bluuebunny/crossref_metadata_2025 huggingface dataset:
Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism
Authors: Gawthorne Jeffrey M. , Smith Richard M.
Published: August 2015

1. A study was made of the effects of injected l-methionine on the activity of several enzymes of folate metabolism, and on the transport of methotrexate in liver preparations from vitamin B12-deficient ewes and their pair-fed controls receiving vitamin B12. 2. The activities of dihydrofolate reductase (EC 1.5.1.3) and 5-methyltetrahydrofolate–homocysteine transmethylase were significantly decreased in the liver of vitamin B12-deficient animals, but were unaffected by l-methionine. 3. The concentration of S-adenosyl-l-methionine in the liver of deficient animals was about one-half of that in normal animals, and was restored to normal by either vitamin B12 or l-methionine. 4. Methylenetetrahydrofolate reductase (EC 1.1.1.68) from sheep liver was inhibited by S-adenosyl-l-methionine in vitro, but not by concentrations of S-adenosyl-l-methionine found in the liver of vitamin B12-deficient animals after injection of physiological amounts of l-methionine. 5. Pteroylpolyglutamate synthetase activity was significantly increased in the liver of vitamin B12-deficient animals, and was decreased by intravenous injections of l-methionine. 6. l-Methionine injections increased the initial rate of uptake of methotrexate in liver slices from deficient animals and acted synergistically with vitamin B12 to increase the quantity taken up in 40min. The failure of folate metabolism in vitamin B12 deficiency can be satisfactorily explained if l-methionine similarly affects the membrane transport of naturally occurring folates. 7. Further details of the results have been deposited as Supplementary Publication SUP 50028 (4 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
The transport of sulphate and sulphite in rat liver mitochondria
Authors: Crompton M. , Palmieri F. , Capano Michela , Quagliariello E.
Published: August 2015

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO2 or an exchange of the sulphite anion for hydroxyl ion(s).
Cyclitol glucosides and their role in the synthesis of a glucan from uridine diphosphate glucose in Phaseolus aureus. Characterization of some cyclitol glucoside and their synthesis
Authors: Kemp Jennifer , Loughman Brian C.
Published: August 2015

Short-chained sugar compounds, thought to be involved in the synthesis of callose, were formed in small amounts from UDP-glucose by soluble extracts from hypocotyls of seedlings of Phaseolus aureus. The properties of the glycosides were investigated by treatment with various chemicals and analysis by paper chromatography, g.l.c. and mass spectrometry. The data obtained support the characterization of these compounds as myoinositol-β-glucoside and diglucosylmyoinositol. The cyclitol moiety was provided by the enzyme extract. Free myoinositol was not the immediate substrate but a compound containing myoinositol, isolated from the enzyme extract, may be involved. The method of synthesis of these glucosides is compared with that of other cyclitol glycosides.
Glycosyltransferases in the Golgi membranes of onion stem
Authors: Powell Janet T. , Brew Keith
Published: August 2015

Cell fractions consisting largely of Golgi membranes were prepared from the meristematic region of the onion. Several enzyme activities were found to be localized in these fractions: inosine diphosphatase, galactosyltransferases and glucosyltransferases. The fractions catalysed the transfer of [14C]galactose from UDP-galactose to endogenous and cell-sap acceptors, to N-acetylglucosamine and to ovalbumin. In the presence of bovine α-lactalbumin, transfer to glucose (lactose synthesis) was catalysed. [14C]Glucose was transferred from UDP-glucose to endogenous and cell-sap acceptors, to cellobiose and to fructose (sucrose synthesis). All these activities were latent, being potentiated by detergents (Triton X-100 or sodium deoxycholate). The characteristics of some of these enzyme activities are described and their biological significance is discussed.
Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine
Authors: Bauer Christian H. , Lukaschek Rainer , Reutter Werner G.
Published: August 2015

1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100–120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose–N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-14C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of galactosamine by: (a) inhibition of protein synthesis leading to a diminution of the endogenous acceptor pool of the galactosyltransferase; (b) inhibition of the galactosyltransferase activity by galactosamine metabolites and (c) replacement of UDP-galactose by UDP-galactosamine.
Mode of inhibition of mitochondrial energy transduction by chlorophenoxyisobutyrate
Authors: Panini S. R. , Kurup C. K. Ramakrishna
Published: August 2015

1. α-p-Chlorophenoxyisobutyric acid, the ethyl ester of which is widely used as an antihypercholesterolaemic drug, is an inhibitor of energy-transfer reactions in isolated rat liver mitochondria. 2. The compound at lower concentrations (<4.0μmol/mg of mitochondrial protein) inhibits state 3 oxidation, stimulates state 4 oxidation, abolishes respiratory control and stimulates the latent adenosine triphosphatase activity of mitochondria. The inhibition imposed on state 3 oxidation is relieved by dinitrophenol. 3. At higher concentrations it inhibits coupled phosphorylation as well as dinitrophenol-stimulated adenosine triphosphatase activity. The inhibition of state 3 oxidation under these conditions is not reversed by uncouplers. 4. The three coupling sites of phosphorylation exhibit differential susceptibility to inactivation by this compound. Coupled phosphorylation at the first site is abolished at a drug concentration of 3.0μmol/mg of protein. The third site is inactivated when the concentration of the drug reaches 5.0μmol/mg of protein. The second site is the most refractory and drug concentrations of the order of 10.0μmol/mg of protein are required effectively to inhibit phosphorylation at this site. 5. The compound also inhibits ATP-dependent reversal of electron transport as well as the adenosine triphosphatase activity in submitochondrial particles. 6. The oxidation of NADH and succinate in these particles is not inhibited. 7. These properties indicate that the compound acts as an ‘inhibitory uncoupler’ of energy-transfer reactions in isolated mitochondria.
The role of adenosine 3′:5′-cyclic monophosphate in the division of WI 38 cells. The cellular response to prostaglandin E1 and the effects of an cyclic adenosine 3′:5′-cyclic monophosphate analogue and prostaglandin E1 on cell division
Authors: Kurtz Mary J. , Polgar Peter , Taylor Linda , Rutenburg Alexander M.
Published: August 2015

Inhibition of growth and DNA synthesis was observed in WI 38 cells incubated with 8-methylthioadenosine 3′:5′-cyclic monophosphate or prostaglandin E1. The effect of both compounds on cell growth was reversible. On removal of these compounds from culture media the cells initiated DNA synthesis and divided. In addition, prostaglandin E1 stimulated cyclic AMP formation in these cells to over 40 times the normal basal value. The increase in cyclic AMP concentration in WI 38 cells after addition of prostaglandin E1 showed a marked variation. Cells that had recently been treated with trypsin and plated at a lower cell density exhibited a smaller response to addition of prostaglandin E1 than cells that had divided and reached confluence.
Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria
Authors: Danks Susan M. , Chappell J. B.
Published: August 2015

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD+-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.
Lomofungin as an inhibitor of nucleic acid synthesis in Saccharomyces cerevisiae
Authors: Cannon Michael , Jimenez Antonio
Published: August 2015

1. The antibiotic lomofungin was found to be a potent inhibitor of both DNA and RNA synthesis in Saccharomyces cerevisiae. Under selected growth conditions inhibition of DNA synthesis by the drug preceded inhibition of RNA synthesis. 2. Although in general lomofungin inhibited synthesis of ribosomal RNA and polydisperse RNA more effectively than that of low-molecular-weight RNA, under certain conditions the drug inhibited almost completely synthesis of both 4S and 5S RNA. 3. Inhibition of both RNA and DNA synthesis may be explained if RNA synthesis is required for DNA synthesis in yeast. Alternatively, lomofungin, in addition to interacting with DNA-dependent RNA polymerase, might interfere with a component(s) of the DNA-synthetic apparatus. The drug may thus prove to be of considerable value in studies of DNA synthesis in eukaryotes.
A re-evaluation of energy-independent calcium-ion binding by rat liver mitochondria
Authors: Reed Ken C. , Bygrave Fyfe L.
Published: August 2015

The impermeability of the mitochondrial inner membrane to the chelator ethanedioxybis(ethylamine)tetra-acetic acid permits discrimination between Ca2+which has been transported to the internal (matrix) phase and Ca2+which binds to the external surfaces of the mitochondrion. With this technique, it is shown that ‘energy-independent high-affinity’ binding is a measure of carrier-mediated active Ca2+transport in respiration-inhibited mitochondria; the carrier also transports Ca2+to the internal phase after treatment with carbonyl cyanide m-chlorophenylhydrazone, but in this case the active-transport component is inhibited. The Ca2+-binding sites associated with the external membrane surfaces are similar in concentration and affinity for both inhibited and uncoupled mitochondria; it was not possible to measure external Ca2+binding which could be identified as carrier specific. The results are discussed in relation to the mechanism of mitochondrial Ca2+transport, and to previous studies of energy-independent Ca2+binding.
Phosphatidylinositol cleavage catalysed by the soluble fraction from lymphocytes. Activity at pH5.5 and pH7.0
Authors: Allan David , Michell Robert H.
Published: August 2015

Phosphatidylinositol breakdown by subcellular preparations of small lymphocytes from pig mesenteric lymph nodes was investigated. Activity was higher than in preparations from the tissues studied previously; it was recovered largely in the soluble fraction, which showed pH optima at both 5.4–5.6 and 7.0–7.3. As in other tissues, phosphatidylinositol cleavage produced 1,2-diacylglycerol and a mixture of myo-inositol 1:2-cyclic phosphate and myo-inositol 1-phosphate. It was stimulated by addition of CaCl2 and, less effectively, by MgCl2. On sucrose-density-gradient ultracentrifugation at pH7.0 two peaks of activity were observed (approx. sedimentation coefficients 8S and 10S); the activity profiles on the gradients were similar when assayed at pH7.0 and 5.5. Activity at pH7.0 (and 0.4mm-CaCl2) was decreased by agents, such as salts and lipophilic cations, which tend to neutralize the negative charge of phosphatidylinositol; at pH5.5 these agents slightly stimulated activity. It is suggested that the same enzyme(s) may be responsible for activity at both pH optima and that previous workers may have underestimated the pH7.0 activity because of the inhibitory influence of cations under the usual assay conditions.
Thymidine kinase activity in cardiac muscle during embryonic and postnatal development
Authors: Gillette Paul C. , Claycomb William C.
Published: August 2015

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a Km value for thymidine of 1.6μm. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.
The reconstitution of functional respiratory chains in membranes from electron-transport-deficient mutants of Escherichia coli as demonstrated by quenching of atebrin fluorescence
Authors: Haddock Bruce A. , Downie J. Allan
Published: August 2015

Measurements were made of electron-transport-dependent quenching of atebrin fluorescence in particles prepared from mutants of Escherichia coli unable to synthesize either ubiquinone or haem. Such quenching was either absent (in haem-deficient particles) or decreased (in ubiquinone-deficient particles), but was restored under conditions used to reconstitute oxidase activities. It is concluded that the reconstitution of oxidase activity, in both cases, is associated with the formation of a functional, proton-translocating, respiratory chain.
Polyribosomal and particulate distribution of lysyl- and phenylalanyl-transfer ribonucleic acid synthetases
Authors: Moline Gale , Hampel Arnold , Enger M. Duane
Published: August 2015

1. Only two aminoacyl-tRNA synthetases from Chinese hamster ovary cells are found associated with ribosomes and polyribosomes. 2. Phenylalanyl-tRNA synthetase activity is found with the 60S subunit, 80S monoribosome and individual polyribosomes. An additional 15S form of the enzyme is also seen. 3. Lysyl-tRNA synthetase activity is found in a form of about 20S and associated with ribosomal subunits and polyribosomes. The ribosomal subunits having lysyl-tRNA synthetase activity are about 6S larger than the bulk of the ribosomal subunits. 4. The lysyl- and phenylalanyl-tRNA synthetases found in different complexes have differential sensitivity to EDTA and centrifugation properties.
Separation and properties of human brain hexosaminidase C
Authors: Braidman Isobel , Carroll Mark , Dance Norman , Robinson Donald
Published: August 2015

Hexosaminidase C was separated from human brain supernatant by immunoadsorption of the A and B forms on to a column of immobilized antibody followed by preparative starch-block electrophoresis. There were some differences in the properties of hexosaminidase C preparations after each of these stages, shown by comparison of their heat-inactivation characteristics and filtration through Bio-Gel P-200. The C form prepared by both separation steps had properties which differed markedly from those of the A and B isoenzymes; its molecular weight was much larger, greater than 200000, it had optimum activity between pH6 and 7 and could not be successfully eluted from DEAE-cellulose, even with high salt concentrations, or from Sephadex G-200. These results seem to support the proposal that the C form is under a separate genetic control from the others.
Kinetic studies of oxidized nicotinamide–adenine dinucleotide-facilitated reactions of d-glyceraldehyde 3-phosphate dehydrogenase
Authors: Harrigan Patricia J. , Trentham David R.
Published: August 2015

The kinetics of the acylation of d-glyceraldehyde 3-phosphate dehydrogenase from pig muscle by 1,3-diphosphoglycerate in the presence of NAD+ has been analysed by using the relaxation temperature-jump method. At pH7.2 and 8°C the rate of acylation of the NAD+-bound (or holo-) enzyme was 3.3×105m−1·s−1 and the rate of phosphorolysis, the reverse reaction, was 7.5×103m−1·s−1. After a temperature-jump perturbation the equilibrium of NAD+ binding to the acyl-enzyme was re-established more rapidly than that of the acylation. The rate of phosphorolysis of the apoacylenzyme from sturgeon muscle and of aldehyde release from the d-glyceraldehyde 3-phosphate–apoenzyme complex were ≤40m−1·s−1 and ≤12s−1 respectively at pH8.0 and 22°C, which means that both processes are too slow to contribute significantly to the reaction pathway of the reversible NAD+-linked oxidative phosphorylation of d-glyceraldehyde 3-phosphate. Phosphorolysis of both acyl-apoenzyme and acyl-holoenzyme was first-order in Pi up to 100mm-Pi and more. PO43− could be the reactive species of the phosphorolysis of the acyl-holoenzyme, in which case phosphorolysis is a diffusion-controlled reaction, although other kinetically indistinguishable rate equations for the reaction are possible.
Evaluation of equilibrium constants by affinity chromatography
Authors: Nichol Lawrence W. , Ogston Alexander G. , Winzor Donald J. , Sawyer William H.
Published: August 2015

Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100–lysozyme–d-glucose system in acetate–chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m−1 and 1.2×106m−1 are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 105 glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.
Nuclear protein kinase activity in glucagon-stimulated perfused rat livers
Authors: Palmer Warren K. , Castagna Monique , Walsh Donal A.
Published: August 2015

Nuclei isolated from glucagon-stimulated perfused rat livers contained 2–3 times as much protein kinase activity as did nuclei from control animals. In the presence of either the heat-stable inhibitor or the protein kinase regulatory subunit the elevated cyclic AMP-independent enzyme activity from stimulated nuclei was inhibited to an activity equivalent to that found in controls.
Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins
Authors: Moore Graham , Crichton Robert R.
Published: August 2015

Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH4. After incorporation of either 3H or 14C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex.
Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5′-phosphate
Authors: Evans Robert W. , Holbrook J. John
Published: August 2015

1. The α and β subforms of aspartate aminotransferase were purified from pig heart. 2. The α subform contained 2mol of pyridoxal 5′-phosphate. The apo-(α subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(α subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The β subform had approximately half the specific enzyme activity of the α subform, and contained an average of one active pyridoxal 5′-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5′-phosphate the protein fluorescence of the apo-(β subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.
Inhibition of the soluble adenosine triphosphatase from mitochondria by adenylyl imidodiphosphate
Authors: Philo Roger D. , Selwyn Michael J.
Published: August 2015

1. Adenylyl imidodiphosphate is an inhibitor with high affinity for the soluble ATPase (adenosine triphosphatase) from mitochondria. 2. The reaction of the inhibitor with the ATPase is slow and estimates for the association and dissociation reaction rate constants are given. 3. The number of binding sites for the inhibitor appears to be doubled in the presence of 2,4-dinitrophenol. 4. Adenylyl imidodiphosphate is less effective as an inhibitor of the ATPase activity of this enzyme than of the inosine triphosphatase activity. It is also less effective on the ATPase of frozen-thawed or intact mitochondria and did not inhibit ADP-stimulated respiration by intact mitochondria.
Energy-linked reduction of nicotinamide–adenine dinucleotide in membranes derived from normal and various respiratory-deficient mutant strains of Escherichia coli K12
Authors: Poole Robert K. , Haddock Bruce A.
Published: August 2015

1. Assay conditions are described for the ATP-dependent, uncoupler-sensitive, energy-linked reduction of NAD+ by succinate, dl-α-glycerophosphate or d-lactate in membranes from aerobically grown Escherichia coli. 2. The reaction may be demonstrated in electron-transport particles (ET particles) from cells grown in glycerol, but not in depleted particles washed in low-ionic-strength buffer, or in ET particles from cells grown in glucose. 3. The latter two classes of particles have low specific activities of ATPase (adenosine triphosphatase), succinate dehydrogenase, dl-α-glycerophosphate dehydrogenase and d-lactate dehydrogenase relative to undepleted ET particles from cells grown in glycerol. 4. Reconstitution of energy-linked NAD+ reduction in particles from cells grown in glucose was done by: (a) addition of the high-speed supernatant fraction from sonicates of the same cells; (b) addition of a protein fraction, precipitated by (NH4)2SO4 from this supernatant, or (c) addition of an (NH4)2SO4-precipitated fraction from the low-ionic-strength wash of particles from cells grown in glycerol. 5. The use of (NH4)2SO4-precipitated fractions from ATPase- or succinate dehydrogenase-deficient mutants grown in glycerol in the above reconstitution indicated that failure to demonstrate the reaction in particles from cells grown in glucose was a result of inadequate activities of appropriate dehydrogenases, rather than of ATPase. 6. Energy-linked NAD+ reduction could be demonstrated in particles from a ubiquinone-deficient mutant only after restoration of NADH oxidase activity by adding ubiquinone-1. 7. The measured rate of the energy-linked reaction in particles from a haem-deficient mutant, however, was not stimulated after the ATP- and haematin-dependent acquisition of functional cytochromes. 8. Results are interpreted as evidence of the ubiquinone-dependent, but cytochrome-independent, nature of the site I region of the respiratory chain in E. coli.
Caution in the interpretation of plant ribosome studies (Short Communication)
Authors: Leaver Christopher J. , Dyer John A.
Published: August 2015

Published methods of ribosome isolation from plant tissues result in a partial and selective recovery of the total ribosome population. The enrichment of polyribosomes obtained may give a false impression of the change in translational capacity of the tissue under study.
Synthesis of phosphoenolpyruvate carboxykinase (guanosine triphosphate) by isolated liver polyribosomes
Authors: Ballard F J , Hopgood M F , Reshef L , Tilghman S , Hanson Richard W.
Published: August 2015

1. Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was synthesized by postmitochondrial supernatants of rat liver in the presence of appropriate salts, an energy supply and [3H]leucine. Synthesis of enzyme released from polyribosomes was detected by immunoprecipitation with specific antibody followed by electrophoresis of the dissolved antibody–antigen precipitates on sodium dodecyl sulphate–polyacrylamide gels in the presence of a14C-labelled enzyme marker. 2. Enzyme synthesis in vitro occurs predominantly on free rather than bound polyribosomes. 3. Starved animals in which de-induction of phosphoenolpyruvate carboxykinase (GTP) had been initiated by re-feeding for 2h had a markedly decreased rate of enzyme synthesis, whether the measurements were made after injection of radioactive leucine into the intact animal or if synthesis was determined in vitro. 4. The low rate of enzyme synthesis by liver polyribosomes from re-fed animals was not due to the absence of soluble factors, nor could it be increased by the addition of cyclic AMP to the protein synthesis system. 5. Phosphoenolpyruvate carboxykinase (GTP) synthesis in vitro is diminished relative to total protein synthesis when the postmitochondrial supernatant is kept at 0°C for several hours before measurement of protein synthesis. Since this effect is blocked by heparin, it is probably caused by selective ribonuclease attack on enzyme mRNA. 6. De-induction of phosphoenolpyruvate carboxykinase (GTP) is tentatively explained as being due to a transcriptional block in specific mRNA synthesis, followed by rapid degradation of existing message.
Alternative pathways of zeaxanthin biosynthesis in a Flavobacterium species. Experiments with nicotine as inhibitor
Authors: McDermott J C B , Brown D J , Britton G , Goodwin T W
Published: August 2015

In Flavobacterium R1519, nicotine blocks zeaxanthin biosynthesis by specifically inhibiting the cyclization reaction. Lycopene (at high nicotine concentrations, e.g. 7.5mm) and rubixanthin (at low nicotine concentration, e.g. 1mm) replace zeaxanthin as the main carotenoid. On removal of the nicotine lycopene is converted into β-carotene under anaerobic conditions and into zeaxanthin in the presence of O2. The conversion in vivo of β-carotene into zeaxanthin was also demonstrated. Cyclization (an anaerobic process) thus precedes hydroxylation (O2-requiring) in the biosynthesis of zeaxanthin. The conversion in vivo of rubixanthin into β-cryptoxanthin and into zeaxanthin was demonstrated, thus indicating the operation of alternative pathways of zeaxanthin biosynthesis. Several alternative biosynthetic pathways are considered and the results are also discussed in terms of reaction sequences of carotenoid ‘half-molecules’.
Inducibility and some properties of the threonine dehydratase of sheep liver
Authors: Doonan S , Koerner D H , Schmutzler W , Vernon Charles A.
Published: August 2015

The threonine dehydratase extracted from sheep liver is in an essentially inactive form, referred to here as the precursor, but can be activated by incubation at high temperature in alkaline solution. A method for purification of the activated enzyme to a state approaching homogeneity has been devised. The activated enzyme catalyses the deamination of serine but rapidly loses activity during the process. The enzyme inactivated by incubation with serine can be reactivated under the same conditions as those described for the precursor, the kinetic parameters being the same in both cases. Individual sheep livers differ widely in their content of threonine dehydratase, and the possible role of dietary factors in this variability has been examined. The average amount of the enzyme in the livers of sheep fed on high-plant-protein diets did not differ significantly from that in livers obtained from slaughterhouses, but was higher than that in livers from animals fed only on grass or normal concentrates. Large increases in enzyme concentration were, however, obtained when sheep were injected intraperitoneally with a protein hydrolysate. The enzyme from the livers of these sheep could be purified by the same technique and to the same specific activity as the enzyme from the livers of control sheep.
Intranuclear localization and receptor proteins for 1,25-dihydroxycholecalciferol in chick intestine
Authors: Lawson D E M , Wilson P W
Published: August 2015

1. The intranuclear distribution of cholecalciferol and its metabolites was studied in the intestine of rachitic chicks. 2. At high doses of cholecalciferol the nuclei contain the vitamin and its 25-hydroxy metabolite, but over 80% of this is localized on the nuclear membranes. The hormone, 1,25-dihydroxycholecalciferol, is found within the cell nuclei irrespective of the intake of cholecalciferol, but significant amounts could not be found with chromatin isolated free of nuclear membranes. 3. 1,25-Dihydroxycholecalciferol is associated in the nucleus with an acidic protein. Since one of the actions of 1,25-dihydroxycholecalciferol is to control the synthesis of mRNA for calcium-binding protein it was to be expected that the hormone would be bound to chromatin, as with the other steroid hormones. It is suggested that the hormone–receptor complex exists as part of an equilibrium mixture of the complex bound to the DNA and in a free form. 4. A protein extract of nuclei was obtained, which when incubated at 4°C for 1h took up the 1,25-dihydroxycholecalciferol. The nature of this binding was studied. 5. There appear to be two nuclear proteins able to bind the hormone one of which is the intestinal nuclear receptor. The binding sites on this protein are saturable with the hormone, have an association constant of 2×109m-1and show a high chemical specificity for the 1,25-dihydroxycholecalciferol. The number of nuclear binding sites for the hormone provided by this receptor is similar to the maximum intestinal hormone concentration so far observed. Its sedimentation coefficient is 3.5S, and is very close to that observed for the nuclear protein to which is attached the 1,25-dihydroxycholecalciferol formed in vivo from vitamin D. 6. The cytoplasmic protein has an association constant of 1×109m-1and a sedimentation coefficient of 3.0S, but its relation with the nuclear receptor is not yet clear.
Inhibition of mitochondrial phosphate transport by p-diazobenzenesulphonate (Short Communication)
Authors: Dawson Alan P.
Published: August 2015

The impermeant protein-modifying reagent p-diazobenzenesulphonate inhibits phosphate transport in rat liver mitochondria. The rate of inhibition is increased by the presence of oxidizable substrate, and decreased by uncoupling agents. These results are compared with the results on inhibition of phosphate transport by N-(N-acetyl-4-sulphamoylphenyl)maleimide (Klingenberg et al., 1974).
The nature of the collagen cross-links in bone in the chronic uraemic state
Authors: Russell J E , Avioli L V , Mechanic G
Published: August 2015

The cross-links from NaB3H4-reduced bone collagen of chronically uraemic rats and pairfed controls were compared. The ratio of the reduced cross-links deltadelta'-dihydroxylysinonorleucine to delta-hydroxylysinonorleucine was significantly increased in the uraemic animals. The observed increment in the dihydroxylysinonorleucine:hydroxylysinonorleucine ratio was accentuated as the uraemic state advanced. The data indicate that osteodystrophy of chronic renal insufficiency is characterized by an alteration of the quantitative relations between cross-links and aldehydic precursors of bone collagen.
Mechanistic, inhibitory and stereochemical studies on cytoplasmic and mitochondrial serine transhydorxymethylases
Authors: Akhtar M , El-Obeid H A , Jordan P M
Published: August 2015

By using cytoplasmic and mitochondrial serine transhydroxymethylase isoenzymes from rabbit liver, it was shown that both enzymes exhibited similar ratios of serine transhydroxymethylase/threonine aldolase activities. Both enzymes catalysed the removal of the pro-S hydrogen atom of glycine, which was greatly enhanced by the presence of tetrahydrofolate. The cytoplasmic as well as the mitochondrial enzyme catalysed the synthesis of serine from glycine and [3H2]formaldehyde in the absence of tetrahydrofolate. The results are consistent with our previous suggestion that a role of tetrahydrofolate in the serine transhydroxymethylase reaction is to transport formaldehyde in and out of the active site (Jordan & Akhtar, 1970). The isoenzymes, however, showed remarkable differences in their inactivation by inhibitors. The serine transhydroxymethylase as well as the threonine aldolase activities of the cytoplasmic enzyme were inactivated in a similar fashion by chloroacetaldehyde, iodoacetamide, bromopyruvate and glycidaldehyde (2,3-epoxypropionaldehyde). These inhibitors had no effect on the two activities of the mitochondrial enzyme. The rate of inactivation of the cytoplasmic enzyme by glycidaldehyde was enhanced by the presence of glycine but decreased by the presence of serine. The implications of these results to the mechanism of catalysis and the nature of the active site of the enzymes are discussed.
Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes
Authors: Craig R K , Keir H M
Published: August 2015

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5′-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was “activated” DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to “activated” DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.
The subunit structure of β-glucosidase from Botryodiplodia theobromae Pat
Authors: Umezurike G M
Published: August 2015

1. A homologous series of beta-glcosidase (beta-D-glcoside glcohydrolase, EC 3.2.1.21), which varied in relative amounts in different preparations from cultures of similar and different age, was observed in cultures od Botryodiplodia theobromae Pat grown for 4-8 week on cotton flock (cellulose) as carbon source. 2. Aging of the purified high-molecular-weight species led to some amount of siddociation into a homolous series of lower-molecular-weight speices. 3. Rough molecular-weight estimates, by gel filtration, of the various species derived from the purifeid high-molecular-weight enzyme were 350000-3800000, 170000, 180000, 83000-87000 and 45000-47000. 4. Electron micrographs of the negatively stained 350000-380000-molecular-weight enzyme showed that the molecule is an octamer in which each roughly spherical monomer occupies a corner of a cube with each side about 7.14nm long. 5. Carboxamidomethylation of the reduced form of each molecular-weight species of the enzyme led to irreversible dissociation of the molecules into electrophoretically identical polypeptides with a moleclar weight of 10000-12000. 6. These results suggest a slow association-dissociation of the type (8n)in equilibrium 2 (4n) in equilibrium 4(2n) in equilibrium 8(n), where n is defined as the monomer. The monomer is in turn made up of four polypeptide a subunits whi-ch are non-catalytic. 7. The Michaelis constants (Km) and heat stability of the four wnzymically active molecular species derived from the purified enzyme increased with molecular complexity, whereas all four species were inhibited by glycerol (100nM) at low concentrations of substrate (o-nitrophenyl beta-D-glucopyranoside) but activated at high substrat concentrations. 8. Only the lowest-molecular-weight species (45species (45,000-47000 mol. wt.) showed substrate inhibition.
Nitrogenase of Klebsiella pneumoniae. A stopped-flow study of magnesium-adenosine triphosphate-induce electron transfer between the compeonent proteins
Authors: Thorneley R N
Published: August 2015

Stopped-flow kinetic data have been obtained for a rapid electron-transfer reaction between the component proteins of nitrogenase from Klebsiella pneumoniae, which was induced by MgATP. Up to three equivalents of the Fe-containing protein were rapidly oxidized by one equivalent of the Fe-Mo-containing protein in a unimolecular reaction, k2 = 2 × 10(2)S-1. Evidence for a tight complex between the component proteins, KD(complex) less than 0.5 muM, which was formed with a rate k1 greater than 1 × 10(7)M-1-S-1, has been obtained. MgATP bound to either the Fe-containing protein or to the two-protein complex with a rate k3 greater than 2.5 × 10(6)M-1-S-1 and with KD(MgATP) = 0.4mM, before the electron-transfer reaction.
Electrostatic effects on the kinetics of bound enzymes
Authors: Engasser J M , Horvath C
Published: August 2015

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.
Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator
Authors: Kakiuchi S , Yamazaki R , Teshima Y , Uenishi K , Miyamoto E
Published: August 2015

1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 μM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 μM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.
Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan
Authors: Tynecka Z , Ward J B
Published: August 2015

The synthesis of peptidoglycan by an autolysin-deficient β-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with β-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.
Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes
Authors: Van Heyningen S , King C A
Published: August 2015

Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes.