bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of synthesis, recycling and body mass of glucose in rats and rabbits in vivo with 3H- and 14C-labelled glucose
Authors: Katz Joseph , Dunn Arnold , Chenoweth Maymie , Golden Sybil
Published: August 2015

1. Glucose labelled with 3H in position 2 and uniformly with 14C was administered simultaneously to rabbits and rats either as a single injection or by continuous infusion. Plasma glucose specific radioactivity and the yield of 3H in the plasma water were monitored. 2. The rates of synthesis, recycling of carbon and total body mass of glucose were calculated, without assuming a multicompartmental model and without fitting data by exponential expressions. 3. The rate of synthesis of glucose in starved-overnight rabbits was 4mg/min per kg (range 3–4.5mg/min per kg) and 25–35% of the glucose carbon was recycled. The mass of total body glucose in starved rabbits was 290mg/kg (range 220–390mg/kg). About one-third of the total body glucose equilibrates nearly instantaneously with plasma glucose. 4. In rats starved overnight, glucose synthesis was about 10mg/min per kg and recycling of carbon ranged from 30–40%. Total body mass (per kg body weight) is similar to that in rabbits. 5. The activity in plasma water after injection of [2-3H]glucose was determined. The initial rate of 3H2O formation is rapid, indicating that the major site of glucose catabolism is in the rapidly mixing pool. The curve of total body glucose radioactivity was obtained from the 3H2O yield, and total mass of glucose was calculated. This agrees with that obtained from the 3H specific-radioactivity curve.
The action of local anaesthetics on lipolysis and on adenosine 3′:5′-cyclic monophosphate content in isolated rat fat-cells
Authors: Siddle Kenneth , Hales C. Nicholas
Published: August 2015

1. Local anaesthetics inhibited hormone-stimulated lipolysis in isolated rat fat-cells. The most potent anaesthetic was dibucaine, which inhibited adrenaline-stimulated lipolysis by 50% at a concentration of 0.16mm. 2. The amount of inhibition produced by a given concentration of anaesthetic was very similar with adrenaline, theophylline and dibutyryl cyclic AMP, at submaximal and maximal concentrations. 3. The inhibitory effect of dibucaine on lipolysis was apparent within 5 min and was constant over 1h. 4. Dibucaine inhibited basal, adrenaline-stimulated and insulin-stimulated glucose uptake at concentrations 6–10-fold higher than those inhibiting lipolysis. 5. The effects of dibucaine on lipolysis and glucose uptake were reversed after removal of anaesthetic and washing of cells. 6. Dibucaine further elevated the concentration of cyclic AMP in the presence of adrenaline or adrenaline plus theophylline. 7. Dibucaine had no effect on ATP content at concentrations causing 80% inhibition of lipolysis, but lowered ATP content at higher concentrations. 8. The relative potency of different local anaesthetics as inhibitors of hormone-stimulated lipolysis paralleled their potency as inhibitors of ion movements in other systems. 9. The possibility is discussed that Ca2+ions are involved in the regulation of lipolysis, and that local anaesthetics inhibit lipolysis by interfering with Ca2+translocation.
Hormonal regulation of adipose-tissue acetyl-coenzyme A carboxylase by changes in the polymeric state of the enzyme. The role of long-chain fatty acyl-coenzyme A thioesters and citrate
Authors: Halestrap Andrew P. , Denton Richard M.
Published: August 2015

1. Acetyl-CoA carboxylase activity was measured in extracts of rat epididymal fat-pads either on preparation of the extracts (initial activity) or after incubation of the extracts with citrate (total activity). In the presence of glucose or fructose, brief exposure of pads to insulin increased the initial activity of acetyl-CoA carboxylase; no increase occurred in the absence of substrate. Adrenaline in the presence of glucose and insulin decreased the initial activity. None of these treatments led to a substantial change in the total activity of acetyl-CoA carboxylase. A large decrease in the initial activity of acetyl-CoA carboxylase also occurred with fat-pads obtained from rats that had been starved for 36h although the total activity was little changed by this treatment. 2. Conditions of high-speed centrifugation were found which appear to permit the separation of the polymeric and protomeric forms of the enzyme in fat-pad extracts. After the exposure of the fat-pads to insulin (in the presence of glucose), the proportion of the enzyme in the polymeric form was increased, whereas exposure to adrenaline (in the presence of glucose and insulin) led to a decrease in enzyme activity. 3. These changes are consistent with a role of citrate (as activator) or fatty acyl-CoA thioesters (as inhibitors) in the regulation of the enzyme by insulin and adrenaline; no evidence that the effects of these hormones involve phosphorylation or dephosphorylation of the enzyme could be found. 4. Changes in the whole tissue concentration of citrate and fatty acyl-CoA thioesters were compared with changes in the initial activity of acetyl-CoA carboxylase under a variety of conditions of incubation. No correlation between the citrate concentration and the initial enzyme activity was evident under any condition studied. Except in fat-pads which were exposed to insulin there was little inverse correlation between the concentration in the tissue of fatty acyl-CoA thioesters and the initial activity of acetyl-CoA carboxylase. 5. It is suggested that changes in the concentration of free fatty acyl-CoA thioesters (which may not be reflected in whole tissue concentrations of these metabolites) may be important in the regulation of the activity of acetyl-CoA carboxylase. The possibility is discussed that the concentration of free fatty acyl-CoA thioesters may be controlled by binding to a specific protein with properties similar to albumin.
Synthesis of fatty acids in the perfused mouse liver
Authors: Salmon D. Michael W. , Bowen Neil L. , Hems Douglas A.
Published: August 2015

1. Fatty acid synthesis de novo was measured in the perfused liver of fed mice. 2. The total rate, measured by the incorporation into fatty acid of3H from3H2O (1–7μmol of fatty acid/h per g of fresh liver), resembled the rate found in the liver of intact mice. 3. Perfusions with l-[U-14C]lactic acid and [U-14C]glucose showed that circulating glucose at concentrations less than about 17mm was not a major carbon source for newly synthesized fatty acid, whereas lactate (10mm) markedly stimulated fatty acid synthesis, and contributed extensive carbon to lipogenesis. 4. The identification of 50% of the carbon converted into newly synthesized fatty acid lends further credibility to the use of3H2O to measure hepatic fatty acid synthesis. 5. The total rate of fatty acid synthesis, and the contribution of glucose carbon to lipogenesis, were directly proportional to the initial hepatic glycogen concentration. 6. The proportion of total newly synthesized lipid that was released into the perfusion medium was 12–16%. 7. The major products of lipogenesis were saturated fatty acids in triglyceride and phospholipid. 8. The rate of cholesterol synthesis, also measured with3H2O, expressed as acetyl residues consumed, was about one-fourth of the basal rate of fatty acid synthesis. 9. These results are discussed in terms of the carbon sources of hepatic newly synthesized fatty acids, and the effect of glucose, glycogen and lactate in stimulating lipogenesis, independently of their role as precursors.
Lipid and basic protein interaction in myelin
Authors: Banik Naren L. , Davison Alan N.
Published: August 2015

1. Purified myelin labelled with [3H]myo-inositol or [1-14C]acetate was incubated with trypsin or acetylated trypsin at 37°C, pH8.0 for 30min. 2. After incubation and centrifugation analysis of the myelin pellet showed marked digestion of basic protein on polyacrylamide-gel electrophoresis. Proteolipid and Wolfgram proteins remained unchanged. 3. A loss of 15% of total protein and loss of all classes of lipids was also found. Most significant lipid losses were phosphoinositides, phosphatidylserine and sulphatide. 4. A low-density material containing more phospholipid than cholesterol and galactolipid was isolated from the supernatant obtained after centrifugation of trypsin-treated myelin. 5. Interaction of sulphatide and myelin basic protein was shown to take place in a biphasic system. Basic protein does not form any complex either with cerebroside or cholesterol in the same solvent system. 6. The release of acidic lipids from myelin suggests that they may be linked to basic protein by ionic forces and the neutral lipids may be by lipid–lipid interactions. 7. The relevance of these studies as a model of brain degeneration is discussed.
The isolation of an azobilirubin β-d-monoglucoside from dog gall-bladder bile
Authors: Gordon Ellen R. , Dadoun Molly , Goresky Carl A. , Chan Tak-Hang , Perlin Arthur S.
Published: August 2015

An ethyl anthranilate azopigment of bilirubin conjugated to β-d-monoglucoside was isolated from dog gall-bladder bile. Glucose was cleaved from the azopigment by treatment with β-glucosidase and β-glucuronidase. Mild alkaline hydrolysis of the compound by sodium methoxide yielded two kinds of compounds, water-soluble and organic-soluble. The former were shown, by enzymic analysis, t.l.c., nuclear magnetic resonance, and combined g.l.c. and mass spectrometry, to contain glucose. No evidence was obtained from these data that a disaccharide was present in this fraction. The organic-soluble compounds formed during this methanolysis were shown, by t.l.c. and mass spectrometry, to be the isomeric dipyrrole azopigments of bilirubin. These findings contribute further evidence to the controversy surrounding the nature of conjugated bilirubin.
The amino acid sequence of plastocyanin from Cucurbita pepo L. (vegetable marrow)
Authors: Scawen Michael D. , Boulter Donald
Published: August 2015

The amino acid sequence of plastocyanin from marrow was determined. It consists of a single polypeptide chain of mol.wt. 10284 containing 99 amino acid residues. The sequence was determined by using a Beckman 890C automatic sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. The sequence is in good agreement with the amino acid composition, except that fewer residues of glutamic acid were found in the sequence than were suggested by the composition. Evidence for histidine-37 was weaker than for the rest of the sequence. A ‘tree’ of phylogenetic affinities was constructed by using several higher-plant plastocyanin sequences.
The release of iron from horse spleen ferritin by reduced flavins
Authors: Sirivech Somjai , Frieden Earl , Osaki Shigemasa
Published: August 2015

Ferritin-Fe(III) was rapidly and quantitatively reduced and liberated as Fe(II) by FMNH2, FADH2 and reduced riboflavin. Dithionite also released Fe(II) from ferritin but at less than 1% of the rate with FMNH2. Cysteine, glutathione and ascorbate gave a similar slower rate and yielded less than 20% of the total iron from ferritin within a few hours. The reduction of ferritin-Fe(III) by the three riboflavin compounds gave complex second-order kinetics with overlapping fast and slow reactions. The fast reaction appeared to be non-specific and may be due to a reduction of Fe(III) of a lower degree of polymerization, equilibrated with ferritin iron. The amount of this Fe3+ ion initially reduced was small, less than 0.3% of the total iron. Addition of FMN to the ferritin–dithionite system enhanced the reduction; this is due to the reduction of FMN by dithionite to form FMNH2 which then reduces ferritin-Fe(III). A comparison of the thermodynamic parameters of FMNH2–ferritin and dithionite–ferritin complex formation showed that FMNH2 required a lower activation energy and a negative entropy change, whereas dithionite required 50% more activation energy and showed a positive entropy change in ferritin reduction. The effectiveness of FMNH2 in ferritin–Fe(III) reduction may be due to a specific binding of the riboflavin moiety to the protein portion of the ferritin molecule.
The copolymeric structure of pig skin dermatan sulphate. Characterization of d-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate
Authors: Fransson Lars-Åke , Cöster Lars , Malmström Anders , Sjöberg Ingrid
Published: August 2015

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C4 fragment in the reducing terminal, ΔUA-GalNAc-(-SO4)-R; (b) monosulphated, unsaturated disaccharide, ΔUA-GalNAc-SO4 when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO4-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.
The reaction of phosphoglycolipids and other lipids with hydrofluoric acid
Authors: Shaw N. , Stead A.
Published: August 2015

1. The use of HF as a dephosphorylating reagent for phospholipids was examined. 2. Hydrolysis of phosphatidylethanolamine at 0°C for 24h with 60% HF gives a good yield of diglyceride. Under similar conditions phosphatidyldiglucosyl diglyceride gives diglyceride and diglucosyl diglyceride. 3. The glycolipid is also obtained from hydrolysis of glycerylphosphoryldiglucosyl diglyceride. No lyso derivative of the glycolipid could be detected and the glycosidic linkage was also stable. 4. Triglycerides, unsaturated and cyclopropane fatty acids were unaffected by the reagent. 5. 1,2-Diglycerides and 1,3-diglycerides were partially isomerized and also gave small amounts of free fatty acid and monoglyceride. 6. Monoglycerides underwent extensive rearrangement to form 1,2- and 1,3-diglycerides. 7. Lysophosphatidylethanolamine also gave 1,2- and 1,3-diglycerides as well as monoglycerides. 8. The application of this procedure to the structure determination of various phosphoglycolipids is discussed.
The hybridization of glyceraldehyde 3-phosphate dehydrogenases from rabbit muscle and yeast. Kinetics and thermodynamics of the reaction and isolation of the hybrid
Authors: Osborne Hillman H. , Hollaway Michael R.
Published: August 2015

A kinetic and thermodynamic study was made of the formation of the hybrid (R2Y2) glyceraldehyde 3-phosphate dehydrogenase from the yeast (Y4) and rabbit (R4) enzymes. The values of the thermodynamic parameters for the equilibrium between R4, Y4 and R2Y2 suggest that the R2–R2 and Y2–Y2 interactions are similar. However, the failure to observe the RY3 and R3Y hybrids is interpreted in terms of differences at the interfaces of the R–R and Y–Y interactions (the glyceraldehyde 3-phosphate dehydrogenase molecule being regarded as a dimer of dimers). The kinetics of formation of the R2Y2 hybrid were studied and a model was proposed to account for the results. Best-fit values for the rate constants of the individual steps were evaluated by computer simulation, and the rate-limiting steps were identified as the dissociation of tetramers to dimers. It is proposed that the cleavage plane for dissociation of the tetramers corresponds to the region of low electron density through the centre of the molecule in the X-ray-crystallographic structure for human glyceraldehyde 3-phosphate dehydrogenase (Watson et al., 1972), which is probably the plane containing the Q and R axes in the lobster enzyme (Buehner et al., 1974). The R2Y2 hybrid was isolated in milligram amounts by ion-exchange chromatography and its rate of reversion to the native enzyme was shown to be consistent with the kinetic model proposed from the hybrid-formation experiments.
Immunological evidence to show that the N-acetylglucosaminidase and N-acetylgalactosaminidase activities of Dictyostelium discoideum reside in the same protein molecule (Short Communication)
Authors: Every D. , Ashworth J. M.
Published: August 2015

Antibodies were produced against purified β-N-acetylhexosaminidase from Dictyostelium discoideum. Ouchterlony tests produced a single precipitin band with both β-N-acetylglucosaminidase and β-N-acetylgalactosaminidase activities. Both activities were co-precipitated in exactly the same proportions in quantitative immunoprecipitation reactions. We conclude that one enzyme protein catalyses the hydrolysis of both N-acetylglucosaminides and N-acetylgalactosaminides.
Effects on protein synthesis of injecting synthetic polyribonucleotides into living cells
Authors: Woodland Hugh , Ayers Sarah E.
Published: August 2015

Micro-injection into the oocytes and eggs of Xenopus laevis was used to ascertain the effects of synthetic polyribonucleotides on protein synthesis in living cells. Poly(U) and poly(A) were not translated detectably, nor did they change the rate of endogenous protein synthesis. The same was true of poly(G,U), poly(A,G,U), poly(A,C,G,U), G-U-G-(U)n, A-(U)n and AUG. In contrast, A-U-G-(U)n was a potent inhibitor of protein synthesis in the cell. This might be because it is initiated normally but lacks a termination codon, or because it inhibits the translation of other molecules in some way not dependent on its normal initiation. Poly(G,U), poly(A,G,U) and poly(A,C,G,U) inhibited haemoglobin synthesis when they were injected into the oocyte with haemoglobin mRNA. The synthetic polyribonucleotides did not inhibit the translation of the natural mRNA when the two sorts of molecules were injected at different times. It is suggested that the synthetic RNA molecules compete with the natural mRNA for a pre-initiation factor in limited supply.
Regulatory effects of polyamines on membrane-bound acetylcholinesterase
Authors: Kossorotow A. , Wolf H. U. , Seiler N.
Published: August 2015

The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte ‘ghosts’ and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na+, K+, Ca2+, Mg2+ and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed.
Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes
Authors: Bajwa S. S. , Sastry P. S.
Published: August 2015

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca2+had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of α- and β-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of α-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by β-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.
The relative stability of liver cytosol enzymes incubated in vitro
Authors: Hopgood M F , Ballard F J
Published: August 2015

1. Relative rates of enzyme inactivation were measured in liver slices, homogenates and cytosol fractions as well as in the presence of trypsin and at acid pH. The enzymes chosen are all present in the cytosol fraction of rat liver, and have widely different degradation rate constants in vivo. 2. The inactivation rates of lactate dehydrogenase, fructose bisphosphate aldolase, glucose 6-phosphate dehydrogenase, glucokinase, phosphoenolpyruvate carboxykinase (GTP), l-serine dehydratase and thymidine kinase in liver preparations at neutral pH are in a similar order to the rate constants of degradation of these enzymes in the intact animal. 3. The two exceptions of this general correlation were tyrosine aminotransferase, which was stable in vitro but not in vivo, and glyceraldehyde phosphate dehydrogenase, which shows the reverse pattern. 4. These findings generally support the concept that the same factors are responsible for enzyme inactivation in vitro as occur in the intact tissue.
Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platelets
Authors: Holmsen H , Setkowsky C A , Day H J
Published: August 2015

1. Human platelet-rich plasma prelabelled with [3H]adenine was incubated at 37°C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and Pi, and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [3H]ATP to [3H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP–hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [3H]ADP and [3H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [3H]ATP disappeared rapidly during the combined shape change–aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.
Fractionation of the proteins of plant microbodies
Authors: Brown R H , Lord J M , Merrett M J
Published: August 2015

1. Glyoxysomes and peroxisomes have been isolated from dark- and light-grown seedlings of pumpkin (Cucurbita pepo) by sucrose-density-gradient centrifugation. 2. Pumpkin microbodies and castor-bean (Ricinus communis) glyoxysomes may be fractionated, by a combination of osmotic shock and treatment with KCl, into three distinct groups of proteins: readily soluble (matrix enzymes), solubilized in the presence of KCl (membrane-bound enzymes) and relatively insoluble (membrane ‘ghost’ proteins). 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of ‘ghost’ fractions indicated that the membrane proteins were generally of low molecular weight; one gel band (mol.wt. 27000–28000) was common to all three microbodies. 4. Although there were major differences in the soluble protein components of pumpkin glyoxysomes and peroxisomes, electrophoresis of the pumpkin microbody ‘ghosts’ indicated that the membrane proteins were similar, four main components being common to each class of microbody (monomer molecular weights 42000, 34000, 27000 and 17000).
Early changes in the messenger ribonucleic acid concentration of amino acid-starved cells of Escherichia coli are not dependent on the state of the rel gene
Authors: Midgley John E. M. , Smith R. John
Published: August 2015

Measurements of the concentration of mRNA in rel+and rel-strains of Escherichia coli shortly after the imposition of amino acid deprivation indicate that there is a temporary fall in the amount of this fraction relative to the total cellular RNA.
Preparation of the lactate oxidase apoenzyme and studies on the binding of flavin mononucleotide to the apoenzyme
Authors: Choong Y S , Shepherd M G , Sullivan P A
Published: August 2015

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.
Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule
Authors: Jansson L , Ogren S , Lindahl U
Published: August 2015

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.
Molecular charcteristics of chicken kidney arginase
Authors: Traniello S , Barsacchi R , Magri E , Grazi E
Published: August 2015

Chicken kidney contains two arginases with different sedimentation coefficients and substrate specificity. The ligher of these arginases, which hydrolyses only L-arginine, has been purified about 3000-fold. Like the “ureotelic” arginase, developed in chicken liver after starvation, it displays many of the properties of the arginase of the “ureotelic” species. This seems to exclude the possibility that ureotelism and uricotelism are characterized by a specific type of arginases. Both liver and kidney arginases are located in the mitochondrial matrix. The rate of hydrolysis of arginine thus not only depends on the arginase activity but also on the rate of transport of arginine into the matrix. This last process therefore is of regulatory significance.
Differences in the protein fluorecence of the two iron(III)-binding sites of ovotransferrin
Authors: Evans R W , Holbrook J J
Published: August 2015

1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+
Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans
Authors: Jeffcoat R
Published: August 2015

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide “map”. 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.
Rat liver mitochondrial monoamine oxidase. A change in the reaction mechanism on solubilization
Authors: Houslay M D , Tipton K F
Published: August 2015

1. The kinetics of benzylamine oxidation by a soluble preparation of rat liver mitochondrial monoamine oxidase were investigated and were shown to conform to adouble-displacement (or Ping Pong) mechanism. 2. The pathway differs in detail from that followed by other amine oxidases, including the membrane-bound enzyme in rat liver mitochondrial outer membranes. 3. It is suggested taht the conformation of the protein in the soluble state differs from that in the membrane-bound state. 4. The full rate equations for this mechanism have been deposited as Supplementary Publication SUP 50039 (5pages) at the British Library (lending Division) (formely the National Lending Library for Science and Technology), Boston Spa, Yorks, LS237BQ, U.K.. from whom copies can be obtained on the terms indicated in Biochem. J (1975) 145,5.
The amino acid sequence of rabbit muscle triose phosphate isomerase
Authors: Corran P H , Waley S G
Published: August 2015

The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 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 can be obtained on the terms given in Biochem. J. (1975) 145, 5.
Deoxyribonucleic acid-dependent ribonucleic acid polymerases from normal and polyoma-transformed BHK-21/C13 cells
Authors: Cooper R J , Keir H M
Published: August 2015

DNA-dependent RNA polymerase (EC 2.7.7.6) ACTIVITIES FROM NORMAL BHK-21/C13 cells and from BHK-21/C13 cells transformed by polyoma virus (PYY cells) were solubilized and fractionated on columns of DEAE-Sephadex. Various properties of the A and B enzymes from the two types of cell were compared. 1. The yields of polymerase relative to the DNA content of the nuclear preparations are similar for both cell types. 2. The ionic-strength optima of polymerases A and B are 12.5 mM and 100mM with respect to (NH4)2SO4 for both cell types. 3. The Mn2+/Mg2+ activity ratio (measured at the respective optimum for each cation) for polymerase A from BHK-21/C13 cells was 1.48 and for the polymerase A from PYY cells was 0.55. The corresponding ratios for polymerase B were 10.11 for BHK-21/C13 cells and 22.75 for PYY cells. 4. Minor differences in the ability of the A polymerases to transcribe native and denatured DNA templates were observed; such differences were not apparent when the B polymerases were compared. 5. All the polymerases were inhibited completely by actinomycin D and by rifampicin AF/013, but not markedly so by rifampicin. Alpha-amanitin inhibited polymerase B but not polymerase A.
Interaction of cartilage proteoglycans with collagen-substituted agarose gels
Authors: Greenwald R A , Schwartz C E , Cantor J O
Published: August 2015

1. Rat tail-tendon collagen was coupled to activated Sepharose 4B at 2.5 mg of collagen/ml of gel. Chromatographic columns of this gel were calibrated with T2 virus (Vo) and Dnp-alanine (Vt). 2. The chromatographic behaviour of cartilage proteoglycans on the collagen-substituted gel was studied under conditions of varying ionic strength. Proteoglycan subunit obtained from bovine nasal cartilage, the proteoglycan obtained after digestion with chondroitnase ABC and purified chondriotin sulphate were all retarded on the collagen gel by an interaction that abolished at I0.17. Purified keratan sulphate and hyaluronic acid were not retarded. 3. A strong ionic interaction between cartilage proteoglycan and collagen was demonstrated to depend on the structure of the protein core of the proteoglycan.
Effects of γ-irradiation on biosynthesis of different types of ribonucleic acids in normal and regenerating rat liver
Authors: Markov G G , Dessev G N , Russev G C , Tsanev R G
Published: August 2015

1. The effect of γ-irradiation (4000rd) on the synthesis of ribosomal (pre-rRNA) and heterogeneous nuclear RNA (pre-mRNA) in normal and in regenerating rat liver was studied by using 40 min labelling with [6(-14)C]orotic acid. 2. Partial hepatectomy caused a sharp transient increase in the specific radioactivity of the endogenous low-molecular-weight RNA precursors in the livers of both normal and irradiated rats. Irradiation of intact animals did not affect the pool. 3. Irradiation enhanced the synthesis of pre-rRNA for at least 12h. The synthesis of pre-mRNA was also enhanced, but only in the first 3h after irradiation. 4. Partial hepatectomy strongly stimulated the synthesis of both pre-rRNA and pre-mRNA. 5. The synthesis of pre-rRNA was enhanced also in regenerating liver of animals irradiated before or after the operation. The conclusion can be drawn that the early increase in the synthesis of ribosomal RNA is a non-specific cellular response to different injuring factors. 6. The only case where irradiation caused an early inhibition of RNA synthesis was that of pre-mRNA in regenerating liver. This supports the hypothesis that ionizing radiation does not suppress the transcription per se but affects the mechanisms of activation of new genes (cellular programming).
Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.)
Authors: Suzuki T , Takahashi E
Published: August 2015

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.
Microbial metabolism of the pyridine ring. Metabolic pathways of pyridine biodegradation by soil bacteria
Authors: Watson G K , Cain R B
Published: August 2015

1. Two bacteria, a Bacillus sp. and a Nocardia sp. (strain Z1) were isolated from soil by enrichment with 0.1 percent (v/v) pyridine and grew rapidly on this compound as sole C, N and energy source. The monohydroxypyridines, tetrahydropyridine, piperidine and some other analogues were not utilized for growth or oxidized by washed suspensions of either bacterium. 2. Cell-free extracts were unable to metabolize pyridine even after supplementation with a variety of cofactors or protecting agents. Treatment of cells with toluene led to rapid loss of the ability to oxidize pyridine. 3. In the presence of 10mM-semicarbazide at pH 6.0, Nocardia Z1 accumulated a semialdehyde idenditied as its 2,4-dinitrophenylhydrazone by chromatography, mixed melting point, mass spectrometry and isotope trapping from [2,6(-14)C]pyridine as glutarate semialdehyde. 4. Extracts of this bacterium prepared from cells grown with pyridine or exposed to the gratuitous inducer 2-picoline, contained high activities of a specific glutarate semialdehyde dehydrogenase. 5. Cells grown with pyridine or glutarate also contained a glutaric dialdehyde dehydrogenase, an acyl-CoA synthetase and elevated amounts of isocitrate lyase but no glutaryl-CoA dehydrogenase. 6. Bacillus 4 accumulated in the presence of 10mM-semicarbazide several acidic carbonyl compounds from pyridine among which was succinate semialdehyde. Extracts of this bacillus after growth of the cells with pyridine contained an inducible succinate semialdehyde dehydrogenase in amounts at least 50-fold over those found in succinate-grown cells. 7. Two mutants of this bacillus, selected for their inability to grow on pyridine were deficient in succinate semialdehyde dehydrogenase. 8. In the presence of 0.2mM-KCN, washed suspensions of Bacillus 4 accumulated formate and possibly formamide from pyridine. The use of [14C]pyridine showed that formate was derived from C-2 of the pyridine ring. 9. The organism had a specific formamide amidohydrolase cleaving formamide quantitatively to formate and NH3. 10. Formate was further oxidized by the particle fraction. There was no soluble formate dehydrogenase in extracts.
Evidence against an involvement of cyclic nucleotides in the induction of βcyanin synthesis by cytokinins
Authors: Elliott D C , Murray A W
Published: August 2015

1. A wide range of purine bases, nucleosides and cyclic nucleotides were shown to induce βcyanin synthesis in Amaranthus seedlings. 2. The induction of pigment by benzyladenine, dibutyryl cyclic AMP or cyclic AMP was not potentiated by aminophylline. Aminophylline was shown to inhibit Amaranthus cyclic AMP phosphodiesterase activity. 4. Incubation of seedlings with aminophylline inhibited the conversion of 6-[G-3H]benzyladenine into presumed 9- and 7-glucosylbenzyladenine. 5. Induction of βcyanin synthesis by 6-benzyladenine or by exposure to red light was not accompanied by changes in the total cyclic AMP content in seedlings. 6. It is concluded that the inducers tested act as cytokinin analogues; no evidence was obtained to support cyclic AMP as an intermediate in the induction process.
The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro
Authors: Shires T K , McLaughlin C M , Pitot H C
Published: August 2015

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.
Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system
Authors: Wheeler K P , Walker J A , Barker D M
Published: August 2015

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.
Short Communications. A comparative study of some kinetic and spectral properties of guanidinated and native cytochrome c
Authors: Brittain T , Greenwood C
Published: August 2015

An investigation of the spectral and some kinetic properties of a chemically modified cytochrome c is presented. The kinetics of reduction by chromous ion and ascorbate are shown to be unchanged from that of the native molecule, as are the kinetics of NO binding. The effects of pH on the visible spectrum are discussed in terms of a possible change in the pattern of co-ordination of the molecule with changing pH.
The interaction between bovine serum albumin and surfactants
Authors: Jones M N , Skinner H A , Tipping E
Published: August 2015

1. Potassium n-decyl phosphate binds exothermically to bovine serum albumin at pH 7.0 to form a specific complex containing approx. 60 phosphate anions. 2. The formation of the complex is accompanied by changes in the u.v. difference spectrum of the protein. 3. At higher phosphate concentrations (above 0.4mM) surfactant molecules continue to be bound, and the protein undergoes a gross change in conformation. 4. n-Dodecyltri-methylammonium bromide binds endothermically to bovine serum albumin at pH7.0 but the extent of binding for a given free surfactant concentration is less than for the phosphate surfactant. 5. Binding is accompanied by a small change in the specific viscosity and by changes in the u.v. difference spectrum of the protein. 6. It is suggested that over the surfactant concentration ranges studied n-decyl phosphate ions first bind to the C-terminal part of the protein and then to the more compact N-terminal part whereas n-dodecyltrimethylammonium ions bind only to the C-terminal part of bovine serum albumin.