bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor
Authors: Sariaslani F. Sima , Harper David B. , Higgins I. John
Published: August 2015

1. Nocardia salmonicolor grew on a variety of alkanes, 1-phenylalkanes and 1-cyclo-hexylalkanes as sole carbon and energy sources. 2. Growth on 1-phenyldodecane in batch culture was diauxic. Isocitrate lyase activity was induced during lag phase, reaching a maximum activity in the first growth phase, during which both the aromatic ring and the side chain were degraded. However, 4-phenylbutyrate, 4-phenylbut-3-enoate, 4-phenylbut-2-enoate, 3-phenylpropionate, cinnamate and phenylacetate accumulated in the growth medium. These compounds disappeared at the onset of diauxic lag and four hydroxylated compounds accumulated; one was 4-(o-hydroxyphenyl)but-3-enoate and another was identified as 4-(o-hydroxyphenyl)butyrate. These compounds were utilized during the second growth phase. 3. Washed 1-phenyldodecane-grown cells oxidized acetate, cinnamate, 3,4-dihydroxyphenylacetate, homogentisate, o-, m- and p-hydroxyphenylacetate, phenylacetate, and 4-phenylbutyrate rapidly without lag. 4. Extracts of such cells rapidly oxidized homogentisate,3,4-dihydroxyphenylacetate, catechol and protocatechuate. 5. The organism grew readily on 4-phenylbutyrate, phenylacetate, o-hydroxyphenylacetate, homogentisate and 3,4-dihydroxyphenylacetate as sole carbon energy sources, but growth was slow on cinnamate and 4-phenylbut-3-enoate. 6. When cinnamate and phenylacetate were sole carbon sources for growth, phenylacetate and o-hydroxyphenylacetate respectively were detected in culture supernatants. 4-Phenylbut-3-enoate and 4-phenylbutyrate both yielded a mixture of cinnamate and phenylacetate. 7. It is proposed that 1-phenyldodecane is catabolized by ω-oxidation of the terminal methyl group, side-chain β-oxidation to 4-phenylbutyrate, both β- and α-oxidation to phenylacetic acid, hydroxylation to homogentisate via o-hydroxyphenylacetate and ring cleavage to maleylacetoacetate. Catabolism via 3,4-dihydroxyphenylacetate may also occur. 8. Growth on 1-phenylnonane was also diauxic and cinnamic acid, phenylpropionic acid, benzoic acid and hydroxyphenylpentanoic acid accumulated in the medium. Respirometric data and ring-cleavage enzyme activities showed similar patterns to those obtained after growth on 1-phenyldodecane. The results suggest that the main catabolic routes for 1-phenyldodecane and 1-phenylnonane may converge at cinnamate. 9. Possible reasons for diauxie are discussed.
Sterol structural requirements for inhibition of streptolysin O activity
Authors: Watson Kenneth C. , Kerr Eric J. C.
Published: August 2015

Reduced streptolysin O, a toxin produced by certain β-haemolytic streptococci, lyses human erythrocytes. The reaction is inhibited by cholesterol at concentrations of about 1.0μg/ml. Other sterols inhibit the lysin and there is a specific requirement for a 3β-hydroxyl group. Inhibition was obtained with 3β-hydroxychol-5-en-24-oic acid, containing a hydrophilic group at C-24. The mode of inhibition is likely to involve attachment to the fixation site of the lysin which attaches the molecule to cell membranes, probably to membrane cholesterol. A second streptolysin site, concerned in the final haemolytic event, may also be involved. Inhibitors of the latter site have not been characterized, other than antibody with specificity for the site.
Metabolic studies on myelin. Evidence for a precursor role of a myelin subfraction
Authors: Agrawal Harish C. , Trotter John L. , Burton Robert M. , Mitchell Raymond F.
Published: August 2015

Myelin prepared from brain tissue of the developing rat (15 days post partum) can be separated into several subfractions. These are (1) ‘myelin-like’ and ‘purified myelin’, by the technique of Davison and co-workers (Agrawal et al., 1970b) and (2) ‘membrane fraction,’ ‘light myelin’ and ‘heavy myelin’ by the discontinuous-sucrose-gradient procedure described in the present paper. ‘Myelin-like’ and ‘membrane-fraction’ subfractions appear to be similar in chemical properties, but different in detailed morphology by electron microscopy. Both fractions are related to myelin, on the basis of demonstrable myelin basic protein by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate and the presence of a myelin-marker enzyme, 2′:3′-cyclic nucleotide 3′-phosphohydrolase. These two fractions have a low lipid content (17% for ‘myelin-like’ and 40% for ‘membrane-fraction’ subfractions) compared with myelin (67–72%). No cerebroside was detected in these two fractions, whereas cerebrosides are a major component of myelin itself. The administration of [2,3-3H]tryptophan to young rats results in more rapid incorporation into proteins of the ‘myelin-like’ and ‘membrane-fraction’ subfractions when compared with incorporation into myelin. Data are presented which are consistent with a precursor–product relationship for conversion of ‘myelin-like’ and ‘membrane-fraction’ subfractions into myelin.
Microbial metabolism of the pyridine ring. The hydroxylation of 4-hydroxypyridine to pyridine-3,4-diol (3,4-dihydroxypyridine) by 4-hydroxypyridine 3-hydroxylase
Authors: Watson G. Keith , Houghton Charles , Cain Ronald B.
Published: August 2015

1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O2 and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH4)2SO4 the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pKm versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.
Comparative effect and fate of non-entrapped and liposome-entrapped neuraminidase injected into rats
Authors: Gregoriadis Gregory , Putman Daphne , Louis Loizos , Neerunjun Diane
Published: August 2015

Non-entrapped and liposome-entrapped Clostridium perfringens neuraminidase (0.5–0.6 unit) was injected into rats and its fate as well as its effect on plasma and erythrocyte N-acetylneuraminic acid was investigated. The following observations were made. (1) Although removal of both non-entrapped and liposome-entrapped neuraminidase from the circulation was completed within 5h after injection, their recovery in tissues was distinctly different; 7–10% of the injected non-entrapped enzyme was found in the liver and none in the liver lysosomal fraction or the spleen. In contrast, 20–26% of the liposome-entrapped enzyme was found in the liver of which 60–69% was in the lysosomal fraction. Spleen contained 3.6–5.0% of the enzyme. (2) The presence of the non-entrapped neuraminidase in blood led to the extensive desialylation of plasma and to a decrease in the concentration or total removal from the circulation of some of the plasma glycoproteins. (3) Injection of non-entrapped neuraminidase also led to the partial desialylation of erythrocytes the life span of which was diminished and their uptake by the liver and spleen augmented. (4) Entrapment of neuraminidase in liposomes before its injection prevented the enzyme from acting on its substrate in plasma or on the erythrocyte surface, and values obtained for plasma glycoproteins and erythrocyte survival were similar to those observed in control rats. (5) Entrapment in liposomes of therapeutic hydrolases intended for the degradation of substances stored within the tissue lysosomes of patients with storage diseases could prevent the potentially hazardous enzymic action of hydrolases in blood and at the same time direct the enzymes to the intracellular sites where they are needed.
Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes
Authors: Butters Terry D. , Hughes R. Colin
Published: August 2015

1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.
Alkaline phosphatase from pig kidney. Method of purification and molecular properties
Authors: Wachsmuth Ernst D. , Hiwada Kunio
Published: August 2015

Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH4)2SO4 precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000–156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25°C of 2600s-1 per tetramer. Its concentration in kidney was estimated to be 8.5–8.8mg/kg.
The specificity of the S1′ subsite of papain
Authors: Alecio M. Robert , Dann Malcolm L. , Lowe Gordon
Published: August 2015

The specificity of the S1′ subsite of the proteolytic enzyme papain was investigated by studying the effect of l-α-amino acid amides on the enzyme-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester and by determining the kinetic parameters for the enzyme-catalysed hydrolysis of some N-benzyloxycarbonylglycyl-l-amino acid amides. These studies showed that the S1′ subsite has a predilection for hydrophobic residues, in particular l-leucine and l-tryptophan. The specificity for these residues is manifest in both the binding and acylation steps. N-Benzyloxycarbonylglycine amide is not hydrolysed under comparable conditions, indicating that the amide group adjacent to and on the C-terminal side of the peptide bond about to be cleaved makes an important contribution to the rate of the papain-catalysed hydrolysis of peptides.
Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf
Authors: Whiting Alan H. , Sykes Bryan C. , Partridge S. Miles
Published: August 2015

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.
Fidelity of deoxyribonucleic acid polymerases from normal and leukaemic human cells in polydeoxynucleotide replication
Authors: Srivastava B. I. Sahai
Published: August 2015

Although the relative incorporation of incorrect nucleotide (dCTP or dGTP) into poly(dA-dT)·poly(dA-dT) by partially purified 3–4S DNA polymerase from normal or leukaemic human cells was four to five times higher than that by the 6–7S DNA polymerase, no significant differences in the infidelity of these polymerases between normal and leukaemic cells were noted.
Identical 3′-terminal octanucleotide sequence in 18S ribosomal ribonucleic acid from different eukaryotes. A proposed role for this sequence in the recognition of terminator codons
Authors: Shine John , Dalgarno Lynn
Published: August 2015

The 3′-terminal sequence of 18S ribosomal RNA from Drosophila melanogaster and Saccharomyces cerevisiae was determined by stepwise degradation from the 3′-terminus and labelling with [3H]isoniazid. The sequence G-A-U-C-A-U-U-AOH was found at the 3′-terminus of both 18S rRNA species. Less extensive data for 18S RNA from a number of other eukaryotes are consistent with the same 3′-terminal sequence, and an identical sequence has previously been reported for the 3′-end of rabbit reticulocyte 18S rRNA (Hunt, 1970). These results suggest that the base sequence in this region is strongly conserved and may be identical in all eukaryotes. As the 3′-terminal hexanucleotide is complementary to eukaryotic terminator codons we discuss the possibility that the 3′-end of 18S rRNA may have a direct base-pairing role in the termination of protein synthesis.
The formation of iron-binding fragments of hen ovotransferrin by limited proteolysis
Authors: Williams John
Published: August 2015

1. Iron was added to hen ovotransferrin to 30% saturation and the protein was digested with trypsin or chymotrypsin. 2. Iron-binding fragments were isolated. They carried one atom of iron/mol (mol.wt. 35000) and consisted of a single polypeptide chain derived from the N-terminal half of the protein. Carbohydrate was not present. 3. The fragments were able to bind a variety of metals and to donate iron to reticulocytes.
Mutationally altered rate constants in the mechanism of alkaline phosphatase
Authors: Halford Stephen E. , Schlesinger Milton J.
Published: August 2015

The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.
The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species
Authors: Milman Harry A. , Cooney David A.
Published: August 2015

A survey was conducted of the distribution of l-asparagine synthetase and of l-asparaginase in the principal organs of representative mammals and birds. Although a radiometric assay was used as a routine, several additional criteria, including enzymic and chromatographic ones, were used to verify that the product of the synthetase was l-asparagine. Recoveries of exogenous l-asparagine were assessed in the presence of a number of mouse organs and found to be about 85%. In addition, evidence is presented for the existence in mouse liver of a thermolabile activity capable of destroying l-asparagine and stimulated by high concentrations of NH4+ ions. Of the organs surveyed, pancreas was generally found to synthesize l-asparagine at the most rapid rate, whereas extracts of liver catalysed the decomposition of this amide at the greatest velocity. Of the species studied, guinea pig had the highest activities of pancreatic l-asparagine synthetase and also of hepatic l-asparaginase. The pancreas of mouse and ox also were good sources of l-asparagine synthetase.
Effect of glucagon on phenylalanine metabolism and phenylalanine-degrading enzymes in the rat
Authors: Brand Larry M. , Harper Alfred E.
Published: August 2015

Glucagon administered subcutaneously to rats for 10 days had no significant effect on liver phenylalanine hydroxylase activity, but induced liver dihydropteridine reductase more than twofold. In rats administered a phenylalanine load orally, glucagon treatment stimulated oxidation and depressed urinary phenylalanine excretion. These responses could not be related to an effect of glucagon on hepatic tyrosine–α-oxoglutarate aminotransferase activity. Even in rats with phenylalanine hydroxylase activity depressed to 50% of control values by p-chlorophenylalanine administration, glucagon treatment increased the phenylalanine-oxidation rate substantially. Although hepatic phenylalanine–pyruvate aminotransferase was increased tenfold in glucagon-treated rats, glucagon treatment did not increase urinary excretion of phenylalanine transamination products by rats given a phenylalanine load. Glucagon treatment did not affect phenylalanine uptake by the gut or liver, or the liver content of phenylalanine hydroxylase cofactor. It is suggested that dihydropteridine reductase is the rate-limiting enzyme in phenylalanine degradation in the rat, and that glucagon may regulate the rate of oxidative phenylalanine metabolism in vivo by promoting indirectly the maintenance of the phenylalanine hydroxylase cofactor in its active, reduced state.
The effect of lysine on gluconeogenesis from lactate in rat hepatocytes
Authors: Cornell Neal W. , Lund Patricia , Krebs Hans A.
Published: August 2015

1. In freshly prepared isolated rat liver cells there is a lag in gluconeogenesis from lactate. The magnitude of the lag increases with increasing lactate concentration. 2. The lag is virtually abolished by lysine. 3. A few other amino acids (tyrosine, arginine, asparagine, ornithine) and NH4Cl had effects similar to, but less pronounced than, lysine during the early stage of incubation. Lysine was unique in accelerating gluconeogenesis beyond the lag period. 4. The effects of the accelerators are not additive. 5. Glycine, serine, threonine, cysteine, tryptophan and histidine at 2mm markedly inhibit (>20%) gluconeogenesis from lactate. 6. Oleate, which promotes gluconeogenesis from lactate by supplying acetyl-CoA required for the pyruvate carboxylase reaction, had no effect on the lag, yet oleate oxidation showed no lag. 7. Preincubation of cells decreased the lag and decreased the magnitude of the lysine effect. 8. Pyruvate (added at 1mm to give an initial [lactate]/[pyruvate] ratio of 10) also abolished the lag and decreased the lysine effect by about 50%. 9. Lysine reversed the inhibition by ethanol of gluconeogenesis from lactate. 10. All accelerators increased the rate of re-oxidation of cytosolic NADH as shown by a rapid re-adjustment of the [lactate]/[pyruvate] ratio on addition of 10mm-lactate. 11. The accelerated rates of gluconeogenesis are associated with an increased formation of aspartate and glutamate and especially alanine. 12. The existence of the lag period can be explained on the basis of the fact that the accumulation of pyruvate during the lag diverts oxaloacetate from gluconeogenesis to malate formation, i.e. that the re-oxidation of cytosolic NADH takes precedence over gluconeogenesis. This means that much oxaloacetate formed by the pyruvate carboxylase reaction has to be transferred twice from the mitochondria to the cytosol by the aspartate shuttle. Under these conditions the operation of the shuttle limits the rate of gluconeogenesis from lactate. Lysine and other accelerators may increase the effectiveness of the shuttle by providing components of the aspartate aminotransferases involved. The question of why lysine specifically accelerates gluconeogenesis beyond the lag period is discussed.
Insulin release from the perfused rat pancreas. Mode of action of tolbutamide
Authors: Bowen Vyvyan , Lazarus Norman R.
Published: August 2015

Dextran-linked tolbutamide causes first-phase insulin release when perfused through the isolated rat pancreas. The linked sulphonylurea is also able to stimulate the membranal adenylate cyclase present in mouse islets. These facts make it highly likely that sulphonylureas exert their pharmacological action by interaction with the plasma membrane. Their action may be mediated via the adenylate cyclase enzyme.
Golgi β-glucuronidase of androgen-stimulated mouse kidney
Authors: Marsh Charles A. , Lin Chi-Wei , Fishman William H.
Published: August 2015

Subcellular fractions were prepared from mouse kidney homogenates by differential and sucrose-gradient centrifugation. A fraction enriched in Golgi apparatus was obtained, which had considerably enriched galactosyltransferase and thiamin pyrophosphatase activities, and was morphologically typical of Golgi material. This preparation also had high β-glucuronidase activity, which increased concomitantly with microsomal β-glucuronidase activity during the specific stimulation of the enzyme in male mouse kidney after androgen administration. The degree of stimulation was much greater in the Golgi fraction. Gel-electrophoretic patterns of Golgi β-glucuronidase resembled more closely those of the enzyme located within lysosomes, but contained minor bands similar to those described previously (Swank & Paigen, 1973) as characteristic of the microsomal enzyme. It was concluded that the Golgi complex is involved in the distribution of the enzyme after its synthesis to both lysosomal and microsomal fractions.
The isolation and identification of the P-component of normal human plasma proteins (Short Communication)
Authors: Binette Paul , Binette Margaret , Calkins Evan
Published: August 2015

A normal human plasma protein called the P-component, which has a reaction of identity with the pentagonal structure found in amyloid-laden organs, has been isolated and identified with a recently characterized protein, the 9.5S α1-glycoprotein.
Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence
Authors: Ashby Barrie , Wootton John C. , Fincham John R. S.
Published: August 2015

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.
Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enzyme by sulphide and by selenide
Authors: Cleere William F. , Coughlan Michael P.
Published: August 2015

1. Turkey liver xanthine dehydrogenase engaged in catalysing the oxidation of xanthine by dichlorophenol–indophenol was progressively inactivated by methanol. This inactivation was reversible by NAD+. 2. Reaction with arsenite and with cyanide, in each case first-order with respect to enzyme, resulted in characteristic alterations in the visible absorption spectrum of the enzyme. The rate of spectral change on reaction with either agent paralleled the rate of loss of enzyme activity. 3. Cyanide inactivation was accompanied by elimination from the enzyme of sulphur as thiocyanate. Partial restoration of activity was effected by incubation with sulphide or with selenide. The results suggest that turkey liver xanthine dehydrogenase, like milk xanthine oxidase (Massey & Edmonson, 1970), contains at the active centre a cyanolysable persulphide group essential to catalytic activity and that selenium may replace sulphur in this group to give an active enzyme. 4. Incubation of the native enzyme with sulphide or with selenide resulted in the rapid loss of half of the xanthine-oxidizing activity, apparently by disrupting the molybdenum and (Fe/S)II loci. This may indicate non-equivalence of the intramolecular electron-transfer systems.
The enthalpy change for the reduction of nicotinamide–adenine dinucleotide
Authors: Burton Kenneth
Published: August 2015

The heat of the reaction NAD++propan-2-ol=NADH+acetone+H+ was determined to be 42.5±0.6kJ/mol (10.17±0.15kcal/mol) from equilibrium measurements at 9–42°C catalysed by yeast alcohol dehydrogenase. With the aid of thermochemical data for acetone and propan-2-ol the values of ΔH=−29.2kJ/mol (−6.99kcal/mol) and ΔG0=22.1kJ/mol (5.28kcal/mol) are derived for the reduction of NAD (NAD++H2=NADH+H+). These values are consistent with analogous but less accurate data for the ethanol–acetaldehyde reaction. Thermodynamic data for the reduction of NAD and NADP are summarized.
Metabolism of biphenyl. Structure and physicochemical properties of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage product from 2,3-dihydroxybiphenyl by Pseudomonas putida
Authors: Catelani Danilo , Colombi Antonio
Published: August 2015

The structure of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid for the meta-cleavage product of 2,3-dihydroxybiphenyl by a Pseudomonas putida strain was demonstrated on the basis of its chemical and physicochemical properties and those of its derivatives.
Proteolysis of rabbit immunoglobulin M by papain
Authors: McIlroy Barbara M. , Stevenson George T.
Published: August 2015

1. Digestion of rabbit immunoglobulin M (IgM) by papain for 5h, in the absence of reducing agent, gives Fabμ and Fc5μ fragments in high yield. Shorter periods give fragment Fc5μ with one or more Fabμ fragments still attached. 2. Reducing agent in the absence of a denaturant cleaves rabbit IgM into half-subunits, each containing one μ chain and one light chain. Digestion by papain in the presence of such a reducing agent destroys the Fcμ domains.
Chemically modified nylons as supports for enzyme immobilization. Polyisonitrile-nylon
Authors: Goldstein Leon , Freeman Amihay , Sokolovsky Mordechai
Published: August 2015

Four-component condensations between amine, carboxyl, isocyanide and aldehyde lead to the formation of N-substituted amides (Ugi, 1962). The present paper describes the use of such condensations for the introduction of chemically reactive groups on to the polyamide backbone of nylon. Polyisonitrile-nylon was synthesized by partial hydrolysis of nylon-6 powder, followed by resealing of the newly formed −CO2... NH2− pairs via a four-component condensation, by using acetaldehyde and 1,6-di-isocyanohexane. Polyisonitrile-nylon could also be converted into a diazotizable arylamino derivative, polyaminoaryl-nylon, by a four-component condensation by using a bifunctional amine, pp′-diaminodiphenylmethane, in the presence of an aldehyde and a carboxylate compound. The versatility of four-component condensations involving the isocyanide functional group of polyisonitrile-nylon allowed coupling of proteins, in an aqueous medium at neutral pH, through either their amino or carboxyl groups. Trypsin and papain were bound to polyisonitrile-nylon through their amino groups by a four-component condensation by using acetaldehyde and acetate; conversely, succinyl-(3-carboxypropionyl-)trypsin, pepsin and papain were coupled through their carboxyl groups in the presence of acetaldehyde and an amine (Tris). Diazotized polyaminoaryl-nylon could be utilized for the immobilization of papain, via the tyrosine residues of the enzyme.
Purification and properties of the trimethylamine dehydrogenase of Bacterium 4B6
Authors: Colby John , Zatman Leonard J.
Published: August 2015

1. The trimethylamine dehydrogenase of bacterium 4B6 was purified to homogeneity as judged by analytical polyacrylamide-gel electrophoresis. The specific activity of the purified enzyme is 30-fold higher than that of crude sonic extracts. 2. The molecular weight of the enzyme is 161000. 3. The kinetic properties of the purified enzyme were studied by using an anaerobic spectrophotometric assay method allowing the determination of trimethylamine dehydrogenase activity at pH8.5, the optimum pH. The apparent Km for trimethylamine is 2.0±0.3μm and the apparent Km for the primary hydrogen acceptor, phenazine methosulphate, is 1.25mm. 4. Of 13 hydrogen acceptors tested, only Brilliant Cresyl Blue and Methylene Blue replace phenazine methosulphate. 5. A number of secondary and tertiary amines with N-methyl and/or N-ethyl groups are oxidized by the purified enzyme; primary amines and quaternary ammonium salts are not oxidized. Of the compounds that are oxidized by the purified enzyme, only trimethylamine and ethyldimethylamine support the growth of bacterium 4B6. 6. Trimethylamine dehydrogenase catalyses the anaerobic oxidative N-demethylation of trimethylamine with the formation of stoicheiometric amounts of dimethylamine and formaldehyde. Ethyldimethylamine is also oxidatively N-demethylated yielding ethylmethylamine and formaldehyde; diethylamine is oxidatively N-de-ethylated. 7. The activity of the purified enzyme is unaffected by chelating agents and carbonyl reagents, but is inhibited by some thiol-binding reagents and by Cu2+, Co2+, Ni2+, Ag+ and Hg2+. Trimethylamine dehydrogenase activity is potently inhibited by trimethylsulphonium chloride, by tetramethylammonium chloride and other quaternary ammonium salts, and by monoamine oxidase inhibitors of the substituted hydrazine and the non-hydrazine types. 8. Inhibition by the substituted hydrazines is time-dependent, is prevented by the presence of trimethylamine or trimethylamine analogues and in some cases requires the presence of the hydrogen acceptor phenazine methosulphate. The inhibition was irreversible with the four substituted hydrazines that were tested.
The β-galactosidase-catalysed hydrolyses of β-d-galactopyranosyl pyridinium salts. Rate-limiting generation of an enzyme-bound galactopyranosyl cation in a process dependent only on aglycone acidity
Authors: Sinnott Michael L. , Withers Stephen G.
Published: August 2015

1. β-d-Galactopyranosyl pyridinium salts are well-behaved substrates for the β-galactosidase of Escherichia coli, catalysis occurring by the interaction of the salt itself with the normal active site of the protein. 2. logkcat. values for seven such salts show a linear relationship (correlation coefficient=−0.997) with the pKa of the parent pyridine. 3. The β-d-galactopyranosyl derivatives of pyridine and 4-bromoisoquinoline exhibit α-deuterium kinetic isotope effects of 1.136±0.040 and 1.187±0.046 on their enzymic hydrolysis, indicating formation of a galactopyranosyl cation in the rate-limiting step. 4. This behaviour of the pyridinium salts contrasts with the behaviour of aryl galactosides and this contrast can be accommodated by the β-galactosidase mechanism of Sinnott & Souchard (1973). 5. The α-deuterium kinetic isotope effect for the hydrolysis of β-d-galactopyranosyl azide is 1.098±0.033; comparison of the kcat. value of the azide with that of a pyridinium salt of the same aglycone pKa enables a maximum factor of 70 to be ascribed to the acceleration of the departure of azide by intracomplex general acid catalysis. 6. The possibility of the rate-limiting process in the glycosidase-catalysed hydrolysis of aryl glycosides being a conformation change is considered for a number of glycosidases where correlations of kcat. with aglycone acidity, reported in the literature, have been unsuccessful.
The nature and properties of the inducible sodium-plus-potassium ion-dependent adenosine triphosphatase in the gills of eels (Anguilla anguilla) adapted to fresh water and sea water
Authors: Sargent John R. , Thomson Alison J.
Published: August 2015

1. Gill tissue from eels adapted to fresh water or to sea water was disrupted in 0.32m-sucrose containing 0.1% (w/v) sodium deoxycholate and the subcellular distribution of (Na++K+)-dependent adenosine triphosphatase was determined. 2. About 70% of the recovered enzyme was in a fraction sedimenting between 225000gav.-min and 6000000gav.-min; the specific activities of enzymes from tissues of freshwater and seawater eels were 16 and 51 μmol of phosphate/h per mg of protein respectively. 3. The enzymes from gills of freshwater and seawater eels were indistinguishable on the basis of a number of parameters. These included phosphorylation by [γ-32P]ATP, the binding of [3H]ouabain, the extent to which bound [3H]ouabain was displaced by increasing concentrations of KCl and pH optima. 4. Electrophoresis on polyacrylamide gels in sodium dodecyl sulphate showed that enzyme preparations from both sources had an identical number of protein components. 5. The higher specific activity of (Na++K+)-dependent adenosine triphosphatase from tissue of seawater eels was accompanied by increased amounts of two protein components. One of these proteins retained 32P after treatment of the enzyme with [γ-32P]ATP and had mol.wt. 97000; the other component was a glycoprotein with mol.wt. approx. 46000. 6. The results are discussed in terms of the nature of the transepithelial NaCl pumps in the gills of freshwater and seawater fish.
Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids
Authors: Jeffreys Alec , Craig Ian
Published: August 2015

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [35S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human–mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.
Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development
Authors: Galand G , Forstner G G
Published: August 2015

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase–glucoamylase identical with membrane-bound maltase–glucoamylase in molecular weight, heat-sensitivity, substrate specificity, Km for maltose and Ki for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase–glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its Km for maltose was 1.5mm. It was inhibited by turanose (Ki=7.5mm) and Tris (Ki=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50°C for 10min. The acid maltase closely resembled β-glucuronidase and acid β-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.
Water-soluble metabolites of p-nitrophenol and 1-naphthyl N-methylcarbamate in flies and grass grubs. Formation of glucose phosphate and phosphate conjugates
Authors: Heenan M P , Smith J N
Published: August 2015

Metabolites isolated from houseflies dosed with 1-napththol or p-nitrophenol were identified as the phosphate and glucose phosphate conjugates of these phenols by titrations, hydrolysis, ionophoresis, i.r. spectra and mixed melting point. [3H]Carbaryl (1-naphthyl N-methylcarbamate) was metabolized by houseflies, blowflies and grass grubs to water-soluble metabolites which had chromatographic and ionophoretic behaviour similar to those of the conjugates of 1-naphthol with glucose, sulphate, phosphate and glucose 6-phosphate.
Simulation of biogenic amine metabolism in the brain
Authors: Turner A J , Illingworth J A , Tipton K F
Published: August 2015

The metabolism of a number of biogenic amines has been simulated by using data obtained from studies of the individual enzymes from pig brain. It is shown that β-hydroxylated amines such as noradrenaline and octopamine are metabolized primarily to the alcoholic metabolite whereas amines lacking this group [e.g. dopamine (3,4-dihydroxyphenethylamine) and 5-hydroxytryptamine] are metabolized at low concentrations to give the corresponding acid. Increase in the amine concentration results in an increase in the proportion of the alcoholic metabolite formed and this may in part account for the effects of the drug reserpine on amine metabolism. The effects of disulfiram (Antabuse) and ethanol (acting through its metabolite acetaldehyde) on amine metabolism may be understood in terms of this simulated model. It is shown that drugs that affect this system also cause alterations in the steady-state concentrations of the intermediate aldehydes and the possible implications of this are discussed.
Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy
Authors: Hill J M , Ab G , Malt R A
Published: August 2015

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20–40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-3H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-3H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-3H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9–48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.
The purification of fructose 1,6-diphosphatase from ox liver and its activation by ethylenediaminetetra-acetate
Authors: Nimmo H G , Tipton K F
Published: August 2015

1.A procedure for the purification of ox liver fructose 1,6-kiphosphatase is described. A number of criteria indicate that the enzyme was not subjected to any significant degree of proteolytic attack during the purification. 2. The molecular weight, amino acid composition and subunit molecular weight are reported. 3. The activation by EDTA was shown to be due to the chelation heavy metals rather than by a more complex interaction with the enzyme as had previously been suggested.
Hybridization properties of nucleolar ribonucleic acid
Authors: Peres C S , Brentani R R
Published: August 2015

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 × SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.
The properties of hydrogen peroxide production under hyperoxic and hypoxic conditions of perfused rat liver
Authors: Oshino N , Jamieson D , Chance B
Published: August 2015

The properties of H2O2 production in the “haemoglobin-free”, “non-circulatory” perfused liver of rats were examined. The H2O2 production with 1 mM-lactate and 0.15 mM-pyruvate was 82nmol/min per g of liver or 333nmol/min per 100g body wt. in the liver of fed rats at 30 degrees C. This rate decreased to almost half in the livers of starved and phenobarbital-pretreated rats. When H2O2 production was stimulated by urate infusion, almost all of the H2O2 produced by the uricase reaction was decomposed by the catalase reaction. During the demethylation reaction of aminopyrine, no change in H2O2 production was detected by the present method; thus microsomal H2O2 production observed in isolated subcellular fractions appeared not to contribute significantly to the H2O2 production in the whole organ. Whereas the rate of the glycolate-dependent H2O2 production was halved at an intracellular O2 concentration that caused a 10 percent increase in the reduction state of cytochrome c, the half-maximal rate of H2O2 production with lactate and pyruvate was observed at an O2 concentration that caused a 40 percent increase in the reduction state of cytochrome c in the liver. No further increase in the rates of H2O2 production was obtained by increasing O2 pressure up to 5 times 10(5) Pa. The rate of ethanol oxidation through the catalase “peroxidatic” reaction varied, depending on the substrate availability. The maximal capability of this pathway in ethanol oxidation reached approx. 1.5 μmol/min per g of liver, when a mixture of urate, glycollate and octanoate was infused to enhance H2O2 production.
Studies on a wide-spectrum intestinal dipeptide uptake system in the monkey and in the human
Authors: Das M , Radhakrishnan A N
Published: August 2015

1. The intestinal transport of glycine and leucine residues of glycyl-L-leucine was studied in the monkey and in the human in vitro. Uptake of both [14C]glycyl-L-leuine and glycyl-L-[14C]leucine show similar Kt values, but there is a marked difference in the Vmax. values. Preliminary studies suggest that this anomalous difference in the Vmax. values may be due to the greater efflux rate of glycine from the tissue. 2. Arrhenius plots of both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake in the monkey intestine show a discontinuity at about 20 degrees C. The activation energies above and below the discontinuity are similar for both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake. These similarities in uptake characteristics suggest that the dipeptide glycyl-L-leucine is transported as one unit. 3. In the monkey intestine, glycyl-L-leucine uptake is inhibited by a wide variety of dipeptides, including those containing acidic and basic amino acids. The inhibition was shown to be competitive by using four representative dipeptides namely: L-alanyl-L-alanine, L-alanyl-L-leucine, L-glutamyl-L-glutamic acid and L-lysyl-L-lysine. The results strongly suggest that in the monkey intestine there may be a dipeptide-uptake system with an extremely broad specificity. These results were also confirmed in the human in a limited way.
Increased synthesis of low-molecular-weight nuclear ribonucleic acids of rat liver after γ irradiation and hepatectomy
Authors: Fónagy A , Hidvégi E J
Published: August 2015

Incorporation of [3H]orotic acid into low-molecular-weight nRNA of rat liver, fractionated on polyacrylamide gels, increased 6-12h after partial hepatectomy and 6h after γ-irridation at 2000 R. The incorporation of orotic acid was particularly increased into the 4.5S, 5S and approx. 10S nRNA fractions. If the irradiation was given after 6h of regeneration and RNA was isolated from the nucleus 12h after hepatectomy then the incorporation of orotic acid into these low-molecular-weight nRNA components was greater than after hepatectomy or irradiation alone.
Subcellular localization of pulmonary antiotensin-converting enzyme (kininase II)
Authors: Ryan J W , Ryan U S , Schultz D R , Whitaker C , Chung A , Dorer F E
Published: August 2015

Goat antibodies to pig lung angiotensin-converting enzyme (kininase II) were conjugated to microperoxidase. Rat lung tissue, previously incubated with non-immune goat serum, was incubated with the antibody-microperoxidase conjugate and then with H2O2 and 3,3-diaminobenzidine. Electron microscopy revealed reaction product on the plasma membrane and caveolae of endothelial cells, especially those of capillaries and venules. These results support the hypothesis that angiotensin I and bradykinin are metabolized by enzymes on the luminal surface of pulmonary endothelial cells.