetase. By differential extraction of glutamylhydroxamate synthetase and glutamyltransferase activities from water homogenates of several rat tissues I obtained an extract, rich in glutamylhydroxamate synthetase activity but nearly devoid of glutamyltransferase activity, and a fraction, solubilized by deoxycholate from the pellet, which contained virtually no glutamylhydroxamate synthetase activity but most of the original glutamyltransferase activity. Synthesis of glutamine, quantitatively similar to the γ-glutamylhydroxamate formed by glutamylhydroxamate synthetase, is catalysed in the water extract but not in the particulate fraction. γ-Glutamylhydroxamate formation by glutamylhydroxamate synthetase and glutamyltransferase shows discrepant substrate and metal specificities and can be differentially inhibited by l-methionine sulphoximine, phosphate and adenine nucleotides. The concordance between the formation of glutamine and γ-glutamylhydroxamate by glutamylhydroxamate synthetase but not by glutamyltransferase and the different solubilities of the glutamylhydroxamate synthetase and glutamyltransferase enzyme activities demonstrate that these two activities are not inextricably associated; they therefore cannot be catalysed exclusively by the same protein.
Methods of determining rate constants in single-substrate–single-product enzyme reactions. Use of induced transport: limitations of product inhibition
Authors: Britton H. G.
Published: August 2015

1. The calculation of the rate constants from steady-state kinetics of a single-substrate–single-product enzyme reaction in which there is an isomerization of the enzyme is described. 2. It is shown that even with the use of isotopically labelled substrates a set of solutions for the constants is obtained rather than a unique solution. However, limits are derived within which they must lie. 3. The most appropriate observations to determine the rate constants are measurements of Vmax. and Km for both substrate and product, and measurement of the degree of countertransport in an induced-transport test. 4. Experimental procedures for induced-transport tests and the quantitative interpretation of the results obtained are discussed. 5. Product inhibition is shown to be an ambiguous and imprecise means of determining the rate constants. Further, the absence of a [substrate]×[product] term in the denominator of the steady-state rate equation does not necessarily mean that the isomerization of the enzyme is rapid, since the term also disappears when the isomerization is very slow. 6. Similar considerations apply to carrier mechanisms.
Isolation and characterization of cyanogen bromide peptides from the collagen of bovine articular cartilage
Authors: Deshmukh Kalindi , Nimni Marcel E.
Published: August 2015

Significant amounts of native collagen can be extracted from bovine articular cartilage after removal of the acid mucopolysaccharides by controlled proteolysis. The fraction thus solubilized upon denaturation gives rise to three identical α chains. Cleavage of these chains with CNBr generated nine peptides, all of which contain glycine as one-third of their total amino acid residues. Two of the smaller peptides CB-1 and CB-2 contain partially hydroxylated proline. A similar CNBr digest of intact cartilage also gives a series of peptides identical with those obtained from the soluble cartilage collagen. The absence of cross-linking peptides, the fact that only few β components are seen in articular cartilage collagen and the similarity in peptide pattern between the two collagen fractions investigated, suggests that this collagen is stabilized by a different cross-linking mechanism, possibly involving an association with the tissue proteoglycans.
Investigation of the active site of papain with fluorescent probes
Authors: Allen Geoffrey , Lowe Gordon
Published: August 2015

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) and 7-(2′-hydroxyethylthio)-NBD (obtained from NBD chloride and mercaptoethanol) undergo a reversible spectral change in alkaline solution that depends respectively on a single apparent pKa 9.76 (at 25°C) and 8.81 (at 32°C). In acid solution however no spectral change was observed. NBD chloride reacts slowly with papain at pH7, but the rate of inhibition increases at lower pH and depends on an apparent pKa of 3.7 (at 35°C), which has been tentatively assigned to the carboxyl group of aspartic acid-158. The spectral properties of NBD-papain indicate that the thiol group of cysteine-25 is the site of reaction. The intensity of the fluorescence-emission spectrum of NBD-papain depends on a single pKa of 4.2 (at 26.7°C). The intensity of the fluorescence-emission spectrum of the mixed disulphide formed from papain and 7-(2′-mercaptoethylamino)-NBD (obtained from NBD chloride and cysteamine) depended on a single pKa of 3.94 in water and 3.89 in aq. 19.2% (v/v) dioxan (at 27°C). This small change to lower pKa value in a medium of lower dielectric constant is characteristic of a cationic acid. The only acid of this type in the active-site region is the conjugate acid of histidine-159.
The effects of environmental temperature on the properties of myofibrillar adenosine triphosphatase from various species of fish
Authors: Johnston Ian A. , Frearson Neil , Goldspink Geoffrey
Published: August 2015

1. Myofibrillar adenosine triphosphatase (ATPase) activities were measured for white myotomal muscle of 19 species of fish. 2. The activity was measured at different temperatures and after periods of preincubation at 37°C. 3. The inactivation half-life at 37°C depended on environmental temperature, increasing as the temperature increased. 4. Cold-water fish had higher myofibrillar adenosine triphosphatase activity at low temperatures than had warm-water fish. 5. The significance of these results is discussed.
The kinetics of hydrolysis of synthetic glucuronic esters and glucuronic ethers by bovine liver and Escherichia coli β-glucuronidase
Authors: Tomašić J. , Keglević D.
Published: August 2015

1. The relative rates of hydrolysis of synthetically prepared β-d-glucuronic esters [aglycone: benzoic acid, veratroic (3,4-dimethoxybenzoic) acid, indol-3-ylacetic acid and ethylbutyric acid], and β-d-glucuronic ethers (aglycone: phenolphthalein, p-nitrophenol, 3,4-dimethoxyphenol, 3,4-dimethoxybenzyl alcohol) by commercial preparations of β-glucuronidase from bovine liver and Escherichia coli were investigated. The rates of hydrolysis of all compounds tested were followed by measuring the formation of glucuronic acid under conditions which do not affect the glycosidic ester bond. 2. The pH profiles of the substrates in reaction with the enzyme from both sources were determined, and substrate-saturation curves at the optimal pH for each substrate were constructed; double-reciprocal plots of activity against concentration were linear. 3. Comparison of kinetic data indicates that neither the type of sugar–aglycone linkage, nor the aglycone structure alone can explain the observed Km and Vmax. values. 4. α-d-Glucuronic esters of benzoic and veratroic acid resisted hydrolysis by β-glucuronidase from both sources.
Deoxyribonucleic acid-dependent ribonucleic acid polymerases from murine spleen cells. Increased amounts of the nucleolar species in leukaemic tissue
Authors: Babcock Donner F. , Rich Marvin A.
Published: August 2015

1. In the spleens of infected mice, the Friend leukaemia virus induces a sharp increase in the ability of subsequently isolated nuclei to incorporate exogenous UTP into an acid-insoluble product. Inhibitor studies indicate that the incremental RNA synthesis proceeds from a DNA template and that both nucleolar and nucleoplasmic activities are involved. 2. The partially purified DNA-dependent RNA polymerases from control and virus-infected tissue are indistinguishable with respect to chromatographic mobility, dependence on bivalent cations, ionic strength, pH and their susceptibility to α-amanitin. The RNA polymerases of the murine spleen resemble the enzymes of other mammalian tissue in these properties. 3. A comparison of the amount of polymerase solubilized from normal and infected tissue correlates with the activity observed in assays of the respective nuclei. These experiments indicated that the increase in nucleolar RNA synthesis after infection is mediated by increased extractable polymerase I activity whereas the change in nucleoplasmic RNA synthesis results from an alteration of chromatin or a chromatin-associated factor.
The binding of haptens by the polypeptide chains of rabbit antibody molecules
Authors: Stevenson G. T.
Published: August 2015

1. The binding of haptens by the polypeptide chains derived from two rabbit immunoglobulin G antibodies was examined by gel chromatography and equilibrium dialysis. 2. The γ chains were examined in a dilute sodium acetate buffer, pH5.4, in which they exist as a monodisperse solution of dimers; aggregation of the protein promoted by some haptens had to be avoided. These chains exhibited variable extents of binding, reflecting the specificities of the parent antibody molecules, usually with only small increments above the binding by γ chains from normal immunoglobulin G. 3. The light chains existed as an interconverting mixture of monomers and dimers in all buffers of near neutral pH that were examined. They bound small amounts of hapten, again broadly reflecting the specificities of the parent antibody molecules. 4. For both the γ and light chains the dimeric state appeared necessary for appreciable binding of hapten. Apparently in each case the partners in the dimer interact in a manner analogous to the γ chain–light chain interaction in the parent antibody molecule, to give a site analogous to the antibody site. This implies that the binding of antigens by isolated chains has a large fortuitous element, providing no reliable indication of their contributions to the original antibody sites.
β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism
Authors: Oeding Volker , Schlegel Hans G.
Published: August 2015

1. β-Ketothiolase was purified 49-fold from fructose-grown cells of Hydrogenomonas eutropha H16 with a yield of 27%; the purification procedure involved precipitation by cetyltrimethylammonium bromide, DEAE-cellulose chromatography and exclusion chromatography on Sephadex G-200; the freeze-dried enzyme is stable. The molecular weight determined by sucrose-gradient centrifugation (8.2S) and by gel filtration is 147000–150000. The optimum pH for the cleavage reaction is 8.1, that for the condensation reaction 7.8, both measured in Tris–HCl buffer. 2. The kinetics of the cleavage reaction are described. Substrate-saturation curves were measured with both acetoacetyl-CoA and CoA as the variable substrates. The concentration of the second substrate was kept constant and was varied during successive experiments. The cleavage reaction is characterized by substrate inhibition by acetoacetyl-CoA, which is partially relieved by free CoA. Hill plots indicate two acetoacetyl-CoA-binding sites. 3. The substrate(acetyl-CoA)-saturation curve for the condensation reaction is hyperbolic. The Km was 3.9×10−4m-acetyl-CoA. In the presence of CoA sigmoidal curves were obtained, with an increasing sigmoidicity from 0.03 to 0.30mm-CoA. The inhibitory action of CoA on the β-ketothiolase condensation reaction and its possible involvement in the regulation of poly-β-hydroxybutyrate synthesis and degradation are discussed.
Lethal effect of rifampicin in Bacillus subtilis as a complicating factor in the assessment of the lifetime of messenger ribonucleic acid
Authors: Coote John G. , Wood David A. , Mandelstam Joel
Published: August 2015

The bactericidal action of rifampicin was compared with that of chloramphenicol in growing and in sporulating cultures of Bacillus subtilis 168. Chloramphenicol kills cells only very slowly, but exposure to rifampicin kills over 95% of cells in a few minutes, causing gross physical damage, which is visible in both phase-contrast and electron microscopy. This is accompanied by a fall in O2 consumption and by lysis. Experiments with synchronized cultures showed that susceptibility to the lethal effect of rifampicin is greater when the cells are dividing. The results suggest that the synthesis of some species of RNA other than mRNA may be necessary for the maintenance of cell integrity, although experiments with actinomycin D do not altogether fit this interpretation. However, we conclude that rifampicin is too toxic to use as an antibiotic for assessing the lifetime of mRNA.
The effect of ethanol on drug oxidations in vitro and the significance of ethanol–cytochrome P-450 interaction
Authors: Cinti Dominick L. , Grundin Robert , Orrenius Sten
Published: August 2015

The effect of ethanol on N-demethylation of aminopyrine in rat liver slices and in the microsomal fraction and on microsomal hydroxylation of pentobarbital and aniline was studied. With liver slices N-demethylation of aminopyrine was stimulated by 35–40% at low ethanol concentrations (2mm), whereas no stimulation occurred at high concentrations (100mm). With the liver microsomal fraction, an inhibitory effect was observed only at high ethanol concentrations (100mm). This was also observed with the other drugs studied. In agreement with these results, only at a high concentration did ethanol interfere with the binding of drug substrates to cytochrome P-450. Further, as previously reported, ethanol produced a reverse type I spectral change when added to the liver microsomal fraction. Evidence that this spectral change is due to removal of substrate, endogenously bound to cytochrome P-450, is reported. A dual effect of ethanol is assumed to explain the present findings; in liver slices, at a low ethanol concentration, the enhanced rate of drug oxidation is the result of an increased NADH concentration, whereas the inhibitory effect observed with the microsomal fraction at high ethanol concentration is due to the interference by ethanol with the binding of drug substrates to cytochrome P-450.
Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities
Authors: Kornberg H. L. , Soutar Anne K.
Published: August 2015

1. A mutant of Escherichia coli, devoid of phosphopyruvate synthetase, glucosephosphate isomerase and 6-phosphogluconate dehydrogenase activities, grew readily on gluconate and inducibly formed an uptake system for gluconate, gluconate kinase and 6-phosphogluconate dehydratase while doing so. 2. This mutant also grew on glucose 6-phosphate and inducibly formed 6-phosphogluconate dehydratase; however, the formation of the gluconate uptake system and gluconate kinase was not induced under these conditions. 3. The use of the Entner–Doudoroff pathway for the dissimilation of 6-phosphogluconate, derived from either gluconate or glucose 6-phosphate, by this mutant was also demonstrated by the accumulation of 2-keto-3-deoxy-6-phosphogluconate (3-deoxy-6-phospho-l-glycero-2-hexulosonate) from both these substrates in a similar mutant that also lacked phospho-2-keto-3-deoxygluconate aldolase activity. 4. Glucose 6-phosphate inhibits the continued utilization of fructose by cultures of the mutants growing on fructose, as it does in wild-type E. coli. 5. The mutants do not use glucose for growth. This is shown to be due to insufficiency of phosphopyruvate, which is required for glucose uptake.
Estimation of the fructose diphosphatase–phosphofructokinase substrate cycle in the flight muscle of Bombus affinis
Authors: Clark Michael G. , Bloxham David P. , Holland Paul C. , Lardy Henry A.
Published: August 2015

1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the 3H/14C ratio in glucose 6-phosphate as well as the rate of 3H release to water after the metabolism of [5-3H,U-14C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4μmol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5°C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4μmol/min per g fresh wt.) and glycolysis was decreased (5.8μmol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27°, 21° and 5°C the rates of substrate cycling were 0, 0.48 and 10.4μmol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca2+-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca2+ concentration associated with the contractile process.
Site of inhibition by 2-amino-1,1,3-tricyanopropene of photosynthetic oxygen evolution by spinach leaf chloroplasts (Short Communication)
Authors: Burden E. Margaret , Horton Alan A.
Published: August 2015

2-Amino-1,1,3-tricyanopropene inhibits photosynthetic O2 evolution, but, unlike 3-(3,4-dichlorophenyl)-1,1-dimethylurea, has little effect on the steady-state fluorescence of chlorophyll. In chloroplasts prepared from spinach leaves and inhibited by 2-amino-1,1,3-tricyanopropene, a 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of ferricyanide may be restored by addition of semicarbazide. It is concluded that 2-amino-1,1,3-tricyanopropene acts at a point close to the photo-oxidation of water.
Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo
Authors: McAllister Anthony , Allison S. P. , Randle Philip J.
Published: August 2015

1. The extractions of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo were calculated from measurements of their arterial and coronary sinus blood concentration. Elevation of plasma free fatty acid concentrations by infusion of intralipid and heparin resulted in increased extraction of free fatty acids and diminished extractions of glucose, lactate and pyruvate by the heart. It is suggested that metabolism of free fatty acids by the heart in vivo, as in vitro, may impair utilization of these substrates. These effects of elevated plasma free fatty acid concentrations on extractions by the heart in vivo were reversed by injection of dichloroacetate, which also improved extraction of lactate and pyruvate by the heart in vivo in alloxan diabetes. 2. Sodium dichloroacetate increased glucose oxidation and pyruvate oxidation in hearts from fed normal or alloxan-diabetic rats perfused with glucose and insulin. Dichloroacetate inhibited oxidation of acetate and 3-hydroxybutyrate and partially reversed inhibitory effects of these substrates on the oxidation of glucose. In rat diaphragm muscle dichloroacetate inhibited oxidation of acetate, 3-hydroxybutyrate and palmitate and increased glucose oxidation and pyruvate oxidation in diaphragms from alloxan-diabetic rats. Dichloroacetate increased the rate of glycolysis in hearts perfused with glucose, insulin and acetate and evidence is given that this results from a lowering of the citrate concentration within the cell, with a consequent activation of phosphofructokinase. 3. In hearts from normal rats perfused with glucose and insulin, dichloroacetate increased cell concentrations of acetyl-CoA, acetylcarnitine and glutamate and lowered those of aspartate and malate. In perfusions with glucose, insulin and acetate, dichloroacetate lowered the cell citrate concentration without lowering the acetyl-CoA or acetylcarnitine concentrations. Measurements of specific radioactivities of acetyl-CoA, acetylcarnitine and citrate in perfusions with [1-14C]acetate indicated that dichloroacetate lowered the specific radio-activity of these substrates in the perfused heart. Evidence is given that dichloroacetate may not be metabolized by the heart to dichloroacetyl-CoA or dichloroacetylcarnitine or citrate or CO2. 4. We suggest that dichloroacetate may activate pyruvate dehydrogenase, thus increasing the oxidation of pyruvate to acetyl-CoA and acetylcarnitine and the conversion of acetyl-CoA into glutamate, with consumption of aspartate and malate. Possible mechanisms for the changes in cell citrate concentration and for inhibitory effects of dichloroacetate on the oxidation of acetate, 3-hydroxybutyrate and palmitate are discussed.
Effect of high tyrosine content on the determination of tryptophan in protein by the acidic ninhydrin method. Application to chicken ovoinhibitor
Authors: Zahnley James C. , Davis John Gorton
Published: August 2015

Colorimetric determination of tryptophan in intact proteins by the acidic ninhydrin method of Gaitonde & Dovey (1970) gives high apparent tryptophan contents for proteins having high tyrosine/tryptophan ratios. Correction for this interference by tyrosine can be achieved by plotting the ratio of observed to expected tryptophan content as a function of tyrosine/tryptophan ratio for proteins of known composition. The equation of the line is: [Formula: see text] Application of this correction to chicken ovoinhibitor, which contains 17 tyrosine residues per molecule, gave results that agree with tryptophan content determined by other methods.
The kinetics of interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamide–adenine dinucleotide and lactate
Authors: Bennett Nigel G. , Gutfreund Herbert
Published: August 2015

Oxamate competes with pyruvate for the substrate binding site on the ENADH complex of pig skeletal muscle lactate dehydrogenase. When this enzyme was mixed with saturating concentrations of NAD+ and lactate in a stopped-flow rapid-reaction spectrophotometer there was no transient accumulation of enzyme complexes with the reduced nucleotide. The steady-state rate of formation of free NADH was reached within the dead-time of the instrument (3ms). When oxamate was added to inhibit the steady state and to uncouple the equilibration: [Formula: see text] through the rapid formation of ENADHOxamate, the rate of formation of ENADH could be measured by observation of the first turnover. This pH-dependent transient is controlled by the rate of dissociation of pyruvate and the fraction of the enzyme in the form ENADHPyruvate.
The nature of the electrophoretically separable multiple forms of rat liver monoamine oxidase
Authors: Houslay Miles D. , Tipton Keith F.
Published: August 2015

1. Treatment of a partly purified preparation of rat liver monoamine oxidase with the chaotropic agent sodium perchlorate caused the enzyme to migrate as a single band of activity of polyacrylamide-gel electrophoresis, whereas the untreated enzyme separated into a number of bands. 2. Treatment with the chaotropic agent caused no loss of enzyme activity towards benzylamine, dopamine or tyramine. 3. The activities of the untreated preparation towards different substrates were inhibited to different extents by heat treatment and by some inhibitors. No such differences could be detected after the enzyme preparation had been treated with sodium perchlorate. 4. Lipid material, which could be separated by gel filtration, was liberated from the enzyme preparation by sodium perchlorate treatment. 5. The molecular weight of the treated enzyme was found to be 380000±38000. 6. Perchlorate treatment altered the solubility of the enzyme. 7. A continuous assay method for monoamine oxidase is described.
Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase
Authors: Focant B. , Watts D. C.
Published: August 2015

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO3- or Cl- ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by αβ-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO3- ions to the enzyme–creatine–MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme–creatine–NO3-–MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO3- is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme–pyruvate–NO3-–MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or α-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K+-free enzyme. Only the former inhibits the K+-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.
The contractile and regulatory proteins of insect flight muscle
Authors: Bullard Belinda , Dabrowska Renata , Winkelman Lowell
Published: August 2015

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca2+ concentrations. Purified insect regulatory protein produced as much sensitivity to Ca2+ as did the rabbit troponin–tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca2+ sensitivity of actomyosins containing insect myosin or actin, with added troponin–tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.
The formation of ferritin from apoferritin. Catalytic action of apoferritin
Authors: Macara Ian G. , Hoy Terence G. , Harrison Pauline M.
Published: August 2015

The iron-storage protein ferritin consists of a protein shell and has an iron content of up to 4500 iron atoms as a microcrystalline ferric oxide hydrate. A study was made of the uptake of ferrous iron by apoferritin in the presence of an oxidizing agent at very low iron:protein ratios. At ratios of less than about 150 iron atoms per apoferritin molecule hyperbolic progress curves were obtained, whereas at higher ratios the curves became sigmoidal under the conditions used. A computer model, developed previously (Macara et al., 1972), was shown to account for this result. The experimental evidence indicates that apoferritin binds ferrous iron and catalyses the initial stage in the formation of the ferric oxide hydrate inside the protein shell. This stage involves the oxidation of sufficient iron within the protein molecule to form a stable nucleus on which the growth of the microcrystalline iron-core particles can proceed. A possible schematic mechanism for the action of apoferritin is suggested.
Functional groups in the activity and regulation of Escherichia coli citrate synthase
Authors: Danson Michael J. , Weitzman P. David J.
Published: August 2015

1. Citrate synthase has been purified from Escherichia coli and shown to exist at an equilibrium between three forms: monomer (mol.wt. 57000), tetramer (mol.wt. 230000) and, possibly, octamer. Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and α-oxoglutarate. 2. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependence of their rates of photo-oxidation with Methylene Blue suggests that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal and the destruction of activity by diethyl pyrocarbonate. 3. The participation of histidine at the α-oxoglutarate effector site is indicated by photo-oxidation and the participation of cysteine at the NADH effector site suggested by photo-oxidation is confirmed by the desensitization to NADH produced by treatment with 5,5′-dithiobis-(2-nitrobenzoate). Inactivation of the enzyme after modification with this reagent suggests the additional involvement of cysteine in catalytic activity. 4. Amino acid analyses of native and photo-oxidized enzyme are consistent with these conclusions. 5. Modification with 2-hydroxy-5-nitrobenzyl bromide indicates the participation of tryptophan in the activity of the enzyme.
An explanation of the asymmetric binding of sugars to the human erythrocyte sugar-transport systems
Authors: Barnett J. E. G. , Holman G. D. , Munday K. A.
Published: August 2015

6-O-Alkyl-d-galactoses competitively inhibit the erythrocyte sugar-transport system when added to the outside of the cells, but not to the inside. n-Propyl β-d-glucopyranoside competitively inhibits the system on the inside of the cells, but not on the outside. A model for sugar transport is proposed.
The reactive serine residue in phosphoglucomutase of Micrococcus lysodeikticus
Authors: Milstein Celia P. , Clarke J. B. , Britton H. G.
Published: August 2015

1. Phosphoglucomutase of Micrococcus lysodeikticus was labelled at the active site by exchange with 32P-labelled substrates of high specific radioactivity. 2. Partial acid hydrolysis gave rise to radioactive peptides; serine phosphate was identified as one of the derivatives. 3. Comparison of the other 32P-labelled peptides with the peptides obtained from the 32P-labelled rabbit muscle phosphoglucomutase indicates that the sequence around the reactive serine residue is identical in both enzymes.
The subcellular distribution and properties of aldehyde dehydrogenases in rat liver
Authors: Tottmar S. O. C. , Pettersson H. , Kiessling K.-H.
Published: August 2015

1. Kinetic experiments suggested the possible existence of at least two different NAD+-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP+-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The Km for acetaldehyde was apparently less than 10μm for enzyme I and 0.9–1.7mm for enzyme II. The Km for NAD+was similar for both enzymes (20–30μm). The Km for NADP+was 2–3mm and for acetaldehyde 0.5–0.7mm for the NADP+-dependent activity. 4. The NAD+-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.
Aromatic amino acid transaminase in rat intestine
Authors: Nakamura Jun , Noguchi Tomoo , Kido Ryo
Published: August 2015

The transamination of aromatic l-amino acids (5-hydroxytryptophan, tryptophan, tyrosine, phenylalanine and kynurenine) was shown to be catalysed by enzyme preparations from rat small intestine. On the basis of the partial purification and characterization of these aromatic amino acid transaminases, it is suggested that rat small intestine contains several kinds of aromatic amino acid transaminases.
Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds
Authors: Evans W. Howard , Hood Diana O. , Gurd James W.
Published: August 2015

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate–Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose–detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5′-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000–130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD+, UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.
Polymerization of proteins with glutaraldehyde. Soluble molecular-weight markers
Authors: Payne John W.
Published: August 2015

Glutaraldehyde is well known for its ability to react with proteins and to produce insoluble cross-linked aggregates. In contrast with this situation, conditions are described here which yield covalently linked soluble protein oligomers. The procedure is applicable to a wide range of proteins, and by slight variation in the reaction conditions, soluble polymers in the molecular weight range 3×104−2×107were produced. The products are valuable aṡ molecular-weight markers, e.g. in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. The inherent similarities of these oligomers make them superior to commercial molecular-weight protein markers, which may have marked differences in composition and charge.
The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis
Authors: Smith R. J. , Midgley J. E. M.
Published: August 2015

In trimethoprim-inhibited RCstr strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RCrel strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RCstr strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RCrel strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RCstr strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RCstr or RCrel bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.