bluuebunny/crossref_metadata_2025 huggingface dataset:
The number of neurophysins in the rat. Influence of the concentration of Bromophenol Blue, used as a tracking dye, on the resolution of proteins by polyacrylamide-gel electrophoresis
Authors: Burford G. D. , Pickering B. T.
Published: August 2015

1. The concentration of Bromophenol Blue used as tracking dye in polyacrylamide-gel electrophoresis affected the resolution of rat neurophysins. 2. A final dye concentration of 1μg/ml in the tris–glycine running buffer was found to give the best results. 3. The presence of two major and one minor neurophysin(s) in the rat was confirmed. 4. The two major proteins were found to re-run as single discrete bands, which had the same mobilities in the absence of dye and different mobilities in its presence.
Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate
Authors: Kather H. , Rivera M. , Brand K.
Published: August 2015

By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.
Metabolism of [14C]methamphetamine in man, the guinea pig and the rat
Authors: Caldwell J. , Dring L. G. , Williams R. T.
Published: August 2015

1. The metabolites of (±)-2-methylamino-1-phenyl[1-14C]propane ([14C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the14C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of14C (45mg/kg) was excreted in the urine and 2–3% in the faeces in 3–4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the14C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.
A novel olefinic rearrangement. The enzymic conversion of cholesta-7,9-dien-3β-ol into cholesta-8,14-dien-3β-ol
Authors: Akhtar M. , Freeman C. W. , Rahimtula A. D. , Wilton D. C.
Published: August 2015

1. [3α-3H]Cholesta-7,9-dien-3β-ol is converted in high yield into cholesterol by a 10000gav. supernatant fraction of rat liver homogenate. 2. Incubation of cholesta-7,9-dien-3β-ol with [4-3H]NADPH and rat liver microsomal fractions under anaerobic conditions resulted in3H being incorporated into the 14α-position of cholest-7-en-3β-ol. 3. Under anaerobic conditions in the absence of NADPH cholesta-7,9-dien-3β-ol was isomerized into cholesta-8,14-dien-3β-ol by rat liver microsomal fractions.
Specificity and kinetics of triose phosphate isomerase from chicken muscle
Authors: Putman Sylvia J. , Coulson A. F. W. , Farley I. R. T. , Riddleston B. , Knowles J. R.
Published: August 2015

The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of kcat. and Km for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis–Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, kcat. is 2.56×105min-1and Km is 0.47mm; with dihydroxyacetone phosphate as substrate, kcat. is 2.59×104min-1and Km is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ‘virtual substrate’ monohydroxyacetone phosphate with solvent2H2O or3H2O was shown. This exchange is about 104-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.
Protein, nucleic acid and starch metabolism in the duckweed Spirodela oligorrhiza, treated with cytokinins
Authors: McCombs P. J. A. , Ralph R. K.
Published: August 2015

Bacteria-free cultures of Spirodela oligorrhiza continue to increase in frond number for 2 to 3 days after transfer to darkness. There is then no further increase in frond number for 3 to 4 weeks, although DNA, RNA and protein synthesis continue at decreased rates and starch accumulates in the plants. We refer to such ‘non-growing’ plants in darkness as dormant. Adding kinetin to dormant Spirodela initiated increased DNA, RNA and protein synthesis within 1h, although new fronds were not detected until 24h after the addition of kinetin. The frond number then continued to increase. Starch accumulated in dormant plants. Accumulation of starch appeared to be a consequence of inhibition of growth rather than the converse. No evidence was obtained for a block in [14C]glucose metabolism that might explain the lack of growth in darkness in the absence of kinetin. In darkness, more ribosomes were membrane-bound in dormant Spirodela than in Spirodela growing with kinetin. Similarities between the response of Spirodela to darkness, stringent control in bacteria and pleiotypic controls in animal cells are discussed. It is suggested that all three processes are ultimately controlled by specific protein kinases that are individually sensitive to different effectors.
Histone phosphatase and cyclic nucleotide-stimulated protein kinase from human lymphocytes
Authors: Murray A. W. , Froscio M. , Kemp B. E.
Published: August 2015

1. Extracts of human peripheral blood lymphocytes contained a histone phosphatase that catalysed the release of Pi from phosphorylated whole thymus histone. 2. Stimulation of the phosphatase was obtained by concentrations of KCl and NaCl of up to 75mm, and by MgCl2; CaCl2 inhibited the enzymic activity. 3. In the absence of MgCl2, phosphoenol-pyruvate inhibited histone phosphatase activity; this inhibition could be partially reversed by adding MgCl2 to assays. 4. Lymphocyte extracts contained a protein kinase activity which was maximally stimulated by 1μm-cyclic AMP (adenosine 3′:5′-cyclic monophosphate) or by 0.1mm-cyclic GMP (guanosine 3′:5′-cyclic monophosphate). 5. Incubation of the enzyme with histone in the absence of ATP or MgCl2 resulted in the dissociation of the enzyme into a lower-molecular-weight species that was not stimulated by cyclic AMP. This effect could be prevented if ATP and MgCl2 were present in reaction mixtures before histone and enzyme were allowed to interact. 6. Cyclic AMP also dissociated the kinase into a lower-molecular-weight species. 7. In the presence of 1μm-AMP, half-maximal activities were obtained with 0.92mm-MgCl2, 6.0μm-ATP and 0.23mg of whole thymus histone/ml.
Studies on the secondary structure of nuclear ribonucleic acids
Authors: Brentani M. , Kubota M. , Brentani R.
Published: August 2015

Rat liver nuclei were separated into two fractions, the nucleolus and the nucleoplasm, by a combined salt–enzymic extraction. RNA was purified from both sources and analysed on sucrose density gradients. In both fractions a prominent heterogeneous RNA class with mean sedimentation coefficient of 18S was found. This material was analysed by measuring the rate of reaction with formaldehyde, the ultraviolet absorbance–temperature profile, the spectrophotometric observation of conformational changes as a function of pH, the spectrophotometric titration of uracil and guanine residues and the effect of both temperature and ionic strength on the spectrophotometric titration of cytosine residues. Nucleoplasmic 18S RNA fraction exhibited, on heating and also by adjustment of the pH to 2.5, a hyperchromicity of about 16 %, close to that observed, in control experiments, for ribosomal RNA (22%). Titration of cytosine residues in solutions containing 1mm-NaCl and 0.1m-NaCl yielded pK values equal to 4.41 and 3.84 respectively. These results suggest that this RNA fraction is composed of structurally complex polymers. The hypochromicity of the nucleolar 18S RNA fraction determined by heating or adjusting the pH to 2.5, was not greater than 6% of the initial value. The rate of reaction with formaldehyde was 88% of that observed for the hydrolysed 18S fraction which suggested only 12% hydrogen bonding. pK values for uracil and guanine residues were 10.1 and 10.05 respectively. Titration of cytosine residues yielded a pK of 4.10, which was found to be independent of temperature and ionic strength variations.
Polyamine biogenesis in the rat mammary gland during pregnancy and lactation
Authors: Russell Diane H. , McVicker Thomas A.
Published: August 2015

Polyamines and RNA accumulate in the rat mammary gland during pregnancy, but the major increases occur after parturition. Therefore the major increases occur after the gland has obtained its maximal complement of epithelial cells. During lactation, the spermidine concentration rises above 5mm and RNA content in the lactating mammary gland reaches a value 16 times that of the unstimulated mammary gland. The ratio of spermidine/spermine, an increase of which initially signals an elevation in biosynthetic activity, is near 1 in the normal mammary gland and is greater than 10 in the lactating mammary gland. Putrescine concentration is very low during the entire course of mammary-gland development, with the exception of early pregnancy. The low putrescine concentration probably reflects the very rapid conversion of putrescine into spermidine. Both ornithine decarboxylase, the enzyme that synthesizes putrescine, and putrescine-stimulated S-adenosyl-l-methionine decarboxylase, the enzyme that synthesizes spermidine, increase in activity during middle and late pregnancy; during lactation, both enzyme activities are elevated until the 21st day of lactation, and then decline. These declines are concomitant with involution. Also, it was found that the amount of ribonuclease activity in the mammary gland was very high during lactation, almost double that in the gland during pregnancy.
Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2
Authors: Kitcher J. P. , Trudgill P. W. , Rees J. S.
Published: August 2015

1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH4)2SO4 fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Δ2-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27×106 and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5–9.5, a Km for 2-furoyl-CoA of 20.2μm and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.
Ribitol and flavinogenesis in Eremothecium ashbyii
Authors: Mehta Smriti U. , Mattoo A. K. , Modi V. V.
Published: August 2015

1. Supplementation of cultures of Eremothecium ashbyii with ribitol leads to a twofold increase in riboflavin formation compared with unsupplemented cultures or those supplemented with ribose or ribulose phosphate. Addition of unlabelled ribitol decreases the incorporation of [1-14C]ribose into riboflavin, indicating that free ribitol is preferred to ribose for incorporation into riboflavin. 2. The enzymes ribitol kinase, d-ribose reductase, d-ribose 5′-phosphatase and GMP nucleosidase were demonstrated in the cell-free extracts. Ribitol induces the formation of ribitol kinase. The enzyme is activated in vitro by the flavinogenic purines, guanine and xanthine. d-Ribose reductase shows a specific requirement for NADPH and forms free ribitol from ribose. 3. The activities of ribitol kinase, ribose 5′-phosphatase and GMP nucleosidase reach their maximal values before riboflavin formation reaches a maximum. 4. [U-14C]GMP is taken up intact by the culture of E. ashbyii and is incorporated into riboflavin as well as into a blue fluorescent compound. The radioactivity from this compound is incorporated into riboflavin by the cell-free extract of E. ashbyii.
A new bacterial sphingophospholipid containing 3-aminopropane-1,2-diol
Authors: Kemp P. , Dawson R. M. C. , Klein R. A.
Published: August 2015

1. A new sphingophospholipid has been isolated from the bacterial fraction of sheep rumen contents. 2. This new lipid has been characterized as a ceramide phosphate moiety esterified to 3-aminopropane-1,2-diol through the primary alcohol group. 3. Mass spectrometry has shown the intact lipid to contain a vicinal hydroxyl amino grouping, and oxidation with periodate converts it quantitatively into a new phospholipid which is probably ceramide phosphorylglycolaldehyde. 4. The sphingophospholipid contains a mixture of saturated long-chain bases mainly with branched-chain alkyl groups, which are typical of a microbial origin. 5. A Gram-negative bacterium isolated from rumen contents contains about 30% of the new lipid in its phospholipids when grown in pure culture.
Localization of d-myoinositol 1:2-cyclic phosphate 2-phosphohydrolase in rat kidney
Authors: Clarke N. , Dawson R. M. C.
Published: August 2015

1. On subcellular fractionation of rat kidney homogenates by differential and density-gradient centrifugation, the bulk of the inositol 1:2-cyclic phosphate 2-phosphohydrolase activity remains with the alkaline phosphatase activity, suggesting localization in the brush borders of the proximal tubules. 2. Histochemical studies with a medium containing inositol 1:2-cyclic phosphate and Escherichia coli phosphomonoesterase show Gomori staining around the brush borders of the proximal tubules in the outer cortex only. 3. Serial sections across the kidney from cortex perimeter to papilla suggest that the inositol 1:2-cyclic phosphate 2-phosphohydrolase has a limited distribution along the proximal tubule of the nephron, probably being limited to the pars convoluta, whereas the alkaline phosphatase extends along the pars recta.
A study of the localization of polynucleotide phosphorylase within rat liver cells and of its distribution among rat tissues and diverse animal species
Authors: See Y P , Fitt P. S.
Published: August 2015

1. Rat liver polynucleotide phosphorylase was localized in the mitochondrion, but may also occur in the nucleus. 2. The mitochondrial enzyme was found in rat heart, kidney, liver, muscle and spleen. 3. Mitochondrial polynucleotide phosphorylase is also present in calf, chicken, guinea-pig and rabbit liver and in goldfish muscle. 4. A possible physiological role for the enzyme in the control of the intramitochondrial ADP concentration is suggested.
Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle
Authors: Britton H. G. , Clarke J. B.
Published: August 2015

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.
Structural investigation of the carbohydrate element of human pituitary follicle-stimulating hormone by methylation analysis
Authors: Kennedy J. F. , Chaplin M. F.
Published: August 2015

A preparation of human follicle-stimulating hormone has been subjected to methylation analysis by using the methyl sulphinyl carbanion–dimethyl sulphoxide–methyl iodide method. The hydrolysis products of the methylated glycoprotein were reduced, acetylated, analysed by gas-phase chromatography and mass spectrometry and identified by comparison with standards. Methylation analysis demonstrated that (1) the d-galactose, mannose, fucose and 2-amino-2-deoxyglucose units exist in the pyranose forms, (2) the 2-amino-2-deoxyglucose units are N-acetylated, (3) the fucopyranose units occupy terminal non-reducing positions, (4) the d-galactopyranose units are linked in the 1- and 2-positions, (5) the mannopyranose units exist in three forms, some as terminal non-reducing residues, some as 1,6-linked residues and some as 1,3,4-linked branch points, and (6) the 2-acetamido deoxyglucopyranose units are 1,6-linked. These structural assignments are compared with other data previously obtained for the carbohydrate moieties of follicle-stimulating hormone.
The effect of cortisol on the synthesis of rat plasma albumin, fibrinogen and transferrin
Authors: Jeejeebhoy K. N. , Bruce-Robertson A. , Ho J. , Sodtke U.
Published: August 2015

A decrease of absolute synthesis of albumin, no change in that of fibrinogen and an increased fractional synthesis of transferrin were observed 3h after intraperitoneal administration of a pharmacological dose of 5 mg of cortisol to 220g rats in the post-absorptive state and previously kept on a diet with 40% protein. The concentration in liver of total free amino acids was practically unchanged at this time. Intraperitoneal administration of a mixture of amino acids with the cortisol raised this concentration and was accompanied by an almost complete de-repression of the synthesis of albumin, with no real effect on that of fibrinogen. In considerable contrast, in rats studied at 24h after intraperitoneal administration of cortisol, and who had been fed once in the interim (but who had received no amino acids intraperitoneally), there was a marked increase in the absolute synthesis of albumin and fibrinogen, with an increase in fractional synthesis that was less proportionately but still very significant and which included transferrin. The amino acid concentrations had risen above the supplemented values at 3h but not as much proportionately as the fractional synthesis rates, and of course not as much as the absolute synthesis rates, of albumin and fibrinogen. These time-dependent effects of cortisol suggest to us that our studies resolve the apparently conflicting results of the effect of cortisol on the synthesis of albumin reported by others.
Metabolism of glutamine and glutamic acid by isolated perfused kidneys of normal and acidotic rats
Authors: Hems D. A.
Published: August 2015

1. When isolated kidneys from fed rats were perfused with glutamine the rate of ammonia release at pH7.4 (110–360μmol/h per g dry wt.) was one to two times that of glutamine removal. Glucose formation from 5mm-glutamine was 16μmol/h per g. If kidneys were perfused with glutamine at pH7.1 (10–13mm-sodium bicarbonate) there was no increase in glutamine removal or in the formation of ammonia or glucose. 2. When isolated kidneys from fed rats were perfused with glutamate at pH7.4, glucose formation was 59μmol/h per g, glutamine formation was 182μmol/h per g and ammonia release was negligible. At pH7.1 glutamine synthesis was inhibited and formation of ammonia and glucose were increased. 3. In perfused kidneys from acidotic rats, which had received 1.5% (w/v) NH4Cl to drink for 7–10 days, gluconeogenesis from glutamine was enhanced (101μmol/h per g). Glutamine removal and ammonia formation were also increased, compared with the rates in perfused kidney from normal rats. The extra glutamine consumed was equivalent to the extra glucose formed. 4. When the kidney from the 7–10-day-acidotic rat was perfused with glutamate gluconeogenesis was increased (113μmol/h per g). Synthesis of glutamine was decreased, and ammonia release was approximately equal to the rate of glutamate removal. 5. The time-course of these metabolic alterations was investigated after the rapid induction of acidosis by infusion of 0.25m-HCl into the right side of the heart. The increase in gluconeogenesis from glutamine developed gradually over several hours. When kidneys from 6h-acidotic rats were perfused with glutamate, formation of glucose and glutamine were both rapid. 6. In acidotic rat kidneys perfused with glutamine, tissue concentrations of glutamate and glucose 6-phosphate were increased compared with those in control perfused kidneys from non-acidotic rats. 7. The results are discussed in terms of control of the renal metabolism of glutamine. In particular, it is suggested that in acidotic rats glucose formation is the major fate of the carbon of the extra glutamine utilized by the kidney, and that inhibition of glutamine synthetase could contribute to the increase in intracellular ammonia concentration in the kidney.
Mode of degradation of the chondroitin sulphate proteoglycan in rat costal cartilage
Authors: Wasteson Å. , Lindahl U. , Hallén A.
Published: August 2015

1. Chondroitin sulphate was isolated from different regions of rat costal cartilage after extensive proteolysis of the tissues. The molecular weight, determined by gel chromatography, of the polysaccharide obtained from an actively growing region (lateral zone) near the osteochondral junction was higher than that of the polysaccharide isolated from the remaining portion of the costal cartilage (medial zone). 2. In both types of cartilage the molecular weight of chondroitin sulphate, labelled with [35S]sulphate, remained unchanged in vivo over a period of 10 days, approximately corresponding to the half-life of the chondroitin sulphate proteoglycan. The molecular-weight distribution of chondroitin [35S]sulphate, labelled in vivo or in vitro, was invariably identical with that of the bulk polysaccharide from the same tissue. It is concluded that the observed regional variations in molecular-weight distribution were established at the time of polysaccharide biosynthesis. 3. In tissue culture more than half of the 35S-labelled polysaccharide–proteins of the two tissues was released into the medium within 10 days of incubation. The released materials were of smaller molecular size than were the corresponding native proteoglycans. In contrast, the molecular-weight distribution of the chondroitin [35S]sulphate (single polysaccharide chains) remained constant throughout the incubation period. 4. A portion (about 20%) of the total radioactive material released from 35S-labelled cartilage in tissue culture was identified as inorganic [35S]sulphate. No corresponding decrease in the degree of sulphation of the labelled polysaccharide could be detected. These findings suggest that a limited fraction of the proteoglycan molecules had been extensively desulphated. 5. It is suggested that the initial phase of degradation involves proteolytic cleavage of the proteoglycan, but the constituent polysaccharide chains remain intact. The partially degraded proteoglycan may be eliminated from the cartilage by diffusion into the circulatory system. An additional degradative process, which may occur intracellularly, includes desulphation of the polysaccharide, probably in conjunction with a more extensive breakdown of the polymer.
The development of the respiratory chain of Saccharomyces carlsbergensis during respiratory adaptation
Authors: Cartledge T. G. , Lloyd D. , Erecińska M. , Chance B.
Published: August 2015

1. Subcellular fractionation of sphaeroplasts produced at different stages during the first 4h of respiratory adaptation of anaerobically grown glucose-de-repressed Saccharomyces carlsbergensis gave mitochondrial fractions that contained all the detectable c- and a-type cytochromes. 2. The rates of cytochrome formation were studied; individual cytochromes were produced at different rates so as to give respiratory chains having widely differing cytochrome ratios. A CO-reacting haemoprotein other than cytochrome a3 also increased throughout 8h of respiratory adaptation. 3. Even after short periods of aeration, organisms contained mitochondria in which cytochrome–cytochrome interactions and the reaction of cytochrome a3 with O2 proceeded at rates almost as fast as in organelles from aerobically grown cells. 4. The technique of flow–flash photolysis enabled kinetic resolution of the reoxidation of cytochromes a3 and a to be achieved and their individual contributions to extinction changes in the Soret region were assessed. The ratio cytochrome a3/cytochrome a increased over the early stages of adaptation.
Regulation of the enzymes converting l-mandelate into benzoate in bacterium N.C.I.B. 8250
Authors: Livingstone A. , Fewson C. A.
Published: August 2015

l-Mandelate is oxidized to benzoate by the enzymes l-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I. Conditions have been established for measuring these three enzymes as well as benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase II and catechol 1,2-oxygenase in a single cell-free extract prepared from bacterium N.C.I.B. 8250. The kinetics of induction of all these enzymes have been measured under a variety of conditions. l-Mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I appear to be co-ordinately regulated because (a) their differential rates of synthesis are proportional to one another under various conditions of induction and repression, (b) they are specifically and gratuitously induced by thiophenoxyacetate and a number of other compounds, and (c) mutant strains have been isolated that lack all three enzymes. Phenylglyoxylate is the primary inducer of the regulon as mutant strains lacking phenylglyoxylate carboxy-lyase form the other two enzymes in the presence of l-mandelate or phenylglyoxylate, whereas in mutant strains devoid of l-mandelate dehydrogenase activity only phenylglyoxylate induces phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I.
Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus
Authors: Glasser S. R. , Chytil F. , Spelsberg T. C.
Published: August 2015

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.
Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase
Authors: Barnett J. E. G. , Rasheed A. , Corina D. L.
Published: August 2015

After removal of tightly bound NAD+ by using charcoal, a preparation of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-3H]NADH to give [5-3H]-glucitol 6-phosphate and [5-3H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [3H]myoinositol 1-phosphate in the presence of [4-3H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH4 in the presence of NAD+. Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD+. There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15×105.
Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration
Authors: Jameson G. W. , Roberts D. V. , Adams R. W. , Kyle W. S. A. , Elmore D. T.
Published: August 2015

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine α-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for α- and β-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for α-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02–3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p′-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N2-acetyl-N1-benzylcarbazate respectively. p-Nitrophenyl N2-acetyl-N1-(9-anthrylmethyl)carbazate has been synthesized; it did not react with α-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.
The nature of polydisperse ribonucleic acid in plants
Authors: Jackson M. , Ingle J.
Published: August 2015

The aggregation of ribosomal RNA species during chromatography on methylated albumin on kieselguhr was decreased from 50 to 15% by the lower salt concentrations made possible by the use of higher pH values. The polydisperse RNA was resolved into two fractions. About 50% was eluted with the rRNA whereas the remainder was bound to the column, and was recovered only by solubilization of the methylated albumin. Both fractions of polydisperse RNA were similar in size range, but the bound fraction was considerably richer in AMP. No D-RNA (DNA-like RNA) peak was resolved under these conditions of column fractionation. However, the properties of the bound RNA were consistent with it containing both D-RNA and TB-RNA (tenaciously bound RNA). The relationship between these two fractions of AMP-rich RNA was considered. The bound RNA and ribosomal RNA responded differently to various treatments. The salt concentration required to elute ribosomal RNA was halved by increasing the pH of the fractionation, but the amount of bound RNA was in fact increased. Denaturation by hot urea decreased the binding of ribosomal RNA to the methylated albumin, but did not facilitate elution of bound RNA. The high affinity between the AMP-rich polydisperse RNA and the methylated albumin does not therefore appear to arise for the secondary structure conferred by the high AMP content.
Stereoconfiguration of amino acids in peptides from a mycobacillin-synthesizing cell-free system (Short Communications)
Authors: Sengupta S. , Bose S. K.
Published: August 2015

The stereoconfiguration of amino acids, as determined by treatment with L-amino acid oxidase, d-amino acid oxidase and l-glutamate decarboxylase (containing l-aspartate decarboxylase activity), in the peptides from a mycobacillin-synthesizing cell-free system is identical with that of the growing mycobacillin peptide chaid if its synthesis starts from l-proline and is interrupted at various points by amino acid deprivation.
Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase
Authors: Holbrook J. John , Ingram V. Ann
Published: August 2015

1. Pig heart lactate dehydrogenase is inhibited by addition of one equivalent of diethyl pyrocarbonate. The inhibition is due to the acylation of a unique histidine residue which is 10-fold more reactive than free histidine. No other amino acid side chains are modified. 2. The carbethoxyhistidine residue slowly decomposes and the enzyme activity reappears. 3. The essential histidine residue is only slightly protected by the presence of NADH but is completely protected when substrate and substrate analogues bind to the enzyme–NADH complex. The protection is interpreted in terms of a model in which substrates can only bind to the enzyme in which the histidine residue is protonated and is thus not available for reaction with the acylating agent. 4. The apparent pKa of the histidine residue in the apoenzyme is 6.8±0.2. In the enzyme–NADH complex it is 6.7±0.2. 5. Acylated enzyme binds NADH with unchanged affinity. The enzyme is inhibited because substrates and substrate analogues cannot bind at the acylated histidine residue in the enzyme–NADH complex.
Intermediates in fatty acid oxidation
Authors: Stewart H. B. , Tubbs P. K. , Stanley K. K.
Published: August 2015

1. Aqueous extracts of acetone-dried liver and kidney mitochondria, supplemented with NAD+, CoA and phenazine methosulphate, efficiently convert fatty-acyl-CoA compounds into acetyl-CoA; the process was followed with an O2 electrode. 2. Label from [1-14C]octanoyl-CoA appears in acetyl-CoA more rapidly than that from [8-14C]octanoyl-CoA. 3. Oxidation of [8-14C]octanoyl-CoA was terminated by addition of neutral ethanolic hydroxylamine and the resulting hydroxamates were separated chromatographically. Hydroxamate derivatives of 3-hydroxyoctanoyl-, hexanoyl-, butyryl- and acetyl-CoA were obtained. 4. These and other observations suggest that oxidation of octanoyl-CoA by extracts involves participation of free intermediates rather than uninterrupted complete degradation of individual molecules to acetyl-CoA by a multienzyme complex. 5. Intact liver mitochondria studied by the hydroxamate technique were also shown to form intermediates during oxidation of labelled octanoates. In addition to octanoylhydroxamate, [8-14C]octanoate gave rise to small amounts of hexanoyl-, butyryl- and 3-hydroxyoctanoyl-hydroxamate. In contrast with extracts, however, where the quantity of intermediates found was a significant fraction of the precursors, mitochondria oxidizing octanoate contained much larger quantities of octanoyl-CoA than of any other intermediate.
The function of teichoic acids in cation control in bacterial membranes
Authors: Hughes A. H. , Hancock I. C. , Baddiley J.
Published: August 2015

1. The effects of teichoic acids on the Mg2+-requirement of some membrane-bound enzymes in cell preparations from Bacillus licheniformis A.T.C.C. 9945 were examined. 2. The biosynthesis of the wall polymers poly(glycerol phosphate glucose) and poly(glycerol phosphate) by membrane-bound enzymes is strongly dependent on Mg2+, showing maximum activity at 10–15mm-Mg2+. 3. When the membrane is in close contact with the cell wall and membrane teichoic acid, the enzyme systems are insensitive to added Mg2+. The membrane appears to interact preferentially with the constant concentration of Mg2+ that is bound to the phosphate groups of teichoic acid in the wall and on the membrane. When the wall is removed by the action of lysozyme the enzymes again become dependent on an external supply of Mg2+. 4. A membrane preparation that retained its membrane teichoic acid was still dependent on Mg2+ in solution, but the dependence was damped so that the enzymes exhibited near-maximal activity over a much greater range of concentrations of added Mg2+; this preparation contained Mg2+ bound to the membrane teichoic acid. The behaviour of this preparation could be reproduced by binding membrane teichoic acid to membranes in the presence of Mg2+. Addition of membrane teichoic acid to reaction mixtures also had a damping effect on the Mg2+ requirement of the enzymes, since the added polymer interacted rapidly with the membrane. 5. Other phosphate polymers behaved in a qualitatively similar way to membrane teichoic acid on addition to reaction mixtures. 6. It is concluded that in whole cells the ordered array of anionic wall and membrane teichoic acids provides a constant reservoir of bound bivalent cations with which the membrane preferentially interacts. The membrane teichoic acid is the component of the system which mediates the interaction of bound cations with the membrane. The anionic polymers in the wall scavenge cations from the medium and maintain a constant environment for the membrane teichoic acid. Thus a function of wall and membrane teichoic acids is to maintain the correct ionic environment for cation-dependent membrane systems.
Interrelationship of islet metabolism, adenosine triphosphate content and insulin release
Authors: Ashcroft Stephen J. H. , Weerasinghe L. Chatra C. , Randle Philip J.
Published: August 2015

The oxidation of some exogenous substrates and their effects on ATP content and insulin release in mouse pancreatic islets were measured. The ATP concentration of islets incubated without exogenous substrate shows a gradual decrease, which can be prevented by glucose or mannose (20mm) or leucine (2.5mm); d-glyceraldehyde (5mm) is as effective as glucose (5mm); fructose or N-acetylglucosamine (20mm), pyruvate (10mm) and dl-3-hydroxybutyrate (2mm) are less effective; galactose (20mm), acetate (10mm), octanoate (2mm) and succinate (10mm) have no ATP-maintaining ability. Islets oxidize glucose, mannose, glyceraldehyde, leucine and, less readily, N-acetylglucosamine and glucosamine; galactose, however, is poorly metabolized. Mannoheptulose inhibits the oxidation of glucose but not of glyceraldehyde. Insulin release, measured over a 2h incubation, is stimulated by glucose, mannose, leucine, glyceraldehyde or glucosamine but not by fructose or N-acetylglucosamine. The latter, however, potentiates the effects of glucose or glyceraldehyde (5mm) or leucine (2.5mm) on release; the potentiating effects are inhibited by mannoheptulose, which also blocks glucose-, but not glyceraldehyde- or leucine-stimulated release. In the presence of glucose (20mm), metabolic inhibitors depress insulin release and islet ATP content in parallel. However, rates of insulin release and ATP content measured after incubation with various combinations of exogenous substrates do not appear to be correlated. Sulphonylureas stimulate insulin release but decrease islet ATP concentrations. These results provide further evidence of a close association between the metabolic activity of exogenous substrates and their ability to initiate insulin release. Glucoreceptor models are formulated in the light of these observations and discussed.
Malate exchange between the cytosol and mitochondria
Authors: Rognstad Robert , Katz Joseph
Published: August 2015

1. By comparing the relative isotopic yields in glucose and CO2 from precursors of mitochondrial and cytosolic malate, it is evident that the rate of isotopic exchange between these compartments is rapid. 2. A variety of potential inhibitors of malate exchange were tested, but no specific and effective inhibitor of the isotopic exchange has been found. 3. Compounds such as n-butylmalonate and p-iodobenzylmalonate, which have been used as inhibitors of the malate–phosphate transport system in isolated mitochondria, do not appear to be sufficiently specific to be useful in studies with intact cells.
Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome
Authors: Germinario Ralph J. , Kahlenberg Arthur , Pinsky Leonard
Published: August 2015

1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na235SO4 and d-[2-3H]glucose, were used and their intracellular fates during uptake and `chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [35S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [3H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased 35S/3H ratio (nmol of [35S]sulphate incorporated/nmol of [3H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of `chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [3H]glucose. 3. After 34h exposure to a `corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [35S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of `corrected' Hurler-syndrome cells.
Inhibition of spermidine formation in rat liver and kidney by methylglyoxal bis(guanylhydrazone)
Authors: Pegg A. E.
Published: August 2015

The effect of methylglyoxal bis(guanylhydrazone), a substance known to inhibit putrescine-dependent S-adenosyl-l-methionine decarboxylase, on polyamine metabolism in liver and kidney was investigated. Almost complete inhibition of the incorporation of putrescine into spermidine was obtained up to 8h after administration of 80mg of methylglyoxal bis(guanylhydrazone)/kg body wt. by intraperitoneal injection. However, by 20h after administration of the inhibitor spermidine synthesis was resumed. Considerable accumulation of putrescine occurred during this period (up to 3 times control concentrations in both tissues), but there was only a slight fall in the spermidine content. These results suggest that the putrescine-activated S-adenosyl-l-methionine decarboxylase plays an essential role in spermidine biosynthesis in rat liver and kidney, and the possibility of using methylglyoxal bis(guanylhydrazone) to study the role of polyamine synthesis in growth is discussed.