bluuebunny/crossref_metadata_2025 huggingface dataset:
The influx of calcium ions into human erythrocytes during cold storage
Authors: Long Cyril , Mouat Barbara
Published: August 2015

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl2, are stored at 3°C, Ca2+ influx into the cells occurs. Simultaneously, efflux of K+, Na+, Cl− and water takes place and cell volume diminishes. 2. The extent of Ca2+ influx increases with duration of cold storage and with increasing concentration of Ca2+ in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca2+ exhibit Ca2+ efflux against a concentration gradient when subsequently incubated at 37°C. 4. Ca2+ influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca2+ influx during cold storage; however, in iso-osmotic choline chloride, Ca2+ influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37°C causes rapid efflux of K+ and Na+ and renders the cell membranes highly permeable to Ca2+ during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37°C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca2+. 8. The experimental results lead to the conclusion that the main prerequisite for Ca2+ influx into erythrocytes is the partial depletion of the cells of their univalent cations.
Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen
Authors: Chain Ernst B. , Sender Peter M.
Published: August 2015

In the absence of glucose, insulin stimulated the incorporation of 14C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on 14C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.
Concentrations of asparagine in tissues of prepubertal rats after enzymic or dietary depletion of asparagine (Short Communication)
Authors: Frankel David L. , Wells Herbert , Fillios Louis Charles
Published: August 2015

Growth of weanling rats was significantly depressed after 8 days of asparagine depletion produced by dietary means or by asparaginase treatment. Moreover, the concentration of free asparagine was significantly lowered in forebrain, skeletal muscle, liver, kidney, spleen and small intestines 3 h after an asparaginase injection, but remained lowered only in forebrain and skeletal muscle after 8 days of enzymic or dietary depletion of asparagine.
Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase
Authors: Cox G. B. , Gibson F. , McCann L. M. , Butlin J. D. , Crane F. L.
Published: August 2015

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg2+,Ca2+-stimulated adenosine triphosphatase. 2. The Mg2+,Ca2+-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg2+,Ca2+-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg2+,Ca2+-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg2+,Ca2+-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA-) could not be re-activated by the purified Mg2+,Ca2+-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg2+,Ca2+-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA-).
Iodoacetamide-induced sensitization of the pancreatic β-cells to glucose stimulation
Authors: Hellman Bo , Idahl Lars-Åke , Lernmark Åke , Sehlin Janove , Täljedal Inge-Bert
Published: August 2015

At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003–0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the Vmax. of glucose-stimulated insulin release without altering the apparent Km for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the β-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.
The effect of methanol and dioxan on the rates of the β-galactosidase-catalysed hydrolyses of some β-d-galactopyranosides: rate-limiting degalactosylation. The pH-dependence of galactosylation and degalactosylation
Authors: Sinnott Michael L. , Viratelle Odile M.
Published: August 2015

1. The effect of methanol on the β-galactosidase-catalysed hydrolysis of some nitrophenyl β-d-galactopyranosides has been studied under steady-state conditions. 2. The initial fractional rate of increase of kcat. as a function of methanol concentration with 2,4- and 3,5-dinitrophenyl β-d-galactopyranosides, but not with the other substrates studied, indicated that degalactosylation of the enzyme was rate-limiting. 3. The decrease in kcat. at high methanol concentrations for these substrates is considered to arise from causes other than galactosylation becoming rate-limiting. 4. Both galactosylation and degalactosylation of the enzyme require protonation of a group of pKa approx. 9.
d-3-Hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetics of radioisotope redistribution at chemical equilibrium catalysed by the enzyme in solution
Authors: Preuveneers M. J. , Peacock D. , Crook E. M. , Clark J. B. , Brocklehurst K.
Published: August 2015

1. The reversible NAD+-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0°C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate–NAD+ oxidoreductase, EC 1.1.1.30), was studied kinetically at chemical equilibrium by monitoring radioisotope redistribution with sodium dl-hydroxy[3-14C]butyrate and [4-3H]NAD+(labelled in the nicotinamide ring). 2. When all substrates are maintained at concentrations approaching saturation (approx. 3–50 times the Km values) the first-order rate constant for the enzyme-catalysed interconversion of NAD+ and NADH is much smaller than that for the enzyme-catalysed interconversion of d-3-hydroxybutyrate and acetoacetate. 3. The rate of interconversion of NAD+ and NADH increases initially with increasing concentrations of d-3-hydroxybutyrate and acetoacetate (ratio of concentrations maintained constant), passes through a maximum and approaches closely to zero at saturating concentrations of the latter substrates. 4. The rates of interconversion of NAD+ and NADH and of d-3-hydroxybutyrate and acetoacetate increase with increasing concentration of NAD+ (up to 66 times its Km value) and NADH (up to 180 times its Km value) (ratio of the concentrations of the nicotinamide nucleotides maintained constant). 5. These findings support the description of this catalysis as an ordered Bi Bi mechanism with no detectable alternative pathway, in which the interconversion of the central ternary complexes is not rate-limiting, and provide no evidence for the formation of dead-end complexes. 6. The solubility of 2,4-dinitrophenylhydrazine in HCl exhibits an acidity optimum, the maximum solubility at 25.0°C (3.8mg/ml, 19mm) occurring at 2.29m-HCl; in solutions of this acidity acetone 2,4-dinitrophenylhydrazone is relatively insoluble (0.098mg/ml, 0.413mm).
A procedure for the rapid purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase (Short Communication)
Authors: Humphries Peter , McConnell David J. , Gordon Robert L.
Published: August 2015

A rapid procedure involving DNA–cellulose chromatography followed either by sedimentation in a high-salt glycerol gradient or by gel filtration is described for the complete purification of Escherichia coli DNA-dependent RNA polymerase.
Kinetics of the reaction between oxygen and haemoglobin bound to haptoglobin (Short Communication)
Authors: Chiancone Emilia , Antonini Eraldo , Brunori Maurizio , Alfsen Annette , Lavialle Françoise
Published: August 2015

The kinetics of the reaction between O2 and haemoglobin bound to haptoglobin 1–1 were investigated by the stopped-flow and temperature-jump techniques. The reaction, which follows second-order kinetics in the lower concentration range, becomes independent of O2 concentration above about 150μm-O2.
A collagenase in extracts of the invertebrate Bipalium kewense
Authors: Phillips J. , Dresden M. H.
Published: August 2015

The localization, isolation and partial characterization of a collagenolytic enzyme from the land planarian Bipalium kewense is described. This enzyme can be obtained by direct extraction of the organism, and can be separated from non-collagenous proteolytic activity by (NH4)2SO4 precipitation and Sephadex-gel chromatography. Its mode of attack on collagen and sensitivity to a variety of inhibitors indicate that this enzyme differs from vertebrate collagenases and a previously described invertebrate collagenase.
The reaction of bilirubin with diazomethane
Authors: Kuenzle C. C. , Weibel M. H. , Pelloni R. R.
Published: August 2015

Dimethoxybilirubin dimethyl ester and monomethoxybilirubin dimethyl ester were prepared by treating bilirubin with diazomethane, and the correctness of the assigned structures was proved by elemental analysis as well as by i.r. and n.m.r. spectroscopy. The phenylazo compounds derived from monomethoxybilirubin dimethyl ester were also prepared and characterized spectroscopically. Dimethoxybilirubin dimethyl ester occurs in solution as a single molecular species, unlike bilirubin dimethyl ester, which in non-polar solvents exists as an equilibrium mixture of conformational isomers. This difference in the behaviour of the two compounds is explained by the absence of intramolecular hydrogen bonds in dimethoxybilirubin dimethyl ester, a situation that allows free rotation about the central methylene bridge, whereas in bilirubin dimethyl ester an internally hydrogen-bonded conformation can be distinguished by n.m.r. spectroscopy from a non-bonded family of rotamers. This finding is regarded as additional evidence for a newly conceived conformational structure of bilirubin and bilirubin dimethyl ester that is maximally stabilized by intramolecular hydrogen bonds. This is discussed in detail in the Appendix (Kuenzle et al., 1973), which also includes a description of the molecular mechanism pertaining to the reaction of bilirubin with diazomethane.
Uridine kinase in embryonic rat liver. Modulation of enzyme activity by 5-azacytidine
Authors: Veselý J. , Čihák A.
Published: August 2015

Uridine kinase activity in rat liver decreases during embryonic and postnatal development. Administration of 5-azacytidine enhances liver uridine kinase activity in adult rats, but depresses it in embryos. The liver enzymes from the foetus and the adult are precipitated at different (NH4)2SO4 concentrations although they are eluted at about the same position on chromatography on a column of Sepharose 6B.
Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis
Authors: Davis J. S. , Balinsky J. B. , Harington J. S. , Shepherd J. B.
Published: August 2015

1. An improved radioassay for glutathione synthetase and γ-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver γ-glutamylcysteine synthetase was purified 324-fold by saline–bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver γ-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver γ-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl–enzyme as intermediate before the addition of cysteine and the release of γ-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.
Purification and properties of arginase of rat kidney
Authors: Kaysen George A. , Strecker Harold J.
Published: August 2015

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn2+. Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.
Microbial metabolism of amino alcohols. 1-Aminopropan-2-ol and ethanolamine metabolism via propionaldehyde and acetaldehyde in a species of Pseudomonas
Authors: Jones A. , Turner J. M.
Published: August 2015

1. Growth and manometric experiments showed that a Pseudomonas sp. P6 (N.C.I.B. 10431), formerly known as Achromobacter sp. P6, was capable of growth on both stereoisomers of 1-aminopropan-2-ol, and supported the hypothesis that assimilation involved metabolism to propionaldehyde, propionate and possibly 2-hydroxyglutarate. A number of alternative intermediary metabolites were ruled out. 2. Accumulation of propionaldehyde from 1-aminopropan-2-ol by intact cells occurred only during active growth, was transitory and was accompanied by morphological changes in the pseudomonad. 3. Enzymic and radioactive tracer evidence showed that 1-aminopropan-2-ol O-phosphate was the intermediate between amino alcohol and aldehyde. The operation of an inducibly formed ATP–amino alcohol phosphotransferase was established by measuring substrate disappearance, ADP formation and amino alcohol O-phosphate formation. This novel kinase had two activity peaks at about pH7 and 9. It acted on both l- and d-isomers of 1-aminopropan-2-ol, and also on l-threonine and ethanolamine, but had only low activity towards choline. The enzyme was partially purified by ion-exchange chromatography. 4. An amino alcohol O-phosphate phospho-lyase (deaminating) produced propionaldehyde from dl- and d-1-aminopropan-2-ol O-phosphate, and also formed acetaldehyde less rapidly from ethanolamine O-phosphate. It had optimum activity at about pH8 in Tris–HCl buffers. The enzyme was partially purified and evidence was obtained that a single enzyme was responsible for both activities. Apparent Km values for the substrates were determined. Activity was inhibited by dl-threonine O-phosphate, dl-serine O-phosphate, choline O-phosphate and Pi. Enzyme formation was induced by growth with either amino alcohol substrate. 5. Radioactive tracer experiments with dl-1-amino[3-14C]propan-2-ol confirmed the operation of the amino alcohol kinase and demonstrated coupling with the phospho-lyase enzyme in vitro to produce [14C]-propionaldehyde. 6. An aldehyde dehydrogenase, found in extracts of the pseudomonad after growth on 1-aminopropan-2-ol, was characterized and concluded to be responsible for propionaldehyde and acetaldehyde oxidation. The enzyme was inactive with methylglyoxal. 7. Propionate and acetate were concluded to be metabolized via propionyl-CoA and acetyl-CoA, and studies were made of a CoA ester synthase found in extracts. 8. Studies of a strain of Pseudomonas putida N.C.I.B. 10558 suggested that 1-aminopropan-2-ols were metabolized via their O-phosphates, propionaldehyde and propionate. Amino alcohol kinase activity was detected and extracts contained a phospho-lyase showing higher activity with the 1-aminopropan-2-ol O-phosphate than with ethanolamine O-phosphate.
Effect of administration of the fructose on the glycogenolytic action of glucagon. An investigation of the pathogeny of hereditary fructose intolerance
Authors: Berghe G. Van Den , Hue L. , Hers H. G.
Published: August 2015

1. The mechanism by which the administration of fructose to patients with hereditary fructose intolerance makes them unresponsive to the hyperglycaemic action of glucagon was studied. In four patients, a 10-fold increase in the urinary excretion of cyclic AMP was induced by glucagon, but this effect was drastically decreased by the previous administration of fructose (250mg/kg). Further, the intravenous injection of 6-N,2′-O-dibutyryl cyclic AMP did not cause an increase in the blood glucose during fructose-induced hypoglycaemia. 2. The administration of a large dose of fructose (5g/kg) to mice decreased markedly both the concentration of ATP and the increase in the concentration of cyclic AMP caused by glucagon in the liver. Other ATP-depleting agents had a similar effect and a linear correlation could be drawn between the concentration of ATP and the change in cyclic AMP concentration; a half-maximal effect was obtained for a concentration of ATP close to the Km value of adenylate cyclase. 3. The administration of fructose to mice caused the inactivation of phosphorylase in the liver, but this effect was easily reversed by glucagon. 4. At a concentration of 10mm-fructose 1-phosphate and 1.5mm-Pi, purified liver phosphorylase a was inhibited by 70%. This inhibition appears to be a likely explanation for the unresponsiveness to glucagon of patients with hereditary fructose intolerance.
The uptake of [45Ca]calcium ions by matrix vesicles isolated from calcifying cartilage (Short Communication)
Authors: Ali S. Y. , Evans Lois
Published: August 2015

Extracellular membranous matrix vesicles, which contain various phosphatases and appear to initiate hydroxyapatite formation in growth cartilage, were isolated and incubated with 45Ca2+ and shown to form mineral in the presence of ATP. There is enhanced calcification in the presence of serum and under alkaline conditions.
Some aspects of carnitine metabolism in avocado (Persea americana) (Short Communication)
Authors: Panter R. A. , Mudd J. B.
Published: August 2015

The fact that palmitoyl-l-carnitine is oxidized by avocado mitochondria at a rate comparable with that of succinate oxidation suggests that there are at least two systems for β-oxidation in higher plants. The carnitine-associated system is located in a mitochondrial fraction, whereas the glyoxylate-cycle-associated system is located in the glyoxysomes.
The transport of inorganic phosphate by the mitochondrial dicarboxylate carrier
Authors: Johnson R. N. , Chappell J. B.
Published: August 2015

1. N-Ethylmaleimide inhibited the influx and efflux of Pi in rat liver mitochondria. 2. The efflux was stimulated by either succinate or malate in the presence of N-ethylmaleimide, and this stimulation was reversed by 2-n-butylmalonate. 2-Oxoglutarate and citrate, even in the presence of low concentrations of malate, were relatively ineffective in stimulating efflux of Pi under these conditions, as was glutamate. 3. By using radioactively labelled Pi and dicarboxylate ions an exchange was demonstrated, the stoicheiometry of which was 1.3±0.5 dicarboxylate ions:1 Pi (n=10). 4. An exchange between unlabelled and labelled Pi in the presence of N-ethylmaleimide was found which was sensitive to 2-n-butylmalonate. 5. It is concluded that the mitochondrial dicarboxylate carrier can transport phosphate by an exchange diffusion with certain penetrant dicarboxylic acids or with phosphate itself. The exchange mechanism is sensitive to 2-n-butylmalonate but is unaffected by N-ethylmaleimide; the action of mersalyl in this context is commented on.
Sialyltransferase: regulation of α-foetoprotein microheterogeneity during development (Short Communication)
Authors: Zimmerman Ernest F. , Madappally Mathew M.
Published: August 2015

The temporal accumulation of the electrophoretic components of mouse α-foetoprotein in foetal plasma and amniotic fluid is reported. To explain the progressive appearance of the sialylated α-foetoproteins, the activity of sialyltransferase in foetal liver and yolk sac was measured. These results indicate that the increase in sialyltransferase activity in these tissues is responsible for the increased sialylation of α-foetoprotein.
Binding of parathyroid hormone to bovine kidney-cortex plasma membranes
Authors: Sutcliffe H. S. , Martin T. J. , Eisman J. A. , Pilczyk R.
Published: August 2015

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with 125I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. 75Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca2+ inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.
Microbial metabolism of amino alcohols. Metabolism of ethanolamine and 1-aminopropan-2-ol in species of Erwinia and the roles of amino alcohol kinase and amino alcohol O-phosphate phospho-lyase in aldehyde formation
Authors: Jones Alan , Faulkner Anne , Turner John M.
Published: August 2015

1. Growth of Erwinia carotovora N.C.P.P.B. 1280 on media containing 1-aminopropan-2-ol compounds or ethanolamine as the sole N source resulted in the excretion of propionaldehyde or acetaldehyde respectively. The inclusion of (NH4)2SO4 in media prevented aldehyde formation. 2. Growth, microrespirometric and enzymic evidence implicated amino alcohol O-phosphates as aldehyde precursors. An inducibly formed ATP–amino alcohol phosphotransferase was partially purified and found to be markedly stimulated by ADP, unaffected by NH4+ ions and more active with ethanolamine than with 1-aminopropan-2-ol compounds. Amino alcohol O-phosphates were deaminated by an inducible phospho-lyase to give the corresponding aldehydes. This enzyme, separated from the kinase during purification, was more active with ethanolamine O-phosphate than with 1-aminopropan-2-ol O-phosphates. Activity of the phospho-lyase was unaffected by a number of possible effectors, including NH4+ ions, but its formation was repressed by the addition of (NH4)2SO4 to growth media. 3. E. carotovora was unable to grow with ethanolamine or 1-aminopropan-2-ol compounds as sources of C, the production of aldehydes during utilization as N sources being attributable to the inability of the microbe to synthesize aldehyde dehydrogenase. 4. Of seven additional strains of Erwinia examined similar results were obtained only with Erwinia ananas (N.C.P.P.B. 441) and Erwinia milletiae (N.C.P.P.B. 955).
Hormonal regulation of glycogen metabolism in neonatal rat liver
Authors: Schwartz Alan L. , Rall Theodore W.
Published: August 2015

1. The development of active and inactive phosphorylase was determined in rat liver during the perinatal period. No inactive form could be found in tissues from animals less than 19 days gestation or older than the fifth postnatal day. 2. The regulation of phosphorylase in organ cultures of foetal rat liver was examined. None of the agents examined [glucagon, insulin or dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate)] changed the amount of phosphorylase activity. 3. Glycogen concentration in these explants were nevertheless decreased more than twofold by 4h of incubation with glucagon or dibutyryl cyclic AMP. Incubation with insulin for 4h increased the glycogen content twofold. 4. Glycogen synthetase activity was examined in these explants. I-form activity (without glucose 6-phosphate) was found to decrease by a factor of two after 4h of incubation with dibutyryl cyclic AMP, whereas I+D activity (with glucose 6-phosphate) remained nearly constant. Incubation for 4h with insulin increased I-form activity threefold, with only a slight increase in I+D activity. 5. When explants were incubated with insulin followed by addition of dibutyryl cyclic AMP, the effects of insulin on glycogen concentration and glycogen synthetase activity were reversed. 6. These results indicate that the regulation of glycogen synthesis may be the major factor in the hormonal control of glycogen metabolism in neonatal rat liver.
The specific radioactivity of the precursor pool for estimates of the rate of protein synthesis (Short Communication)
Authors: Fern Edward B. , Garlick Peter J.
Published: August 2015

Infusion of rats with [U-14C]glycine resulted in labelling of glycine and serine in tissue proteins. The pattern of labelling in protein more nearly resembled that of the free amino acids in the tissue than in the plasma.
The binding of cationic detergent to insulin, zinc–insulin and iso-insulin (Short Communication)
Authors: Birdi Kulbir S.
Published: August 2015

Insulin, zinc–insulin and iso-insulin were precipitated by cetyltrimethylammonium bromide when the detergent/protein molar ratio was less than 10:1, whereas no precipitation was observed at ratios greater than 10:1. It is concluded that the detergent binds as a dimer at each binding site at ratios greater than 10:1. U.v. difference spectra indicated that the tyrosine residues were perturbed by detergent binding.
O-Methyl sugars in lipopolysaccharides of Rhodospirillacea. Identification of 3-O-methyl-d-mannose in Rhodopseudomonas viridis and of 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose in Rhodopseudomonas palustris respectively
Authors: Weckesser Jürgen , Mayer Hubert , Fromme Inge
Published: August 2015

1. This paper deals with the identification of three O-methyl sugars in lipopolysaccharides isolated from strains of the Gram-negative photosynthetic family Rhodospirillaceae. In addition to the previously described 3-O-methyl-l-xylose, a second O-methyl sugar was encountered in the lipopolysaccharide of Rhodopseudomonas viridis F, namely 3-O-methyl-d-mannose. The lipopolysaccharides of two strains of Rhodopseudomonas palustris (strain 1e5 and 8/1) contain two O-methylsugars, 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose (d-acovenose). 4-O-Methyl-d-xylose, but not 3-O-methyl-6-deoxy-d-talose, could be identified in the lipopolysaccharides of the strains K/1 and 2/2 of the same species. 2. The O-methyl sugars described in this communication were isolated by paper chromatography and identified by g.l.c., paper chromatography, high-voltage electrophoresis and mass spectrometry. Besides the genuine sugars, their alditol acetates and their demethylated (parental) forms were investigated. Optical rotation measurements and, in one case, enzymic reactions were used to establish the optical configuration of the sugars under investigation.
The occurrence of two types of synthesis of deoxyribonucleic acid during normal growth in Bacillus subtilis
Authors: Harris William J.
Published: August 2015

A study of the relative utilization of thymine and thymidine as precursors for DNA synthesis during normal growth in Bacillus subtilis showed that thymine serves preferentially as a precursor for ‘repair’ synthesis, whereas thymidine is used preferentially for ‘replicative’ synthesis. Further, evidence was obtained which suggests that during normal growth both ‘replicative’ and ‘repair’ DNA syntheses occur simultaneously. ‘Repair’ synthesis is distinguished not only on the basis of its preferential utilization of thymine but also by its selective inhibition by caffeine. ‘Replicative’ synthesis, however, is selectively inhibited by 6-(p-hydroxyphenylazo)-uracil. ‘Repair’ synthesis would seem to be a ‘pre-fork’ phenomenon and its inhibition is highly lethal to the cell.
Molecular weight and amino acid composition of the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61
Authors: Frère J.-M. , Ghuysen J.-M. , Perkins H. R. , Nieto M.
Published: August 2015

A procedure allowing the purification of milligram amounts of the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 to protein homogeneity (95% purity) is described. The isolated protein has a molecular weight of about 38000 and consists of one polypeptide chain. Its amino acid composition is presented.
Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide
Authors: Barry Standish , O'Carra Pádraig
Published: August 2015

1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+(probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.
The chemistry of the collagen cross-links. The characterization of Fraction C, a possible artifact produced during the reduction of collagen fibres with borohydride
Authors: Robins Simon P. , Bailey Allen J.
Published: August 2015

The present paper describes the isolation and identification of a major radioactive component of borotritide-reduced collagen, previously designated Fraction C. The derived structure for the compound confirms that it is identical with the ‘post-histidine’ component described by Tanzer et al. (1973) and given the trivial name histidino-hydroxymerodesmosine. Detailed studies of the effects of acid pH on the formation of Fraction C after borohydride reduction demonstrated the apparent lability of the non-reduced form, thus confirming our previous findings (Bailey & Lister, 1968). Inhibition of the formation of this component by the acid treatment appears to be due to protonation of the histidine imidazole group. Since the only new component formed on reduction of the acid-treated fibres was the reduced aldol condensation product, these results indicate that neither the histidine nor the hydroxylysine residues can be involved in covalent linkage with the aldol condensation product in the native fibre. It is suggested therefore that the proposed non-reduced aldimine form of Fraction C does not exist as an intermolecular cross-link in vivo. Thus the presence of histidino-hydroxymerodesmosine as a tetrafunctional cross-link in reduced collagen fibres is a result of a base-catalysed reaction promoted by the borohydride-reduction procedure and this component must therefore be considered as an artifact.
Kinetic studies of the acylation of pig muscle–d-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism
Authors: Harrigan P. J. , Trentham D. R.
Published: August 2015

In the presence of NAD+the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD+and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H+indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5–8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results.
Collagen cross-links. A mass-spectrometric and1H- and13C-nuclear-magnetic-resonance study
Authors: Hunt Eric , Morris Howard R.
Published: August 2015

1. The structure of a tetrafunctional cross-link (compound C) isolated from borohydride-reduced cow skin collagen has been determined by the combined use of mass spectrometry and1H and13C n.m.r. spectroscopy. 2. A new technique, of potentially wide applicability, is described for the mass-spectrometric determination of functional groups. 3. Detailed study of protonation shifts in the13C spectrum has allowed an unambiguous assignment of the exact structural relationship of the component parts of the molecule. 4. New interpretations of existing data on this molecule are given.
Purification and properties of rabbit muscle l-glycerol 3-phosphate dehydrogenase
Authors: Bentley Philip , Dickinson F. Mark , Jones Ian G.
Published: August 2015

A modified procedure has been developed for the purification of rabbit muscle l-glycerol 3-phosphate dehydrogenase. The product of the preparation satisfies all criteria of homogeneity. Some physical properties of the enzyme have been re-investigated. The results suggest that previous preparations may have been contaminated with significant amounts of heavy-molecular-weight protein.
The uptake and efflux of l-phenylalanine and l-tyrosine from rat brain cerebral cortex slices
Authors: Joanny Pierre , Natali Jean-Pierre , Hillman Harold , Corriol Jacques
Published: August 2015

The uptake of radioactive l-phenylalanine and l-tyrosine into the tissue water of rat brain cerebral cortex slices was shown to have saturation kinetics. The apparent Km for the uptake of l-phenylalanine was 0.86mm and for l-tyrosine was 1.64mm; for phenylalanine the apparent Vmax. value was 0.64μmol/min per ml of tissue water, and for l-tyrosine it was 0.98μmol/min per ml of tissue water. The accumulation of the two amino acids by the tissue was depressed in the absence of O2, at 0°C, or in the presence of metabolic inhibitors. The influxes and effluxes of both l-isomers were more rapid than those of the d-isomers. Competition between these two amino acids and each with l-tryptophan in respect of uptake into tissue water was shown. Their rates of influx were faster, and rates of efflux were slower, in the presence than in the absence of Na+. It was concluded that these amino acids were taken up by active transport via a carrier mechanism.
Purification of 14C-labelled deoxyribonuclease II from HeLa S3 lysosomes and its use as a marker for the study of nuclear deoxyribonuclease II
Authors: Slor Hanoch
Published: August 2015

Deoxyribonuclease II (DNAase II) in mammalian cells has generally been considered to be located in the lysosomes. Several recent studies have indicated that some DNAase II activity is present in purified nuclei; this, however, could have been due to some contamination of the nuclear fraction by lysosomes, or alternatively, it could have been caused by specific binding of lysosomal DNAase II to the nuclear fraction during isolation. Our previous studies have eliminated the possibility that lysosomal contamination was the cause of the presence of DNAase II in isolated nuclei. In this study I have purified 14C-labelled lysosomal DNAase II and added it to cells during isolation of their nuclei. This study demonstrates that there is no specific binding of lysosomal DNAase II to the nuclear fraction and concludes that DNAase II activity observed in isolated nuclei represents an intrinsic activity that might be involved in nuclear DNA metabolism.
Glycolytic isoenzymes and glycogen metabolism in regenerating liver from rats on controlled feeding schedules
Authors: Bonney Robert J. , Hopkins Harold A. , Walker P. Roy , Potter Van R.
Published: August 2015

Intact rats trained on a controlled feeding and lighting schedule designated ‘8+16’ exhibited diurnal oscillations in liver weight, glucokinase activity and liver glycogen content. Glucokinase activity expressed as units/g of liver decreased to 30% of that from unoperated controls during the first 48h after partial hepatectomy and returned to near normal values in 2 weeks. When the glucokinase activity was expressed as units/liver per 100g body wt., a decrease to 50% of control activity was observed between 24 and 48h after the operation. A similar pattern was found for pyruvate kinase type I. In contrast, pyruvate kinase type III activity increased after partial hepatectomy. It is suggested that the newly divided cells after partial hepatectomy do not synthesize glucokinase and pyruvate kinase I but do synthesize pyruvate kinase III. Glycogen was found to accumulate as early as 24h after partial hepatectomy, and normal concentrations were reached after 48h if the operation was performed at times other than during the feeding periods.
Mechanism of action of a microsomal inhibitor of protein synthesis potentiated by oxidized glutathione
Authors: Rupniak H. T. R. , Quincey R. V.
Published: August 2015

Extracts of microsomal fractions cause an inhibition of protein synthesis that is most pronounced in the presence of 0.1mm-GSSG and 0.01mm-GTP, and is abolished by thiol or 0.4mm-GTP (Scornik et al., 1967). Fractionation of microsomal extracts showed that this inhibition of protein synthesis was caused by an enzyme, nucleoside diphosphate phosphohydrolase. Direct inhibition of protein synthesis on detergent-treated polyribosomes by 0.1mm-GSSG was observed under conditions of GTP limitation induced by omission of a GTP-regenerating system, or addition of a nucleoside triphosphate diphosphohydrolase. Thus GSSG potentiated the inhibition of protein synthesis caused by an enzyme that promoted removal of GTP. The inhibition was abolished by adding 4mm-2-mercaptoethanol or 0.4mm-GTP. Nucleoside diphosphate phosphohydrolase was thought also to act by promoting removal of GTP, thus causing an inhibition of protein synthesis that was potentiated by GSSG.
The effect of magnesium ions on the diemthylaniline oxidation rate and electron transfer in liver microsomal fraction
Authors: Archakov A. I. , Karuzina I. I. , Kokareva I. S. , Bachmanova G. I.
Published: August 2015

1. Reactions of N-demethylation, p-hydroxylation and N-oxidation of one substrate, i.e. dimethylaniline, have been used to show that the activating effect of Mg2+ takes place only in the first two reactions. 2. An increase in Vmax. of N-demethylation of dimethylaniline is accompanied by an increase in Km. In the p-hydroxylation of dimethylaniline Vmax. increases whereas Km does not change. A comparison of the changes in the Km values of these reactions with the change in Ks shows that in both cases Km does not characterize the affinity of cytochrome P-450 for dimethylaniline. 3. The rate-limiting site of N-demethylation and p-hydroxylation of dimethylaniline, as well as the total rate of NADPH oxidation in the presence of dimethylaniline, is between cytochromes b5 and P-450. Addition of Mg2+ to the incubation medium changes the hydrophobic environment of phosphatidylcholine in the membrane, the process being accompanied by a sharp increase in the fluorescence quantum yield of 8-anilinonaphthalene-1-sulphonate.
The effects of starvation, environmental temperature and injury on the rate of disposal of glucose by the rat
Authors: Heath Dennis F. , Corney Patricia L.
Published: August 2015

1. The disposal rate of glucose, R, given by R=kvQ, where Q is the quantity of plasma glucose and kv is a rate coefficient, was determined from the disappearance of [U-14C]-glucose from blood after single intravenous injection. Values of R should be close to the carbohydrate oxidation rate in the states investigated. 2. Normal rats (i) experimental methodology was studied. The best (single sampling) method gave the following results. (ii) The plasma glucose concentration (Cp) and R were temporarily increased by the stress of handling and injection. (iii) R was increased by decreasing the environmental temperature from 29°C to 20°C in line with previously published (Stoner & Marshall, 1971) changes in total body O2 consumption. (iv) Starvation decreased R such that R=constant×Cp2. (v) The results suggested some central control of cell permeability to glucose. 3. Injured post-absorptive rats were studied in the ebb phase after three severe injuries: scalding at 20° and 29°C (non-lethal) and bilateral hind-limb ischaemia at 20°C (85% mortality). (i) Handling and injection did not affect Cp. (ii) The rise in Cp after injury was not closely correlated with its severity. (iii) The value of R was nearly independent of severity. (iv) Unlike in normal rats R varied little with ambient temperature (in line with O2 consumption) or with Cp. Values of kv varied inversely as Cp. (v) The results were explained in terms of a centrally integrated response to injury involving the hypothalamus which over-rode the controls operating in normal rats. Hormonal factors are discussed.
Association of nascent polypeptide with 30S ribosomal subunits
Authors: Cannon Michael , Mirza M. Amin A. , Anderson Margaret L. M.
Published: August 2015

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).