bluuebunny/crossref_metadata_2025 huggingface dataset:
A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi
Authors: Lavelle F , McAdam M E , Fielden E M , Roberts P B , Puget K , Michelson M
Published: August 2015

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.
The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain
Authors: Lumsden J , Coggins J R
Published: August 2015

A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex of about 270000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.
A consideration of the effects of added solutes on the activity of bovine superoxide dismutase
Authors: McAdam M E
Published: August 2015

Pulse radiolysis was used to study the effects of changing the concentration of buffer and added salts on the activity of bovine superoxide dismutase. The results are discussed with reference to other work and are interpreted mainly in terms of ionic-strength effects. The effects are best considered as empirical parameters, which are essential for comparative activity measurements. The specific activity of the enzyme was higher than found in previous pulse-radiolysis studies.
Factors influencing the activity of ornithine aminotransferase in isolated rat liver mitochondria
Authors: McGivan J D , Bradford N M , Beavis A D
Published: August 2015

1. The characteristics of ornithine catabolism by the aminotransferase pathway in isolated mitochondria were determined. 2. Ornithine synthesis from glutamate and glutamate gamma-semialdehyde produced by the oxidation of proline was studied. No ornithine was formed in the absence of rotenone. 3. The mechanism of ornithine transport was reinvestigated, and the existence of an ornithine+/H+ exchange system postulated. 4. The kinetics of ornithine transport, ornithine catabolism in intact mitochondria and ornithine aminotransferase activity in solubilized mitochondria were compared. It is concluded that ornithine aminotransferase activity in liver mitochondria is rate-limited by the transport of ornithine across the mitochondrial membrane, and that this enzyme is involved primarily in ornithine degradation rather than ornithine synthesis.
Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir
Authors: Dalessandro G , Northcote D H
Published: August 2015

A protein fraction [precipitate obtained between 40 and 65% (NH4)2SO4 satn.] prepared from cambial cells, differentiating xylem cells and differentiated xylem cells of pine and fir trees contained all the enzymes required for the nucleoside diphosphate sugar interconversions. By using UDP-D-[U-14C]glucose or UDP-D-[U-14C]galactose, UDP-D-[U-14C-]glucuronic acid and UDP-D-[U-14C]xylose as substrates, the activities of UDP-D-galactose 4-epimerase (DC 5.1.3.2), UDP-D-xylose 4-epimerase(EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D-glucuronate 4-epimerase (EC5.1.3.6), UDP-d-glucuronate decarboxylase (EC 4.1.1.35) were measured at different stages of cell-wall development. The specific activities and the activities per cell of these enzymes varied during differentiation of cambium to xylem according to the type polysaccharide synthesized. Variations were also found between the two species investigated. These data, compared with those obtained in out previous work on angiosperms [see the preceding paper, Dalessandro & Northcote (1977) Biochem. J. 162, 267-279], suggest that some control of polysaccharide synthesis operates at the level of the formation of the precursors of pectin and hemicellulose syntheses.
Re-evaluation of amino-oxyacetate as an inhibitor
Authors: Smith S B , Briggs S , Triebwasser K C , Freedland R A
Published: August 2015

Data are provided which indicate that pyruvate and/or acetaldehyde can reverse the inhibition of alanine aminotransferase and aspartate aminotransferase by amino-oxyacetate. It was shown that acetaldehyde could reverse the inhibition of gluconeogenesis from alanine and that pyruvate could reverse the inhibition of urea synthesis by amino-oxyacetate.
Effects of glucose, glucose metabolites and calcium ions on adenylate cyclase activity in homogenates of mouse pancreatic islets
Authors: Capito K , Hedeskov C J
Published: August 2015

The effects of glucose, a series of glucose metabolites, nicotinamide nucleotides, Ca2+ and p-chloromercuribenzenesulphonate on adenylate cyclase activity in homogenates of mouse pancreatic islets were studied. The basal activity of the adenylate cyclase was approx. 6 pmol of cyclic AMP formed/30 min per microng of DNA at 30 degrees C. The enzyme activity was stimulated by some 150% by fluoride. Starvation of the animals for 48h had no effect on either the basal or the fluoride-stimulated activity. The adenylate cyclase activity was increased by 40-50% when 17 mM-glucose, 10 micronM-phosphoenolpyruvate or 10 micronM-pyruvate was added to the assay medium. The effect of glucose was unchanged in the presence of 17 mM-mannoheptulose, and mannoheptulose alone had no effect. The other glycolytic intermediates, and the coenzymes NAD+, NADH and NADPH, at concentrations up to 1 mM were without any detectable effect on the rate of formation of cyclic AMP. The insulin secretagogue p-chloromercuribenzenesulphonate inhibited the adenylate cyclase markedly even at a concentration of 10 micronM. Calculated concentrations of free Ca2+ of 10 micronM and 0.1 mM inhibited adenylate cyclase by 29 and 71% respectively. It is concluded that both glucose itself and phosphoenolpyruvate and/or pyruvate are true activating ligands for islet and adenylate cyclase and that inhibition of the cyclase by Ca2+ may be of physiological significance.
The influence of lysosomes on glycogen metabolism
Authors: Geddes R , Stratton G C
Published: August 2015

Lysosome-rich fractions were isolated from rat liver homogenates. In some fractions the lysosomes were separated from the contaminating mitchondria by the use of discontinuous Ficoll/sucrose gradients. Some glycogen was associated with lysosome fractions. This glycogen was of very large molecular size and of a quite different molecular-weight distribution from that isolated from the cytosol. It is suggested that appreciably more than 10% of cellular glycogen is located within the lysosome.
Protein chromatography on adsorbents with hydrophobic and ionic groups. Purification of human erythrocyte glycophorin
Authors: Simmonds R J , Yon R J
Published: August 2015

Human erythrocyte glycophorin was purified rapidly by (a) chromatography of a Triton X-100 extract of erythrocyte ‘ghosts’ on N-(3-carboxypropionyl)aminodecyl-Sepharose in buffers containing Triton X-100 or sodium dodecyl sulphate, or (b) chromatography of whole ‘ghosts’, solubilized in sodium dodecyl sulphate, on dodecyl-Sepharose, in buffers containing sodium dodecyl sulphate. The products contained 85-95% glycophorin (electrophoretic band PAS-1) and the major contaminants were glycoproteins PAS-2 (possibly a subunit of glycophorin) and PAS-3.
Partial purification and properties of frog liver tyrosine aminotransferase
Authors: Ohisalo J J , Andersson S M , Pispa J P
Published: August 2015

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5′-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.
Comparison of glucose metabolism in the lactating mammary gland of the rat in vivo and in vitro. Effects of starvation, prolactin or insulin deficiency
Authors: Robinson Alison M. , Williamson Dermot H.
Published: August 2015

1. Measurements of arteriovenous differences across mammary glands of normal and starved lactating rats, and lactating rats made short-term insulin-deficient with streptozotocin or prolactin-deficient with bromocryptine, showed that only in the starved animals was there a significant decrease in glucose uptake. This decrease was accompanied by release of lactate and pyruvate from the gland, in contrast with the uptake of these metabolites by glands of normal lactating rats. 2. There were no marked differences in metabolite concentrations in freeze-clamped glands in the four conditions studied, apart from a decrease in [lactate] and [pyruvate] and an increase in [glucose] in the glands of the streptozotocin-treated group. 3. Acini isolated from the glands of starved, insulin or prolactin-deficient rats had a higher production of lactate and pyruvate from glucose than did glands from normal rats; this is in agreement with the reported decrease in the proportion of active pyruvate dehydrogenase in these situations [Field & Coore (1976) Biochem. J.156, 333–337; Kankel & Reinauer (1976) Diabetologia12, 149–154]. 4. Addition of insulin did not increase the uptake of glucose by acini from normal glands, but it caused a significant increase in the utilization of glucose by acini from glands of starved rats. Insulin did not decrease the accumulation of lactate and pyruvate in any of the experiments. 5. It is concluded that isolated acini represent a suitable model for the study of mammary-gland carbohydrate metabolism in that they reflect metabolism of the gland in vivo.
The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-β-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver
Authors: Khalkhali Z , Serafini-Cessi F , Marshall R D
Published: August 2015

The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats.
Retained folates in the rat
Authors: Barford P A , Staff R J , Blair J A
Published: August 2015

The retention of radioactivity after doses of 14C- and 3H-labelled folic acid is described. Radioactivity was retained in liver, kidney and gut of rats for some time after administration of the dose. The retained radioactivity could not be displaced by large doses of unlabelled folic acid or unlabelled 5-methyltetrahydrofolate. 14C- and 3H-labbelled folates showed similar chromatographic behaviour onion-exchange chromatography to 5-methyltetrahydrofolate, and on ion-exchange and gel-permeation chromatography to synthetic pteroylhepta-gamma-glutamate.
Comparison in different species of biliary bilirubin-IX α conjugates with the activities of hepatic and renal bilirubin-IX α-uridine diphosphate glycosyltransferases
Authors: Fevery J , Van de Vijver M , Michiels R , Heirwegh K P M
Published: August 2015

The bilrubin-IXalpha conjugates in bile and the activities of bilirubin-IX alphax–UDP-glycosyltransferases in liver and kidney were determined for ten species of mammals and for the chicken. 1. In the mammalian species, bilirubin-IX alpha glucuronide was the predominant bile pigment. Excretion of neutral glycosides was unimportant, except in the cat, the mouse, the rabbit and the dog, where glucose and xylose represented 12–41% of total conjugating groups bound to bilirubin-IX alpha. In chicken bile, glucoside and glucuronide conjugates were of equal importance. They probably represent only a small fraction of the total bile pigment. 2. The transferase activities in liver showed pronounced species variation. This was also apparent with regard to activation by digitonin, pH optimum and relative activities of transferases acting on either UDP-glucuronic acid or neutral UDP-sugars. 3. Man, the dog, the cat and the rat excrete bilirubin-IX alpha largely as diconjugated derivatives. In general, diconjugated bilirubin-IX alpha could also be synthesized in vitro with liver homogenate, bilirubin-IX alpha and UDP-sugar. In contrast, for the other species examined, bilirubin pigments consisted predominantly of monoconjugated bilirubin-IX alpha. Synthesis in vitro with UDP-glucuronic acid, UDP-glucose or UDP-xylose as the sugar donor led exclusively to the formation of monoconjugated bilirubin-IX alpha. 4. The transferase activities in the kidney were restricted to the cortex and were important only for the rat and the dog. No activity at all could be detected for several species, including man. 5. Comparison of the transferase activities in liver with reported values of the maximal rate of excretion in bile suggests a close linkage between conjugation and biliary secretion of bilirubin-IX alpha.
Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin
Authors: Pays E
Published: August 2015

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.
Acetyl-coenzyme A and coenzyme A analogues. Their effects on rat brain choline acetyltransferase
Authors: Rossier J
Published: August 2015

Choline acetyltransferase has the same affinity for acetyl-CoA, propionyl-CoA and butyryl-CoA (Km=1.4 micron). Choline acetyltransferase may use the two latter compounds as substrate, but the longer the acyl chain the lower will be Vmax. CoA is an inhibitor (Ki=1.8 micron). The position of the 3′-phosphate is of primary importance. Desphospho-CoA is a weak inhibitor (Ki=500 micron). 5′-AMP is already an inhibitor (Ki=2500 micron). Phosphopantetheine is not an inhibitor. Dextran Blue is a potent inhibitor (Ki=0.05 micron). Choline acetyltransferase binds to hydrophobic affinity columns. Because of its affinity for nucleotides, affinity for Dextran Blue and hydrophobicity, it is proposed that it contains the ‘nucleotide fold’, which is a common structural domain present in several enzymes binding nucleotides.
Evidence for two-step binding of reduced nicotinamide-adenine dinucleotide to aldehyde dehydrogenase
Authors: MacGibbon A K H , Buckley P D , Blackwell L F
Published: August 2015

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.
Inhibition of carbon dioxide fixation by lead acetate in rat liver mitochondria
Authors: Amatruda J M , Staton A J , Kiesow L A
Published: August 2015

These studies were undertaken to determine the mechanism by which intravenously administered lead salts inhibit hepatic gluconeogenesis. Within 1 h after the intravenous administration of lead acetate (10 mg), there is 97% inhibition of CO2 fixation in isolated rat liver mitochondria. This effect is concentration-dependent. The induction of phosphoenolpyruvate carboxykinase activity observed with starvation was also inhibited by intravenously administered lead acetate, but the activities of pyruvate kinase, glucose 6-phosphate dehydrogenase and pyruvate carboxylase were unaffected, as was the oxidation of palmitate and palmitoyl-CoA by mitochondria from Pb2+-treated animals. The addition of reduced glutathione to mitochondria from Pb2+-treated animals had no effect on the inhibited CO2 fixation. ATP concentrations in mitochondria from Pb2+-treated animals are decreased and the dose-response relationships for the effect of Pb2+ on CO2 fixation and ATP concentrations correspond. We conclude that the decrease in mitochondrial ATP in Pb2+-treated animals is probably responsible for the marked inhibition ov CO2 fixation, and hence the impairment of gluconeogenesis from alanine, lactate and pyruvate observed by others.
Faster synthesis and slower degradation of liver protein during developmental growth
Authors: Conde R D , Scornik O A
Published: August 2015

A study is presented of the liver protein gain during the early stages of postnatal development. Fractional rates of protein synthesis and degradation were determined in vivo in livers of 4-day-old mice. At this age, liver protein accumulated at a rate of 18% per day. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was extimated as the balance between synthesis and accumulation of stable liver proteins, or from the disappearance of radioactivity from liver protein previously labelled by the administration of NaH14CO3. We found that the neonatal livers: (1) synthesize 139% as much protein per unit time and unit mass as adult tissue, which is accounted for by a higher ribosome concentration (synthesis per mg of RNA was the same); (2) retain 39% of the newly synthesized protein as stable liver components (compared with 48% in adult mice); (3) degrade protein at 56% of the rate in the adult liver. This lower rate of degradation is quantitatively the most significant difference between the growing and non-growing liver.
Effect of citrate on the activities of 6-phosphofructokinase from nervous and muscle tissues from different animals and its relationships to the regulation of glycolysis
Authors: Newsholme E A , Sugden P H , Williams T
Published: August 2015

1. Citrate inhibits the activities of phosphofructokinase from muscles and nervous tissues from different animals across the Animal Kingdom except for the insects. The enzymes from the flight muscle of nine different insects and the cerebral ganglion of the locust were investigated: no inhibition by citrate was observed. Inhibition was observed with the enzymes from both aerobic (e.g. pectoral muscle of pigeon) and anaerobic (e.g. fish muscle, pectoral muscle of the game birds) muscles. It is suggested that this inhibition is of physiological importance in decreasing the rate of glucose utilization in skeletal muscle of animals during starvation and/or prolonged exercise. 2. The rates of glucose utilization by the sartorius and gastrocnemius muscles of the frog were markedly decreased by ketone bodies. The latter elevated the glucose 6-phosphate and citrate contents of the gastrocnemius muscle, indicating that citrate inhibition of phosphofructokinase could be, in part, responsible for the decreased rate of glycolysis. 3. These findings provide evidence that the concept of the glucose-fatty acid-ketone-body cycle involves both aerobic and anaerobic skeletal muscle and nervous tissue from a wide range of animals except the insects. In the latter the concept of the cycle may not be applicable.
A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae
Authors: Burns G R J , Wynn C H
Published: August 2015

1. The three arylsulphatases of Aspergillus oryzae exhibit pronounced kinetic differences and substrate specificities. Arylsulphatase I hydrolyses all substrates tested, whereas arylsulphatase III will not hydrolyse tyrosine O-sulphate or phenolphthalein disulphate. Arylsulphatase II does not hydrolyse p-nitrophenyl sulphate or phenolphthalein disulphate at appreciable rates in the absence of added phenolic compounds. Phenols such as tyramine increase the rate of hydrolysis of these substances by this enzyme 1000-fold. At pH 6.9 arylsulphatase I exhibits an apparent Km of 0.1 mM for p-nitrophenyl sulphate, whereas the Km of arylsulphatase III for this substrate is 1 mM. 2. These differences were utilized to develop an assay procedure which can be used to determine the separate activities of the three enzymes present in mixtures. This assay has potential use as a means of examining the relative activities of the three enzymes in investigations of the differences in the mechanisms regulating their synthesis.
Regulation of mammalian protein synthesis in vivo. Stimulated liver ribonucleic acid synthesis in vivo after cycloheximide treatment
Authors: Ch'ih J J , Pike L M , Devlin T M
Published: August 2015

1. As shown by a double-radioisotope technique in vivo, at a non-lethal dose of cycloheximide, a stimulation of nuclear RNA synthesis occurred by 12 h after the treatment; the stimulation lasted over 48 h. Analysis of radioactive nuclear RNA by gel electrophoresis demonstrated that most of the cycloheximide-stimulated synthesis could be accounted for by known rRNA precursors (45 S, 41 S, 32 S and 28 S). 2. During the inhibitory phase of protein synthesis, 2 h after cycloheximide treatment, synthesis of the poly(A)-containing mRNA isolated from the cytoplasmic ribonucleoprotein complexes with an oligo(dT)-cellulose column was stimulated, whereas the synthesis of rRNA was slightly inhibited. However, during the stimulatory phase of protein synthesis, 24 h after cycloheximide treatment, the syntheses of both poly(A)-containing mRNA and rRNA were enhanced. 3. Kinetic studies revealed that the newly synthesized RNA species were transported from the nuclei, integrated into the ribonucleoprotein complexes, and associated with both free and membrane-bound polyribosomes. 4. These data corroborate our proposal that the stimulated protein synthesis after cycloheximide administration involves gene transcription.
Skeletal-muscle sarcolemma from normal and dystrophic mice. Isolation, characterization and lipid composition
Authors: de Kretser T A , Livett B G
Published: August 2015

1. Mouse skeletal-muscle sarcolemma was isolated, and the preparations obtained from normal mouse muscle and from muscle of mice with hereditary muscular dystrophy were characterized with respect to appearance under the optical and electron microscopes, distribution of marker enzymes, histochemical properties and biochemical composition. 2. The sarcolemmal membranes from normal and dystrophic muscle were subjected to detailed lipied analysis. Total lipid content was shown to increase in sarcolemma from dystrophic mice as a result of a large increase in neutral lipid and a smaller increase in total phospholipids. Further analysis of the neutral-lipid fraction showed that total acylglycerols increased 6-fold, non-esterified fatty acid 4-fold and cholesterol esters 2-fold, whereas the amount of free cholesterol remained unchanged in sarcolemma from dystrophic muscle. Significant increases were found in lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in dystrophic-muscle sarcolemma; however, the relative composition of the phospholipid fraction remained essentially the same as in the normal case. 3. The overall result of alterations in lipid composition of the sarcolemma in mouse muscular dystrophy was an increase in neutral lipid compared with total phospholipid, and a 4-fold decrease in the relative amount of free cholesterol in the membrane. The possible impact of these changes on membrane function is discussed.
Synthesis of the native copper(II)-transport site of human serum albumin and its copper(II)-binding properties
Authors: Iyer K S , Lau S J , Laurie S H , Sarkar B
Published: August 2015

A derivative of the native-sequence tripeptide of the specific Cu(II)-transport site of human serum albumin, L-aspartyl-L-alanyl-L-histidine N-methylamide, was synthesized, and its binding to Cu(II) was examined to determine the influence of the side-chain groups on the Cu(II) binding. The equilibria involved in the Cu(II)-L-aspartyl-L-alanyl-L-histidine N-methylamide system were investigated by analytical potentiometry. Three complex species were found in the pH range 4-10. The same species were identified in both the visible and circular-dichroism spectra. The main species present in the physiological pH range is shown to have the same ligands around the square-planar Cu(II) ion as those reported for albumin and tripeptides diglycyl-L-histidine and its N-methylamide derivative. The results obtained from competition experiments showed that this tripeptide has a higher affinity towards Cu(II) than has albumin itself. The overall findings are compared with those from albumin. At neutral pH the side chains do not play any important role in the Cu(II) binding, but at low pH the beta-carboxyl group of the N-terminal aspartic residue becomes important. A possible competition site on albumin for Cu(II) at low pH is discussed.
A reappraisal of the reaction pathway of pyruvate carboxylase
Authors: Easterbrook-Smith S B , Wallace J C , Keech D B
Published: August 2015

The reaction pathway catalysed by pyruvate carboxylase was re-examined by using two independent experimental approaches not previously applied to this enzyme. To avoid the variable stoicheiometry associated with oxaloacetate formation, the reaction rate was measured by following release of Pi. Initial velocities, when plotted as a function of varying concentrations of either MgATP2- or HCO3-, at fixed concentrations of pyruvate, gave in double-reciprocal-form families of straight intersecting lines. Further, when the reaction velocity was determined as a function of varying MgATP2- concentrations by using pyruvate, 3-fluoropyruvate and 2-oxobutyrate as alternative carboxyl-acceptor substrates, the slopes of the double-reciprocal plots were significantly different. Both results support a sequential reaction pathway.
Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking
Authors: Bristow A F , Virden R
Published: August 2015

1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.
The binding of porphyrins by ligandin
Authors: Tipping E , Ketterer B , Koskelo P
Published: August 2015

Spectrophotometric and equilibrium-dialysis measurements show that ligandin (glutathione S-transferase B, EC 2.5.1.18) binds monomeric porphyrins at a single site with association constants in the range 10(4)-10(6) litre/mol at pH 7.0. Binding affinities are paralleled by the tendencies of the porphyrins to aggregate, increasing in the order: uroporphyrins I and III less than coproporphyrins I and III approximately haematoporphyrin less than protoporphyrin IX. From this it is deduced that the hydrophobic effect is the predominant driving-force for binding. The porphyrins can be displaced from their binding site on ligandin by bromosulphophthalein and oestrone sulphate. In enzyme inhibition studies, 50% inhibition was brought about by 8 micron-haematoporphyrin and by 1 micron-protoporphyrin IX. In the analysis of the haemotoporphyrin-ligandin system the self-association of haematoporphyrin was studied in detail. It was found to be limited to dimerization in the concentration range 0-200 micron at pH 7.0, 25 degrees C and a dimerization constant of 1.9 × 10(5) litre/mol was determined. Coproporphrin III has a dimerization constant of 5.2 × 10(5) litre/mol under the same conditions.
Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro
Authors: Mezquita C , Teng C S
Published: August 2015

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids.
The effects of hypoglycin on glucose metabolism in the rat. A kinetic study in vivo and [U-14C,2-3H]glucose
Authors: Osmundsen H , Billington D , Taylor J R , Sherratt H S A
Published: August 2015

1. The kinetics of glucose metabolism were evaluated in rats deprived of food 15-21 h after the administration of hypoglycaemic doses of hypoglycin (100 mg/kg body wt.) by following changes in the specific radioactivities of 14C and 3H in blood glucose after an intravenous dose of [U-14C,2-3H]glucose [Katz, Rostami & Dunn (1974) Biochem. J. 142, 161-170]. 2. During this time, recycling of glucose through the Cori cycle was virtually abolished, the rate of irreversible disposal of glucose and its total body mass were both decreased by about 70%, whereas there was little effect on the mean transit time for glucose. 3. It was concluded that hypoglycaemia is due to inhibition of gluconeogenesis.
Intestinal neuraminidase activity of suckling rats and other mammals. Relationship to the sialic acid content of milk
Authors: Dickson J J , Messer M
Published: August 2015

1. The neuraminidase activity of homogenates of the mucosa of the middle and distal thirds of the small intestine of rats increased about 5-fold between birth and 4 to 8 days of age, and then gradually declined to the much lower adult activity by 24 days. No comparable changes occurred in the proximal third. 2. In 8-day-old rats, the neuraminidase activity of the middle and distal thirds of the small intestine was about 10 times greater than that of the proximal third, 20 times greater than that of the colon and at least 100 times greater than that of the liver, brain, gastric mucosa or pancreas. 3. In all other species investigated (mice, rabbits, cats and guinea pigs), the neuraminidase activity of the middle and distal thirds of the small intestine was greater in suckling animals than in adults. 4. The sialic acid content of rat milk increased about 2-fold between birth and 8 days post partum and then declined. 5. There was a highly significant positive correlation between the intestinal neuraminidase activity of suckling animals of various species and ages and the sialic acid content of milk obtained from the corresponding species and stage of lactation. 6. It is suggested that the intestinal neuraminidase of suckling mammals functions primarily to remove sialic acid from various components of milk, thus providing sialic acid for the synthesis of sialoglycoproteins and gangliosides by the young.
Transformation of arachidonate into 6-oxoprostaglandin F1 α, thromboxane B2 and prostaglandin E2 by sheep lung microsomal fraction
Authors: Tai H H , Yuan B , Wu A T
Published: August 2015

In the presence of haemoglobin and isoproterenol, the microsomal fraction of sheep lung catalysed the conversion of arachidonate predominantly into thromboxane B2 and to a lesser extent into 6-oxoprostaglandin F1alpha. Very little prostaglandin E2 and prostaglandin F2alpha were formed. If reduced glutathione was added in combination with haemoglobin and isoproterenol, the synthesis of prostaglandin E2 was favoured over that of thromboxane B2 and 6-oxoprostaglandin F1alpha. The identities of these products were confirmed by t.l.c. and by combined g.l.c.-mass spectrometry. These results indicate that microsomal fraction of sheep lung possesses active prostaglandin synthase, prostacyclin synthase and thromboxane synthase activities.
The biochemical pathway for the breakdown of N4-ethyl-L-asparagine in the bacterium Pseudomonas stutzeri
Authors: Dineen R W , Gray D O
Published: August 2015

N4-Ethyl-L-[u-14C]asparagine and L-[U-14C]aspartate give identical metabolites, mainly intermediates of the tricarboxylic acid cycle and related amino acids, in whole cells of Pseudomonas stutzeri. The labelled asparagine derivative is converted into [14C]-aspartate by cell-free extracts, and this reaction, which has an optimum pH of 8.8 +/- 0.2, is neither inhibited by unlabelled asparagine nor enhanced by unlabelled 2-oxoglutarate. No labelled keto acid corresponding to N4-ethylasparagine was detected in either whole cells or cell-free extracts. Thus N4-ethyl-L-asparagine, like asparagine, must be broken down by hydrolysis, at least in this bacterium.
Proinsulin biosynthesis in broken-cell preparations of islets of Langerhans
Authors: Parry D G , Taylor K W
Published: August 2015

1. Rabbit islets of Langerhans were disrupted by ultrasonic methods and the sonicated preparations were used to study proinsulin biosynthesis. 2. When [3h]leucine is incubated in such preparations, incorporation takes place into proinsulin, as evidenced by characterization on polyacrylamide gels, and by the conversion of this labelled material into insulin, by using trypsin. 3. The labelled proinsulin may also be purified by antiinsulin antibody bound to Sepharose. 4. With the broken-cell preparation it was shown that incorporation of leucine is accelerated by increasing the glucose content of the medium from 2mM to 16mM. However, 16mM-galactose or -sucrose did not stimulate incorporation significantly from basal values. This effect of glucose was abolished by cycloheximide. 5. The significance of these findings in relation to the mechanism of glucose stimulation of proinsulin biosynthesis is discussed.
Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the F1 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12
Authors: Cox G B , Downie J A , Gibson F , Radik J
Published: August 2015

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.
Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose
Authors: Auricchio F , Rotondi A , Sampaolo P , Schiavone E
Published: August 2015

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.
Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1
Authors: Ferenczi M A , Homsher E , Simmons R M , Trentham D R
Published: August 2015

The Mg2+-dependent ATPase (adenosine 5′-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' − 1 E.ATP k' + 2 in equilibrium k' − 2 E.ADP.Pi k' + 3 in equilibrium k' − 3 E.ADP + Pi k' + 4 in equilibrium k' − 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 × 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 × 10(4) M-1.S-1 and 7.4 × 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.
Lysine residue 240 of human serum albumin is involved in high-affinity binding of bilirubin
Authors: Jacobsen C
Published: August 2015

Bilirubin can be coupled covalently to albumin by using water-soluble carbodi-imide as coupling reagent. The optimal specificity in the attachment of bilirubin to the high-affinity site on the albumin molecule was obtained by treating an albumin-bilirubin complex with carbodi-imide in low concentrations and for a short period. The product was reduced, carboxymethylated and digested with trypsin. By fractionation on Sephadex G-50 (superfine grade) a peptide fraction containing most of the bilirubin label was isolated. Further purification by paper chromatography gave one peptide, consisting of residues 240-258. The peptide containined a single lysine residue, 240, and had an intact disulphide bridge. The results indicate that bilirubin is bound to lysine residue 240 at its high-affinity site on human serum albumin.
Affinity chromatography of bacterial lactate dehydrogenases
Authors: Kelly N , Delaney M , O'Carra P
Published: August 2015

The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel.
The specificity of cathepsin B. Hydrolysis of glucagon at the C-terminus by a peptidyldipeptidase mechanism
Authors: Aronson N N , Barrett A J
Published: August 2015

The manner in which human liver cathepsin B (EC 3.4.22.1) digests glucagon was determined. After reaction of the proteinase with the substrate for 24h, more than 15 products were formed. During the first 7 h of reaction, eight products were formed; seven of these were dipeptides that originated from the C-terminal portion of the glucagon molecule, whereas the eighth peptide was the remaining large fragment of the hormone, consisting of residues 1-19. Measurement of the rate of formation of the products showed that cathepsin B degraded glucagon by a sequential cleavage of dipeptides from the C-terminal end of the molecule. Cathepsin B from both rat liver and bovine spleen was shown to hydrolyse glucagon by the same mechanism.
Loss of cell-population-density-dependent incorporation of fucose into the lipid fraction of cultured human tumour cells
Authors: Gacto M
Published: August 2015

The incorporation of radioactively labelled fucose into the lipid fraction of cultured normal human cells and several human tumour-cell lines was examined as a function of the cell population density. Normal cells exhibited a density-dependent pattern of incorporation, whereas in tumour cells the radioactivity incorporated was independent of the cell population density. An exception was found among the tumour cells, which suggests a possible correlation between the loss of this marker and the ability to produce tumours.