bluuebunny/crossref_metadata_2025 huggingface dataset:
Location of an oligomycin-insensitive and magnesium ion-stimulated adenosine triphosphatase in rat spleen mitochondria
Authors: Vijayakumar E K , Weidemann M J
Published: August 2015

1. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration and lowers both the respiratory control and ADP/O ratios; this effect is reversible on addition of excess of EDTA. 2. With [gamma-32P]ATP as substrate, an Mg2+-stimulated ATPase (adenosine triphosphate) was identified in the atractyloside-insensitive and EDTA-accessible space of intact rat spleen mitochondria. 3. Oligomycin has no effect on the activity of the Mg2+-stimulated ATPase at a concentration (2.0mug/mg of protein) that completely inhibits the atractyloside-sensitive reaction. Of the two ATPase activities, only the atracytoloside sensitive reaction is stimulated (approx. 40%) by dinitrophenol. 4. On digitonin fractionation the atractyloside-insensitive Mg2+-stimulated ATPase co-purifies with the outer membrane-fraction of rat spleen mitochondria, whereas (as expected) the atractylosidesensitive activity co-purifies with the inner-membrane plus matrix fraction. 5. Stoicheiometric amounts of ADP and Pi are produced as the end products of ATP hydrolysis by purified outer-membrane fragments; no significant AMP production is detected during the time-course of the reaction. 6. The outer-membrane ATPase is present in rat kidney cortex and heart mitochondria as well as in spleen, but is absent from rat liver, thymus, brain, lung, diaphragm and skeletal muscle.
Reassembly of the peptidyltransferase centre of larger subparticles of rabbit reticulocyte ribosomes from a core-particle and split-protein fraction
Authors: Cox R A , Greenwell P
Published: August 2015

We report the reconstruction, from a core-particle and split-protein fraction, of the larger subribosomal particle of rabbit reticulocytes. The reassembled particle was active in polyphenylalanine synthesis and in the puromycin reaction. The core-particles and split-protein fractions were obtained by treatment of the larger subparticle with salt solutions containing NH4+ and Mg2+ in the molar ratio 40:1 over the range 2.25-2.75 M-NH4Cl/56-69mM-MgCl2 at 0° C. This treatment led to the loss of about eight proteins (approx. 17% of the protein moiety), which were found wholly or largely in the split-protein fraction as shown by two-dimensional gel electrophoresis. The core particle retained 5S rRNA and had much decreased (no more than 10% of control) ability to function in the puromycin reaction or in poly (U)-directed polyphenylalanine synthesis. Activity was recovered when the recombined core-particle and split-protein fractions were dialysed overnight at 4° C against 0.3M-NH4Cl/15mM-MgCl2/1mM-dithiothreitol/15% (v/v) glycerol/20mM-Tris/HCl, pH 7.6, and then heated for 1 h at 37° C. The recovery was 40-80% of the original activity. Raising the concentration of MgCL2 to 300 mM in 2.5 M-NH4CL led to the removal of seven rather than eight proteins, and the core particle remained active in the puromycin reaction. We infer that the protein retained by raising the concentration of Mg2+ is an essential component of the peptidyltransferase centre of the ribosome.
Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis
Authors: Rawat A K
Published: August 2015

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in ‘foetal alcohol syndrome’.
The effects of diet on the esterification of glycerol phosphate, dihydroxyacetone phosphate and 2-hexadecylglycerol by homogenates of rat adipose tissue
Authors: Dodds P F , Brindley D N , Gurr M I
Published: August 2015

1. Male rats were fed for 5 weeks after weaning on a diet containing (by weight) 59% of starch or on diets that contained 39% of starch and 20% of either sucrose, beef tallow or corn oil. 2. The rats fed on the beef tallow consumed more energy than did the rats fed on the starch and sucrose diets. The rats fed on the corn oil drank less water than did the other groups of rats. 3. There were no significant differences between the four groups in terms of body-weight gain, epididymal-fat-pad weight and in the size, number and triacylglycerol content of the adipocytes in the fat-pads. 4. There was a significant correlation (P < 0.001) between the activities of glycerol phosphate acyltransferase and monoacylglycerol acyltransferase in individual rats. Both of these activities were highest in the group fed on the high-starch diet and both correlated with the consumption of glucose by individual rats in the four groups. 5. The percentage of glycerol phosphate converted into diacylglycerol and triacylglycerol was positively correlated with the mean diameters, surface area and triacylglycerol content of the adipocytes for individual rats and was greates in the sucrose-fed rats. 6. The specific activity of dihydroxyacetone phosphate acyltransferase was highest in the rats fed on beef tallow. This activity was positively correlated with the energy intake for all dietary groups over the 5-week feeding period. 7. The results are discussed in terms of the functions of the three routes of glycerolipid synthesis in adipose tissue.
The binding of copper ions to copper-free bovine superoxide dismutase. Copper distribution in protein samples recombined with less than stoicheiometric copper ion/protein ratios
Authors: Rigo A , Viglino P , Calabrese L , Cocco D , Rotilio G
Published: August 2015

Samples of superoxide dismutase containing less than stoicheiometric amounts of Cu2+ were obtained by either partial re-addition of Cu2+ to the Cu2+-free protein or partial removal of Cu2+ by controlled CN-treatment. In these samples the distribution of the metal between the two identical sites on the two subunits was studied by quantitative gel electrophoresis and found to be statistical only in the process of copper removal by CN-. In the other case the distribution fits a model of co-operative interaction between the two sites, where the sites are equivalent for the binding of the first Cu2+ ion, but the occupation of the first site lowers the activation energy of the binding of the second Cu2+ ion. This indicates that binding of Cu2+ ion at its site on one subunit brings about conformational changes that facilitate Cu2+ binding on the other subunit. These results may relate to possible intersubunit interactions during the catalytic activity.
Dolichyl monophosphate and its sugar derivatives in plants
Authors: Brett C T , Leloir L F
Published: August 2015

A glucose acceptor was isolated from soya beans by extraction with chloroform/methanol (2:1, v/v), followed by DEAE-cellulose column chromatography of the extract. This acceptor could not be distinguished from liver dolichyl monophosphate by t.l.c. It could replace dolichyl monophosphate as a mannose acceptor with a liver enzyme and its glucosylated derivative could replace dolichyl monophosphate glucose as a glucose donor in the same system. These results, together with those already reported [Pont Lezica, Brett, Romero Martinez & Dankert (1975) Biochem, Biophys. Res. Commun. 66, 980-987], indicate that the acceptor from soya bean is a dolichyl monophosphate. Gel filtration of its glucosylated derivative on Sephadex G-75 in the presence of sodium deoxycholate indicated that the acceptor contained 17 or 18 isoprene units. An enzyme preparation from pea seedlings was shown to use endogenous acceptors to form lipid phosphate sugars containing mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine. Chromatographic and degradative techniques indicated that the compounds formed were lipid monophosphate mannose, lipid pyrophosphate N-acetylglucosamine, lipid pyrophosphate chitobiose and a series of lipid pyrophosphate oligosaccharides containing both mannose and N-acetylglucosamine. None of these compounds was degraded by catalytic hydrogenation, and so the lipid moiety in each case was probably an alpha-saturated polyprenol. The endogenous acceptors for mannose and N-acetylglucosamine in peas may therefore be dolichyl monophosphate, as has been found in mammalian systems.
Amino acid sequence of the N-terminal forty-two amino acid residues of the C chain of subcomponent C1q of the first component of human complement
Authors: Reid K B M
Published: August 2015

1. The sequence of the N-terminal 42 amino acid residues and the identity of residue 45 of the C chain of subcomponent C1q were established by the use of the automatic protein sequencer. 2. A comparison of the amino acid sequences of the A and C chains of subcomponent C1q shows that they are identical at 18 positions out of the first 45. However, 12 of the amino acid residues in the positions of identity are glycine residues occurring in the repeating triplet sequence Gly-X-Y. 3. Position 36 in the C chain was found to be alanine, which was unexpected since the residue in this position would have to be glycine if the repeating triplet sequence Gly-X-Y were to extend, uniformly, throughout the entire length of the C-chain collagen-like region. This break in the repeating triplet sequence would prevent residue 36 in the C chain from taking part in collagen-like triple-helix formation. 4. The sequence information presented here therefore indicates that there should be a break, or distortion, located approximately half-way along each of the six collagen-like triple-helical regions proposed to be present in subcomponent C1q [Reid & Porter (1976) Biochem. J. 155, 19-23] and this is consistent with what is seen in electron micrographs of intact and pepsin-digested subcomponent C1q.
Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans
Authors: Bamforth C W , Large P J
Published: August 2015

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.
Studies on the purification of rat liver uridine diphosphate glucuronyltransferase
Authors: Burchell B
Published: August 2015

1. A stable, more highly purified, preparation of UDP-glucuronyltransferase was obtained than previously reported. 2. Enzyme activity towards o-aminophenyl and p-nitrophenyl was increased 43- and 46-fold respectively. 3. The final preparation contains only three staining polypeptide bands visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The only known major accompanying protein appears to be epoxide hydratase. 5. The purified enzyme activity towards o-aminophenol can still be activated 3 fold by diethylnitrosamine. 6. On evidence from purification, o-aminophenol and p-nitrophenol appear to be glucuronidated by the same enzyme protein. The possible recognition of the UDP-glucuronyltransferase enzyme is discussed.
Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic β-cells
Authors: Idahl L Å , Lernmark Å , Sehlin J , Täljedal I B
Published: August 2015

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.
Alloxan cytotoxicity in vitro. Microscope photometric analyses of Trypan Blue uptake by pancreatic islet cells in suspension
Authors: Grankvist K , Lernmark Å , Täljedal I B
Published: August 2015

Suspensions of islet cells were prepared by shaking pancreatic islets from non-inbred ob/ob mice in a Ca2+-free buffer. The cells were incubated with or without 20 mM-alloxan, and subsequently with Trypan Blue. The uptake of Trypan Blue by cell nuclei was analysed by microscope photometry and by counting the frequency of cells appearing stained on visual inspection. Cells classified as stained or unstained by inspection showed no overlap in nuclear absorbance. Suspensions not exposed to alloxan contained 70-80% of unstained cells. Alloxan markedly decreased the frequency of unstained cells, an effect counteracted by 5 or 20 mM-D-glucose. The spectrum of Trypan Blue in islet-cell nuclei was red-shifted by about 20 nm. A similar red-shift was observed on adding the dye to solutions of albumin or histones, but not on mixing the dye with DNA. Binding to basic proteins may explain the concentrative uptake of Trypan Blue in dead cells and contribute to the oncogenic transformation of phagocytotically active cells. Beta-Cells in vitro are killed by alloxan and hence represent a valid model for studying the diabetogenic action of the drug.
Proton uptake linked to the 3-deoxy-2-oxo-d-gluconate-transport system of Escherichia coli
Authors: Lagarde A E , Haddock B A
Published: August 2015

Genetic and kinetic evidence is presented to show that the carrier-mediated uptake of the anionic sugars 3-deoxy-2-oxo-D-gluconate and D-glucuronate by Escherichia coli involves the concomitant transport of protons.
Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar
Authors: Dalessandro G , Northcote D H
Published: August 2015

During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.
Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle
Authors: Antoniw J F , Nimmo H G , Yeaman S J , Cowen P
Published: August 2015

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.
Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase
Authors: Snider D E , Parker C W
Published: August 2015

Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.
Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of a nuclear adenylate cyclase
Authors: Wedner H J , Parker C W
Published: August 2015

Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.
Mitochondrial adenosine triphosphatase of the fission yeast Schizosaccharomyces pombe 972h-. Changes in inhibitor sensitivities during the cell cycle indicate similarities and differences in binding sites
Authors: Lloyd D , Edwards S W
Published: August 2015

1. We used 11 different inhibitors of energy conservation as inhibitors of ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe obtained from cells at different stages of the cell cycle. 2. All the inhibitors showed cell-cycle-dependent variations in their I50 values (microng of inhibitor/mg of protein giving 50% inhibition of inhibitor-sensitive ATPase at pH 8.6). 3. From the sensitivity profiles through the cell cycle it was concluded that: (a) oligomycin, venturicidin, triethyltin sulphate and dibutylchloromethyltin chloride all act at closely associated site(s); (b) NN'-dicyclohexylcarbodi-imide and leucinostatin both act at a similar site, which is, however, distinct from that at which other inhibitors of the membrane factor (Fo) act. 4. The variations in I50 values for efrapeptin closely followed changes in specific activity of ATPase, as would be expected for an inhibitor acting at catalytic sites; these fluctuations were different from those for aurovertin, Dio-9, 4-chloro-7-nitrobenzofurazan, quercetin and spegazzinine, all of which show different sensitivity profiles from one another. 5. Anomalous stepwise inhibitor-titration curves were obtained for spegazzinine, NN'-dicyclohexylcarbodiimide, dibutylchloromethyltin chloride and leucinostatin. 6. Possible explanations are proposed for the discontinuous expression of inhibitor-binding sites during the cell cycle.
Acid dissociation constants of glycopeptides
Authors: Austen B M , Marshall R D
Published: August 2015

Glycopeptides containing mainly four amino acid residues in the sequence Asn-Leu-Thr-Ser, with small amounts of additional amino acid residues, were isolated from enzymic hydrolysates of hen's-egg albumin. Heterogeneity of the carbohydrate moiety was confirmed. Acid-base titrations showed that the alpha-amino group has a pKa value of 6.43 at 25 degrees C. The standard free engery and entropy changes associated with the ionization at 25 degrees C were 37.2kJ-mol-1 and -0.014kJ-mol-1- K-1 respectively. The complications arising in the interpretation of titration curves of the glycopeptides, which are heterogeneous with respect to the peptide chain, were considered and discussed in the light of the earlier suggestion that the titration curve of the glycopeptide might be interpreted as being due in part to a structure in which the hydroxyl group of the threonine residue is hydrogen-bonded to the beta-aspartamido oxygen atom [Neuberger & Marshall (1968) in Symposium on Foods - Carbohydrates and their Roles (Schultz, H.W., Cain, R.F. & Wrolstad, R.W., eds.), pp. 115-132, Avi Publishing Co., Westport, CT]. It is concluded that either the glycopeptides do not contain a hydrogen bond of that type, or, if they do, that it cannot be recognized by acid-base-titration studies.
Analysis of nucleosomal deoxyribonucleic acid in a higher plant
Authors: Cheah K S E , Osborne D J
Published: August 2015

Comparisons of nuclease digestions of chromatin in nuclei from rye embryos and rat liver show that their nucleosomal DNA is similar, i.e. DNA subunits consist of 200 base-pair repeats with 140 base-pair cores of identical substructure. The identical nucleosome structure is present in nuclei from cells of rye embryos that have been non-viable (i.e. dead) for more than 7 years. These findings indicate a high degree of stability of the DNA-histone complex and are consistent with conservation of the nucleosomal structure of chromatin during evolution.
The role of the imidazolyl nitrogen atoms of histidine-12 in ribonuclease S
Authors: van Batenburg O D , Voskuyl-Holtkamp I , Schattenkerk C , Hoes K , Kerling K E , Havinga E
Published: August 2015

S-peptide (residues 1--14) analogues in which the active histidine-12 residue is replaced by Npi-methyl-L-histidine, Ntau-methyl-L-histidine and beta-(pyrid-3-yl)-L-alanine were synthesized and tested for their capacity to bind to S-protein and to activate it. The results show that both imidazolyl nitrogen atoms are required for optimal catalytic functioning, Ntau being essential to the catalytic reaction itself, Npi playing a role in keeping the imidazole ring in the correct position.
A comparison of the association of yeast phosphoglycerate mutase (EC2.7.5.3) with that of haemoglobin. An ultracentrifuge study
Authors: Spragg S P , Roche J J , Wilcox J
Published: August 2015

1. Previous work showed that yeast phosphoglycerate mutase (EC 2.7.5.3) has a mol.wt. of between 107000 and 110000. Preliminary examination showed that at dilutions less than 0.1 g/1 the enzyme dissociated into its subunits. 2. This dissociation was quantitatively examined by both equilibrium and velocity centrifugation. 3. The mathematical analysis of the equilibrium records was tested against oxyhaemoglobin in a variety of ionic strengths and at two temperatures. 4. The estimated L2,4 (interaction coefficient) for oxyhaemoglobin generally agreed with published values except at 6 degrees C in 0.9 M-NaCl, when it was 2.5 times larger than the published value. 5. Statistical analysis of ultracentrifugal-equilibrium experiments showed that the predominant reaction for phosphoglycerate mutase was monomer in equilibrium tetramer, to give an L1,4 of 40.3+/-23.4 (S.D.)1(3)-g(-3) at 20 degrees C. Decreasing the temperature decreased the association to given an enthalpy of between 40 and 60kJ/mol. 6. Analysis of velocity experiments carried out with concentrations varying from 0.3 to 17 g/1 gave an L1,4 of 3111(3)-g(-3). Incorporating errors from estimating S20,w into the analysis showed that this estimate could range from 893 to 1421(3)-g(-3). 7. The concentration-dependence of S20,w was 0.95 litre-g-1 and s020,w for the tetramer was 66.9ps. 8. These results are discussed in relation to the activity of the enzyme.
Separation and properties of two arylamidases from rat cardiac-muscle extracts
Authors: Bury A F , Coolbear T , Savery C R
Published: August 2015

Two main arylamidase activities were separated from a particle-free supernatant of rat heart by chromatography on DEAE-Sephadex. Although both enzymes hydrolysed L-leucine 4-nitroanilide, only peak-II enzyme hydrolysed L-lysine 4-nitroanilide. A third minor peak (Ia) contained an enzyme that was active mainly on the L-lysine 4-nitroanilide. The mol.wts. of the enzymes in peaks I and II were approx. 257000 and 105000 respectively. The pH optimum was approx. pH7.0 for peak-I enzyme and 7.0-8.0 for peak-II enzyme. Both enzymes were inhibited by addition of puromycin, p-hydroxymercuribenzoate, o-phenanthroline and bivalent metal ions. Addition of dithiothreitol resulted in stimulation of both activities. Dialysis against o-phenanthroline resulted in inhibition of peak-I and -II enzymes, but after dialysis against EDTA only peak-II enzyme was inhibited.
The reduction of Pseudomonas cytochrome c551 oxidase by chromous ions
Authors: Barber D , Parr S R , Greenwood C
Published: August 2015

The reduction of cytochrome c551 oxidase from Pseudomonas aeruginosa by Cr2+ ions was followed in the stopped-flow apparatus at a number of wavelengths. The c-haem reduction proceeded in a biphasic fashion with second-order rate constants of 2.6 X 10(5)M-1-S-1 and 4.8 X 10(4)M-1-S-1 at 25 degrees C, whereas the biphasic reduction of the d1-haem appeared to be independent of reductant concentration with rate constants of approx. 1.0S-1 and 0.25S-1 respectively. The kinetically determined difference spectra (reduced minus oxidized) for the c- and d1-haems are presented.
Effects of starvation and development on mitochondrial acetoacetyl-coenzyme A thiolase of rat liver
Authors: Reed W D , Ozand P T , Tildon J T , Cornblath M
Published: August 2015

The activity of the putative ketogenic beta-oxoacyl-CoA thiolase from mitochondria of rat liver increases with starvation, during neonatal life, and after the injection of glucagon. These changes are associated with alteration in ketonaemia. The changes in activities of this species of thiolase are not associated with significant alterations in the apparent affinity (Km) for the ketogenic substrate, acetyl-CoA. These results support a role for thiolase in the regulation of ketogenesis.
Incorporation of [14C]shikimate into phenazines and their further metabolism by Pseudomonas phenazinium
Authors: Byng Graham S. , Turner John M.
Published: August 2015

1. During growth of Pseudomonas phenazinium on l-threonine medium, phenazine pigment formation commenced early and 1,6-dihydroxyphenazine 5,10-dioxide (iodinin) was the major component. Growth on l-[U-14C]threonine showed that when growth was complete about 25% of the label had been incorporated into phenazines and 30% into cell substance. 2. The addition of d-[2,3,4,5(n)-14C]shikimate to cultures at different phases of growth showed that the greatest efficiency of incorporation (about 70%) occurred in the mid- to late-exponential phase. Phenazines accounting for most of the 14C supplied were iodinin and 9-hydroxyphenazine-1-carboxylate plus 2,9-dihydroxyphenazine-1-carboxylate. Radioactivity incorporated into cell substance was about one-third of the amount found in phenazines. 3. Kinetic studies showed that radioactivity from a pulse of [14C]-shikimate was incorporated into phenazines immediately, without a discernible lag, and into all detectable phenazines simultaneously rather than sequentially. 4. Radioactive phenazines isolated from culture media were fed to growing cultures and their metabolism was studied. The results supported a scheme for the biosynthesis of iodinin and 1,8-dihydroxyphenazine 10-monoxide by a branched pathway. 5. It is proposed that phenazine-1,6-dicarboxylate is the common precursor of all naturally occurring phenazines.
Control of zinc-thionein synthesis in rat liver
Authors: Squibb K S , Cousins R J , Feldman S L
Published: August 2015

The rate of [35S]cystine incorporation into hepatic zinc-thionein (a metallothionein) was stimulated, with a maximum of 5-6h, after parenteral administration of 2mg of Zn2+ containing 65Zn. The binding of 65Zn to zinc-thionein was measurable by 2-1/2h and reached a plateau by 18h after the injection. A net increase in the hepatic 65Zn content was observed subsequent to the decrease in the rate of zinc-thionein synthesis. The incorporation of both 65Zn and [35S]cystine into zinc-thionein was inhibited by prior administration of either actinomycin D or cordycepin. A second injection of Zn2+, 20h after the initial injection, yielded a 4.9-fold greater increase in zinc-thionein synthesis compared with that after only one injection; however, this synthesis was also inhibitable by actinomycin D. These data support the concept that hepatic zinc-thionein synthesis responds quickly to changes in Zn2+ status and that Zn2+ is bound subsequent to synthesis of nascent thionein chains. The mechanism of control of zinc-thionein synthesis by Zn2+ appears to involve changes in the amounts of a short-lived, poly(A)-containing RNA whose translation can be derepressed by additional exposure to Zn2+.
Comparison of the biliary excretion of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats
Authors: Blanckaert N , Heirwegh K P M , Zaman Z
Published: August 2015

The biliary excretion of the four isomers of bilirubin-IX was studied in Wistar rats (JJ) and homozygous Gunn rats (jj). Synthetic preparations of 14C-labelled pigments were used. 1. After intravenous administration, the alpha-isomer was rapidly excreted in conjugated form in bile of Wistar rats. In Gunn rats excretion was insignificant. In contrast, both rat species promptly excreted the non-alpha-isomers at rates that were comparable with that found for bilirubin-IXalpha in Wistar rats. 2. In normal rats about 16% of the beta- and delta-isomers and at least 50% of the gamma-isomer were excreted as ester conjugates of the injected parent bile pigments. Conjugation of the beta- and delta-isomers had occurred exclusively at the carboxyl groups of pyrrole ring D and C respectively. For bilirubin-IXgamma no preference for any carboxyl group could be established. 3. In homozygous Gunn rats the non-alpha-isomers were apparently excreted chemically unaltered. This suggests that, as for bilirubin-IXalpha, conjugation of the non-alpha-isomers is also deficient in Gunn rats.
Synthesis of cartilage-specific proteoglycan by suspension cultures of adult chondrocytes
Authors: Wiebkin O W , Muir H
Published: August 2015

Chondrocytes isolated from larynges of adult pigs were cultured as cell suspensions for at least 4 days before use. During continuous-labelling experiments in nutrient medium for 18h with 35SO42- and [3H]glucosamine as precursors, some macromolecular polyanionic material was synthesized which behaved on gel chromatography as proteoglycan. Gel chromatography on Sepharose 2B showed that a proportion of the proteoglycans in the medium appeared to be aggregated, and was dissociated in 4M-guanidinium chloride. Moreover the dissociated proteoglycan interacted with hyaluronic acid. Newly synthesized proteoglycan was larger than the average total cetylpyridinium chloride-precipitable material assayed as uronic acid alone.
Phosphatidylinositol-degrading enzymes in liver lysosomes
Authors: Irvine R F , Hemington N , Dawson R M C
Published: August 2015

The major pathway by which liver lysosomal enzymes degrade phosphatidylinositol is through an EDTA-insensitive formation of phosphorylinositol. This is in distinct contrast with the Ca2+-dependent production of phosphorylinositol from phosphatidylinositol, which is located in the cytosol. Lysosomal enzymes can also totally deacylate phosphatidylinositol, producing glycerophosphorylinositol.
Stimulation of calcium accumulation in cardiac sarcolemma by phosphorylase kinase
Authors: Sulakhe P V , St Louis P J
Published: August 2015

After incubation with phosphorylase kinase, calcium accumulation in cardiac sarcolemma was increased from 65+/-2 to 95+/-3nmol/10min per mg of protein. Under these conditions, phosphorylase kinase catalysed phosphorylation of membranes. This phosphorylation was hydroxylamine-insensitive, was stimulated by Ca2+ ions and was unaffected by 3':5'-cyclic AMP.
Some observations on the choice of detergent for solubilization of the human erythrocyte membrane
Authors: Grant D A W , Hjertén S
Published: August 2015

Solubilization of the human erythrocyte membrane by seven detergents is described. Components released into the supernatant or retained in the residue were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two non-ionic detergents exhibiting little u.v. absorption were more efficient than u.v.-absorbing Triton X-100. Evidence is presented of an interchange between protein PAS 1 and protein PAS 2.
Studies on the mechanism of hepatic microsomal N-oxide formation. The role of cytochrome P-450 and mixed-function amine oxidase in the N-oxidation of NN-dimethylaniline
Authors: Hlavica P , Kehl M
Published: August 2015

Evidence is established for the existence of alternative metabolic routes of N-oxidation of NN-dimethylaniline in rabbit liver microsomal fraction. One pathway involves the participation of two types of cytochrome P-450 with different sensitivities towards heat. Both types may represent distinct haemoprotein species or two physical forms of a single pigment. The other pathway is represented by the mixed-function amine oxidase. The enzyme lacks NADPH dehydrogenase activity and is insensitive to treatment with 2-bromo-4'-nitroacetophenone and steapsin: it catalyses N-oxidation of imipramine, trimethylamine and NN-dimethylaniline in molar proportions considerably different from those of the cytochrome P-450-supported reactions. Cytochrome P-450 is estimated to account for the formation of at least 50-60% of the total NN-dimethylaniline N-oxide formed in the intact rabbit liver microsomal fraction, the remainder arising from the action of the mixed-function amine oxidase.
Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions
Authors: Fraser Ian H. , Mookerjea Sailen
Published: August 2015

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn2+ that could not be replaced by Ca2+, Mg2+, Zn2+ or Co2+, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent Km for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.
Changes in surface-membrane components during the differentation of rabbit erythroid cells
Authors: Light N D , Tanner M J A
Published: August 2015

The membrane components of rabbit bone-marrow-bound erythroid cells were characterized and compared with those of circulating rabbit erythroid cells. By the criteria of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, radioiodination with lactoperoxidase and binding of radioiodinated lectins, the two circulating forms of erythroid cells (the reticulocyte and erythrocyte) have the same surface components. In contrast, bone-marrow-bound nucleated erythroid cells have a unique set of membrane surface components which are completely different from those found on circulating cells. Of the ten Coomassie-Blue-staining proteins present in nucleated erythroid-cell plasma-membrane preparations, eight are accessible at the extracellular surface, and all of these are lectin-binding glycoproteins. Bone-marrow erythroid cells separated according to age by velocity sedimentation were also studied. The changeover in surface components occurs after the last nucleated stage of the erythroid cells (the orthochromatic normoblast). We discuss the alterations in membrane surface components observed during the differentiation of the erythroid-cell series in relation to the transition from bone-marrow-bound to circulating forms of these cells. We suggest that the change in membrane surface components may be linked to the loss of the nucleus from the normoblast and the entry of the erythroid cell into the circulation.
The probable site of action of thenolytrifluoracetone on the respiratory chain
Authors: Ingledew W J , Ohnishi T
Published: August 2015

1. It is shown that the electron-transfer inhibitor thenoyltrifluoroacetone abolishes a respiratory-chain electron-paramagnetic-resonance absorbance due to spin-spin interactions of ubisemiquinones at concentrations similar to those required for inhibition of succinate oxidation. 2. A specific site of interaction of thenoyltrifluoroacetone with the respiratory chain is proposed to be on the ubisemiquinone with which succinate dehydrogenase reacts. 3. Our results further demonstrate the close association of the HiPIP (high-potential iron-sulphur) centre of succinate dehydrogenase with ubisemiquinone.
Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro
Authors: Marra E , Doonan S , Saccone C , Quagliariello E
Published: August 2015

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.
Identification and partial characterization of phospholipases in isolated adrenocortical cells. The effects of synacthen [corticotropin-(1–24)-tetracosapeptide] and calcium ions
Authors: Laychock S G , Franson R C , Weglicki W B , Rubin R P
Published: August 2015

Phospholipase A activity was determined in homogenates and subcellular fractions of trypsin-dispersed cat adrenocortical cells. At pH 7.4 homogenate phospholipid hydrolysis was activated by added Ca2+ and inhibited by EGTA. Phospholipid degradation in the presence and absence of Synacthen was completely blocked by EGTA. Ca2+-dependent activation of a membrane-bound phospholipase may be a critical control mechanism for regulating the molecular changes taking place during stimulation by Synacthen.
An automatic method for deriving steady-state rate equations
Authors: Cornish-Bowden A
Published: August 2015

A method is described for systematically deriving steady-state rate equations. It is based on the schematic method of King & Altman [J. Phys. Chem. (1956) 60, 1375-1378], but is expressed in purely algebraic terms. It is suitable for implementation as a computer program, and a program has been written in FORTRAN IV and deposited as Supplementary Publication SUP 50078 (12 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161, 1-2.