bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of alternative molecular forms of xanthine oxidase in the mouse
Authors: Duke E. J. , Joyce P. , Ryan J. P.
Published: August 2015

1. Two major forms of xanthine oxidase are demonstrated for the mouse. On polyacrylamide-gel electrophoresis the duodenal form migrates faster towards the anode than that of the liver. Both forms also differ in their (NH4)2SO4 precipitation patterns and sucrose-density-gradient molecular-weight determinations. 2. The liver form is fully converted into the duodenal form by incubation at 37°C with 2.5mg of crude trypsin/ml for 1½h, without loss of activity. The trypsin-treated liver form behaves like the normal duodenal form as characterized by electrophoresis, (NH4)2SO4 precipitation patterns, and sucrose-density-gradient molecular-weight determinations. 3. Partial conversion is also brought about by purified trypsin and chymotrypsin, but not with β-carboxypeptidase or lipase. The conversion is inhibited by soya-bean trypsin inhibitor. 4. In embryo mice the duodenal form is similar to the liver form on electrophoresis. 5. These studies indicate, as might be expected, that the duodenal form is a modified version of the liver enzyme, probably caused by proteolytic alteration.
The inhibition of pig kidney alkaline phosphatase by oxidized or reduced nicotinamide–adenine dinucleotide and related compounds
Authors: Ramasamy I. , Butterworth P. J.
Published: August 2015

1. The inhibition of alkaline phosphatase by NAD+, NADH, adenosine and nicotinamide was studied. 2. All of these substances except NAD+ act as uncompetitive inhibitors, i.e. double-reciprocal plots are parallel. NAD+, however, is a ‘mixed’ inhibitor of alkaline phosphatase and is less potent than NADH. 3. Inhibition studies with pairs of the inhibitors suggest that, in spite of the difference in type of inhibition, NAD+ and NADH bind to alkaline phosphatase at a common site. Adenosine and nicotinamide also seem to bind at the NAD site and the binding of adenosine is facilitated by nicotinamide, and vice versa. 4. The facilitation may indicate the occurrence of an induced fit for NAD+ and NADH. Attempts to desensitize alkaline phosphatase to NAD+ and NADH inhibition by partial denaturation were unsuccessful. 5. The results are discussed in terms of a two-site model in which separate, but interacting, regions exist on the enzyme to accommodate the adenosine and nicotinamide moieties of NAD, and a single-site model in which the adenosine part of the molecule is bound preferentially and this interacts with the nicotinamide fraction. 6. The activity of alkaline phosphatase can be changed fourfold by alteration of the NAD+/NADH ratio. This sensitivity to the redox state of the coenzyme could be a means of controlling phosphatase activity.
The oestrogen-stimulated synthesis of heterogeneous nuclear ribonucleic acid in the uterus of immature rats
Authors: Knowler J. T. , Smellie R. M. S.
Published: August 2015

An early response to the administration of oestradiol-17β to immature rats is the synthesis of uterine RNA of very high-molecular-weight. This RNA is shown to be heterogeneous nuclear RNA. Increased precursor incorporation into the heterogeneous nRNA is not confined to entities of precise molecular weight but appears to involve much of the size range of the species. These findings are discussed with respect to the mode of action of oestradiol.
Distribution of enzymes that catalyse reactions of glutathione with αβ-unsaturated compounds
Authors: Chasseaud L. F.
Published: August 2015

1. A study of the distribution of glutathione S-alkenetransferases in the livers of vertebrate species suggests that different enzymes may catalyse reactions of GSH with (i) trans-benzylideneacetone, (ii) 2,3-dimethyl-4(2-methylenebutyryl)phenoxyacetic acid, (iii) cinnamonitrile, (iv) o-chlorobenzylidenemalononitrile, (v) methyl vinyl sulphone, and (vi) 3-(β-nitrovinyl)indole. 2. Glutathione S-alkenetransferase activity was generally greatest in rat liver, but the enzyme in hamster liver was more active towards o-chlorobenzylidenemalononitrile, and the enzyme in rabbit, hamster, guinea-pig and mouse livers was more active towards methyl vinyl sulphone. 3. Results from studies of the distribution of activities in rat liver and rat kidney, heat inactivation of rat liver supernatants, and (NH4)2SO4 fractionation and acid-precipitation experiments, differentiated further between some of the enzymes concerned with substrates (i)–(vi). 4. The infrequent detection of mercapturic acids in vivo is discussed.
A method for the simultaneous isolation of Factor X and prothrombin from bovine plasma
Authors: Esnouf M. Peter , Lloyd Peter H. , Jesty Jolyon
Published: August 2015

1. A method is described for the simultaneous isolation of both Factor X and prothrombin from bovine plasma. The proteins are adsorbed on and eluted from barium sulphate and chromatographed on DEAE-Sephadex and are finally purified by rechromatography on DEAE-Sephadex. 2. The proteins can be purified in 48h from the collection of the blood and the method can be used to process large volumes of plasma. 3. The prothrombin has a molecular weight of 70300; the Factor X, on the other hand, is polydisperse, with most of the protein (86%) having a molecular weight of 56000.
Human cathepsin B1. Purification and some properties of the enzyme
Authors: Barrett Alan J.
Published: August 2015

1. Cathepsin B1 was purified from human liver by a method involving autolysis, fractional precipitation with acetone, adsorption on, and stepwise elution from, CM-cellulose and an organomercurial adsorbent, gel chromatography and finally equilibrium chromatography on CM-cellulose. 2. The early stages of the procedure, including the use of the organomercurial adsorbent, were suitable for the simultaneous isolation of cathepsin D. The two cathepsins were sharply separated on the organomercurial column, and particular attention was given to the method for the preparation and use of this adsorbent. 3. A method is described for the staining of analytical isoelectric-focusing gels for cathepsin B1 activity, as well as protein. By this method it was shown that cathepsin B1 was represented by at least six isoenzymes during the greater part of the purification procedure. After the gel-chromatography step this group of isoenzymes was obtained essentially free of other proteins, in good yield. The isoenzymes were resolved from this mixture by chromatography on CM-cellulose. The purified enzyme was stable for several weeks at slightly acid pH values in the absence of thiol compounds; it was unstable above pH7. 4. The pI values of the isoenzymes of cathepsin B1 extended from pH4.5 to 5.5, that of the major isoenzyme tending to increase from 5.0 to 5.2 during the purification procedure. Gel chromatography indicated a molecular weight of 27500 for all of the isoenzymes, whereas polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate gave a value of 24000. 5. An antiserum raised in sheep against the purified enzyme reacted specifically with the alkali-denatured molecule. Purified cathepsin B1 contained no material precipitable by an anti-(human cathepsin D) serum. 6. The enzyme hydrolysed several N-substituted derivatives of l-arginine 2-naphthylamide, as well as haemoglobin, azo-haemoglobin, azo-globin and azo-casein. Greatest activity was obtained near pH6.0. 7. The sensitivity of human cathepsin B1 to chemical inhibitors was generally similar to that of other thiol proteinases. The enzyme was inactivated by the chloromethyl ketones derived from tosylphenylalanine, tosyl-lysine, acetyltetra-alanine and acetyldialanylprolylalanine. 8. The hydrolysis of α-N-benzoyl-dl-arginine 2-naphthylamide by extracts of human liver at pH6 was attributable entirely to cathepsin B1.
Phenethylamines in brain and liver of rats with experimentally induced phenylketonuria-like characteristics
Authors: Edwards David J. , Blau Karl
Published: August 2015

1. Phenethylamines were extracted from brain and liver of rats with phenylketonuria-like characteristics produced in vivo by inhibition of phenylalanine hydroxylase (EC 1.14.3.1) with p-chlorophenylalanine, with or without phenylalanine administration. To protect amines against oxidation by monoamine oxidase, pargyline was also administered. 2. β-Phenethylamine was the major compound found in brain and liver. β-Phenethanolamine and octopamine were also present, in lesser amounts, and the concentrations of these three amines paralleled blood phenylalanine concentrations. By comparison, tissues from control animals had only very low concentrations of these amines. 3. Small amounts of normetadrenaline, m-tyramine and 3-methoxytyramine were also found. 4. The inhibitors used, p-chlorophenylalanine and pargyline, gave rise to p-chlorophenethylamine and benzylamine respectively, the first via decarboxylation, the second probably by breakdown during extraction. 5. Distribution of phenethylamines in different brain regions and in subcellular fractions of rat brain cells was also investigated. The content of phenethylamine was highest in the striatum. 6. These findings are discussed in the light of changes occurring in human patients with uncontrolled phenylketonuria.
Chemostat studies on the regulation of glucose metabolism in Pseudomonas aeruginosa by citrate
Authors: Ng F. M.-W. , Dawes E. A.
Published: August 2015

The effect of the relative concentrations of citrate and glucose on the regulation of key enzymes of the direct oxidative, phosphorylative, Entner–Doudoroff and pentose-cycle pathways of glucose metabolism in Pseudomonas aeruginosa has been investigated in continuous culture under conditions of NH4+-limitation. For comparison isocitrate dehydrogenase and aconitase were also assayed. Measurements were made for steady-state and transient conditions and the effect of growth rate was also studied. When cells grew on 75mm-citrate the glucose concentration had to attain 6–8mm before significant induction of enzymes of glucose metabolism occurred; the specific activities increased further as the result of both raising the glucose concentration to 30mm and then subsequently lowering the citrate to 60mm and then to 45mm. The specific activities of the glucose enzymes increased immediately during the transient period between the steady states characteristic of growth on 6mm- and 8mm-glucose, the increase continuing for about two doubling times. The converse experiment of adding increasing citrate concentrations to 45mm-glucose medium revealed an immediate induction of the citrate-transport system, oxidation of citrate following the increase in citrate concentration up to 8mm. Between 8mm- and 16mm-citrate a marked repression of gluconate, glucose 6-phosphate and 6-phosphogluconate dehydrogenases and the Entner–Doudoroff enzymes occurred. Increased growth rate in citrate medium resulted in decreased specific activities of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase. Increased growth rate in citrate–glucose medium gave decreased specific activities of isocitrate dehydrogenase and aconitase whereas the activities of some of the glucose enzymes decreased initially but then increased at the highest growth rate (0.5h-1), at which a marked increase in glucose utilization occurred. These observations accord with the regulation of glucose enzymes by induction with glucose or its metabolites and repression by citrate or its metabolic products.
The regulation of transport of glucose and methyl α-glucoside in Pseudomonas aeruginosa
Authors: Midgley M. , Dawes E. A.
Published: August 2015

The methyl α-glucoside-transport system of Pseudomonas aeruginosa has been characterized with respect to its specificity, energy-dependence, kinetics and regulation. The uptake of glucose involved two components, one of which transported glucose (Km=8μm) and methyl α-glucoside (Km=2.8mm) whereas the other was more complex, involving the extracellular activity of glucose dehydrogenase. Mutants defective in this enzyme have been isolated and characterized. The methyl α-glucoside–glucose-transport system was repressed when the organism was grown in the absence of glucose; the induction of this transport system by glucose, and its activity once induced, were inhibited by products of citrate metabolism.
Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5α-reductase
Authors: Maynard Paul V. , Cameron Euan H. D.
Published: August 2015

The location and some characteristics of rat adrenal C19-steroid 5α-reductase were investigated by using [7α-3H]androst-4-ene-3,17-dione and [7α-3H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3α- and 3β-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17β-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C19-steroid 5α-reductase, which showed similar values for Km and Vmax. when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5α-androstane-3,17-dione and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5α-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5α-reduction of C19- and C21-steroids in the rat adrenal.
Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes
Authors: Prospero Terence D. , Burge Malcolm L. E. , Norris Kenneth A. , Hinton Richard H. , Reid Eric
Published: August 2015

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl2. Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg2+, there being at least a 12-fold difference between the activity in the presence of Mg2+ and of EDTA. There is, however, a difference in the response of the enzymes to Mg2+ and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl2 and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl2 for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.
Stoicheiometry of lactose–proton symport across the plasma membrane of Escherichia coli
Authors: West Ian C. , Mitchell Peter
Published: August 2015

The rate of [14C]lactose inflow into anaerobic, non-metabolizing, Escherichia coli has been correlated with the rate of the simultaneous effective inflow of H+. It is shown that the initial rates of the two transport processes are essentially the same, and it is concluded that the two processes are strictly coupled with a 1:1 stoicheiometry.
Acetoacetate and brain lipogenesis: developmental pattern of acetoacetyl-coenzyme A synthetase in the soluble fraction of rat brain (Short Communication)
Authors: Buckley Brendan M. , Williamson Dermot H.
Published: August 2015

The existence of acetoacetyl-CoA synthetase in rat brain cytosol is reported. The coupling of this enzyme with cytosolic acetoacetyl-CoA thiolase can provide acetyl-CoA for lipogenesis and cholesterol synthesis without the need for mitochondrial participation. This new route for acetoacetate utilization may be important in developing brain.
The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate by the microsomal fraction of guinea-pig intestinal mucosa
Authors: Brindley David N.
Published: August 2015

1. With microsomal fractions of guinea-pig intestinal mucosa the mean specific activity of palmitoyl-CoA synthetase was approx. 1.3-fold the esterification of sn-glycerol 3-phosphate with palmitoyl-CoA generated by the endogenous synthetase. The latter activity was approx. 2.5- and 5-fold that when palmitoyl-CoA was generated from palmitoylcarnitine or when it was added directly to the assay system. 2. There were significant correlations (P<0.001) between the specific activities of palmitoyl-CoA synthetase and glycerolipid synthesis from either palmitate or palmitoylcarnitine. 3. The mean molar composition of glycerolipid synthesized from palmitate or palmitoylcarnitine was approx. 18% lysophosphatidate, 75% phosphatidate and 7% neutral lipid. 4. Glycerolipid synthesis from palmitate was inhibited by 80–90% after preincubation of microsomal fractions at 37°C for 40min and was caused by inactivation of palmitoyl-CoA synthetase. 5. Addition of 100–400mm-KCl inhibited palmitoyl-CoA synthetase activity and glycerolipid synthesis from palmitate but stimulated glycerol phosphate acyltransferase activity. 6. Diversion of palmitoyl-CoA synthesized by the endogenous synthetase to palmitoylcarnitine resulted in an almost stoicheiometric decrease in glycerolipid synthesis. 7. Addition of rac-1-monopalmitin promoted utilization of palmitoyl-CoA by the monoglyceride pathway but did not inhibit phosphatidate biosynthesis. 8. With rate-limiting concentrations of CoA and Mg2+ the relative decreases in velocity for palmitoyl-CoA synthetase and glycerolipid synthesis from palmitate were almost identical. However, low concentrations of palmitate and ATP produced greater decreases in synthetase activity than in glycerolipid synthesis. 9. There appears to be a fine balance between the activities of palmitoyl-CoA synthetase and glycerol phosphate acyltransferase, with neither activity being in excess with respect to phosphatidate synthesis.
Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage
Authors: Balduini C. , Brovelli A. , De Luca G. , Galligani L. , Castellani A. A.
Published: August 2015

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the Km values for UDP-glucose and NAD+ were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.
The reversibility of adenosine triphosphate cleavage by myosin
Authors: Bagshaw C. R. , Trentham D. R.
Published: August 2015

For the simplest kinetic model the reverse rate constants (k−1 and k−2) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl2, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k−1<0.02s−1 and k−1=16s−1. Apparently, higher values of k−1 and k−2 are found with less-purified protein preparations. The values of k−1 and k−2 satisfy conditions required by previous 18O-incorporation studies of H218O into the Pi moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.
The state of phosphorylation in vivo of membrane-bound phosphoproteins in rat brain
Authors: Weller M. , Rodnight R.
Published: August 2015

The alkali-labile P content of membrane protein prepared from rapidly frozen rat brain was measured, CuSO4 being used to inhibit protein phosphatase activity during subcellular fractionation. The P content of the membrane fraction was significantly increased (+12%) over the control value by incubation of homogenates with ATP before fractionation. This suggests that the membrane protein in rat brain is normally only partially phosphorylated.
Studies of lipid A fractions from the lipopolysaccharides of Pseudomonas aeruginosa and Pseudomonas alcaligenes
Authors: Drewry David T. , Lomax James A. , Gray George W. , Wilkinson Stephen G.
Published: August 2015

Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the β-(1→6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.
Direct measurement of proton binding to the active ternary complex of pig heart lactate dehydrogenase (Short Communication)
Authors: Holbrook J. John
Published: August 2015

With the use of a glass electrode it is shown that one proton is taken up from the solvent when o-nitrophenylpyruvate forms a ternary complex with lactate dehydrogenase and NADH at pH8 and before the complex has rearranged to give products.
A four-iron ferredoxin from Desulfovibrio desulfuricans (Short Communication)
Authors: Zubieta J. A. , Mason R. , Postgate J. R.
Published: August 2015

Ferredoxin from Desulfovibrio desulfuricans was isolated, purified and crystallized. It contains four iron atoms and four sulphido or ‘acid-labile’ sulphur atoms for a molecule of 6000 daltons. The absorption spectrum in the u.v.–visible region and the electron-paramagnetic-resonance signals of the reduced protein are similar to those observed for other four-iron ferredoxins. The amino acid composition is different from that of Desulfovibrio gigas ferredoxin. The redox potential of −0.33V at pH7.0 was determined by dye techniques.
Biosynthesis of glycosoaminoglycans by microsomal preparations from cultured mastocytoma cells
Authors: Lewis Richard G. , Spencer Alice F. , Silbert Jeremiah E.
Published: August 2015

Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized 14C-labelled glycosaminoglycan when incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of 14C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized 14C-labelled glycosaminoglycan when incubated with UDP-[14C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The 14C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The 14C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3′-phosphate 5′-sulphatophosphate, as well as UDP-[14C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.
Relationship between plasma and muscle concentrations of ketone bodies and free fatty acids in fed, starved and alloxan-diabetic states
Authors: Owen Oliver E. , Markus Helene , Sarshik Stuart , Mozzoli Maria
Published: August 2015

1. Concentrations of ketone bodies, free fatty acids and chloride in fed, 24–120h-starved and alloxan-diabetic rats were determined in plasma and striated muscle. Plasma glucose concentrations were also measured in these groups of animals. 2. Intracellular metabolite concentrations were calculated by using chloride as an endogenous marker of extracellular space. 3. The mean intracellular ketone-body concentrations (±s.e.m.) were 0.17±0.02, 0.76±0.11 and 2.82±0.50μmol/ml of water in fed, 48h-starved and alloxan-diabetic rats, respectively. Mean (intracellular water concentration)/(plasma water concentration) ratios were 0.47, 0.30 and 0.32 in fed, 48h-starved and alloxan-diabetic rats respectively. The relationship between ketone-body concentrations in the plasma and intracellular compartments appeared to follow an asymptotic pattern. 4. Only intracellular 3-hydroxybutyrate concentrations rose during starvation whereas concentrations of both 3-hydroxybutyrate and acetoacetate were elevated in the alloxan-diabetic state. 5. During starvation plasma glucose concentrations were lowest at 48h, and increased with further starvation. 6. There was no significant difference in the muscle intracellular free fatty acid concentrations of fed, starved and alloxan-diabetic rats. Mean free fatty acid intramuscular concentrations (±s.e.m.) were 0.81±0.08, 0.98±0.21 and 0.91±0.10μmol/ml in fed, 48h-starved and alloxan-diabetic states. 7. The intracellular ketosis of starvation and the stability of free fatty acid intracellular concentrations suggests that neither muscle membrane permeability nor concentrations of free fatty acids per se are major factors in limiting ketone-body oxidation in these states.
The localization of enzymes of intermediary metabolism in Astasia and Euglena
Authors: Bégin-Heick Nicole
Published: August 2015

Results are presented on the intracellular localization of some of the enzymes of gluconeogenesis, of the tricarboxylic acid cycle and of related enzymes in Astasia and Euglena grown with various substrates. The results indicate the particulate nature of at least part of the malate synthase of Astasia and of part of the malate synthase and isocitrate lyase in Euglena. However, the presence of glyoxysomes (microbodies) in Astasia and Euglena is still open to question, since it has not, so far, been possible to separate the enzymes of the glyoxylate cycle from succinate dehydrogenase in the particulate fraction.
An antimycin A- and cyanide-resistant variant of Candida utilis arising during copper-limited growth
Authors: Downie J. A. , Garland P. B.
Published: August 2015

1. During copper-limited growth of Candida utilis in continuous culture on a non-fermentable carbon and energy source there is a selective pressure favouring the emergence of variants that are less dependent on copper. 2. We describe the properties of such a variant that by-passes cytochrome oxidase (EC 1.9.3.1) by utilizing an alternative oxidase communicating with the respiratory chain at about the level of cytochrome b. 3. Both direct studies of isolated mitochondria and calculations based on growth parameters showed that only one of the normal three phosphorylation sites was active. This site was localized between NADH and the cytochromes. 4. Growth of the variant with copper-supplemented media resulted in the return of cytochrome oxidase but not the loss of the alternative oxidase. 5. The alternative oxidase is inhibited by substituted benzhydroxamic acids. 6. Submitochondrial particles from the variant did not exhibit any novel electron-paramagnetic-resonance-spectroscopy features at about g=2.0 either at 80°K or 12°K.
Regulatory properties of adenosine triphosphate-l-methionine S-adenosyltransferase of rat liver
Authors: Lombardini J. B. , Chou Ting-Chao , Talalay Paul
Published: August 2015

1. Double-reciprocal plots of the reaction velocity of yeast, rat liver and Escherichia coli ATP-l-methionine S-adenosyltransferases (EC 2.5.1.6) as a function of the l-methionine concentrations (under saturating ATP conditions) demonstrate downward deflexions from linearity for the yeast and E. coli adenosyltransferases and an upward deflexion for the rat liver enzyme. 2. The activities of partially purified preparations of rat liver ATP-l-methionine S-adenosyltransferase are enhanced by low concentrations of non-substrate analogues of l-methionine [e.g. 1-aminocyclopentanecarboxylic acid (cycloleucine) and l-2-amino-4-hexynoic acid], or by inorganic tripolyphosphate, an ATP analogue. When the concentrations of these analogues were raised further, the activity decreased. Double-reciprocal plots became linear in the presence of these modifier analogues. The inhibitions are common to all the l-methionine adenosyltransferases examined, but the activation(s) were only found with rat and mouse liver enzymes and not with enzymes obtained from several other tissues of these or other species. 3. The rate of formation of S-adenosyl-l-methionine bears a sigmoidal relation to the l-methionine concentrations when ATP is saturating. The activating effects of the l-methionine analogues and of tripolyphosphate are observed at low l-methionine concentrations, and become obliterated as the l-methionine concentration is raised. These findings are analysed in terms of various regulatory enzyme models.
Heparan sulphate sulphotransferase. Properties of an enzyme from ox lung
Authors: Foley T. , Baker J. R.
Published: August 2015

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH4)2SO4 precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was 35S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [35S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [35S]sulphate by high-voltage electrophoresis at pH5.3. The [35S]sulphate is believed to be derived from N-[35S]sulphated groups of heparan [35S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[35S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [35S]sulphate.
The use of sodium perchlorate in deproteinization during the preparation of nucleic acids
Authors: Wilcockson John
Published: August 2015

Sodium perchlorate in high concentrations will remove from solution the detergent sodium dodecyl sulphate and protein complexed with it. This and the failure of proteins to be precipitated by ethanol from solutions containing a high concentration of sodium perchlorate can be utilized as efficient, rapid and simple deproteinization procedures during the preparation of nucleic acids.
Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of the free acids and their carnitine esters on coenzyme A-dependent oxidations in rat liver mitochondria
Authors: Holland P. C. , Sherratt H. S. A.
Published: August 2015

1. The synthesis of pent-4-enoyl-l-carnitine, cyclopropanecarbonyl-l-carnitine and cyclobutanecarbonyl-l-carnitine is described. 2. Pent-4-enoate strongly inhibits palmitoyl-l-carnitine oxidation in coupled but not in uncoupled mitochondria. Pent-4-enoyl-l-carnitine strongly inhibits palmitoyl-l-carnitine oxidation in uncoupled mitochondria. Prior intramitochondrial formation of pent-4-enoyl-CoA is therefore necessary for inhibition. 3. There was a small self-limiting pulse of oxidation of pent-4-enoyl-l-carnitine during which the ability to inhibit the oxidation of subsequently added palmitoyl-l-carnitine developed. 4. Pent-4-enoate and pent-4-enoyl-l-carnitine are equally effective inhibitors of the oxidation of all even-chain acylcarnitines of chain length C4–C16. Pent-4-enoyl-l-carnitine also inhibits the oxidation of pyruvate and of 2-oxoglutarate. 5. Pent-4-enoate strongly inhibits the oxidation of palmitate but not that of octanoate. This is presumably due to competition between octanoate and pent-4-enoate for medium-chain acyl-CoA ligase. 6. There was less inhibition of the oxidation of pyruvate by pent-4-enoyl-l-carnitine, and of palmitoyl-l-carnitine by cyclopropanecarbonyl-l-carnitine, after pre-incubation with 10mm-arsenate. This suggests that these inhibitions were caused either by depletion of free CoA or by increase of acyl-CoA concentrations, since arsenate deacylates intramitochondrial acyl-CoA. There was little effect on the inhibition of palmitoyl-l-carnitine oxidation by pent-4-enoyl-l-carnitine. 7. Penta-2,4-dienoate strongly inhibited palmitoyl-l-carnitine oxidation in coupled mitochondria; acrylate only inhibited slightly. 8. Pent-4-enoate (0.1mm) caused a rapid and almost complete decrease in free CoA and a large increase in acid-soluble acyl-CoA when incubated with coupled mitochondria. Cyclopropanecarboxylate caused a similar decrease in CoA, with an equivalent rise in acid-soluble acyl-CoA concentrations. n-Pentanoate caused extensive lowering of CoA and a large increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. Octanoate caused a 50% lowering of CoA and an increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. 9. Cyclopropanecarboxylate and n-pentanoate were less potent inhibitors of palmitate oxidation than was pent-4-enoate. 10. It is concluded that pent-4-enoate causes a specific inhibition of β-oxidation after the formation intramitochondrially of its metabolites.
Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The purification and properties of an endogenous activator of the enzyme
Authors: Neuberger Albert , Sandy John D. , Tait George H.
Published: August 2015

1. A low-molecular-weight activator of 5-aminolaevulinate synthetase was detected in extracts of Rhodopseudomonas spheroides. The compound activates the enzyme extracted from oxygenated semi-anaerobically grown organisms by a factor of 6–8. 2. The activator was extensively purified, but owing to the exceedingly small amounts that could be extracted in the active form its structure was not determined. 3. The activator contains an acetylatable amino group; it is more stable at acid than at alkaline pH values; it is stable to treatment with I2–KI or potassium ferricyanide, but irreversibly inactivated by Na2S2O4 or NaBH4. 4. The chromatographic, electrophoretic, chemical and stability properties of the activator are similar to those of pteridines; purified activator preparations contain pteridines, as shown by their fluorescence spectrum. This does not, however, constitute an identification of the activator. 5. The activator enhances the activity of crude and partially purified enzyme and does not appear to require other endogenous factors or a supply of air to produce activation. Activation of the purified enzyme, however, requires the presence of either pyridoxal phosphate or sodium succinate. In the absence of both these factors the activator produces a time- and temperature-dependent decay of activity.
The irreversible disposal rate of free fatty acids in the plasma of fed and starved rats
Authors: Cunningham Vincent J.
Published: August 2015

1. The irreversible disposal rate coefficient for free fatty acids in the plasma of fed and starved rats was determined after a single intravenous injection of [1-14C]palmitic acid into each rat. The dose of labelled palmitic acid was given as a complex with131I-labelled albumin in rat serum. The total amount of [1-14C]palmitic acid remaining in the plasma was determined at short times after injection from the14C/131I ratio in the injected serum and in the collected plasma. The rate coefficient was determined from the area under the curve that describes the disappearance of [1-14C]palmitic acid with time from the plasma. Possible sources of error in these determinations are discussed. 2. The irreversible disposal rate coefficient was significantly higher in fed rats (2.07min−1) than in rats which had been starved for 24h (1.53min−1). The possible relationship between this difference and the processes whereby free fatty acids are removed from the plasma is discussed briefly. 3. An estimate of the irreversible disposal rate for free fatty acids in plasma was made from the concentration of free fatty acids in plasma and from the volume of distribution of131I-labelled albumin. The irreversible disposal rate was significantly lower in the fed state than in the starved.
The reconstitution of oxidase activity in membranes derived from a 5-aminolaevulinic acid-requiring mutant of Escherichia coli
Authors: Haddock Bruce A.
Published: August 2015

1. The reconstitution of oxidase activity in cell-free extracts of a mutant of Escherichia coli K12Ymel, that require 5-aminolaevulinic acid for growth on non-fermentable carbon sources, is described. 2. The reconstitution is dependent on haematin or a haem extract from a prototrophic strain of E. coli, and the product of the reaction has been identified as NADH-reducible cytochrome b. 3. The requirement for haematin cannot be replaced by four other porphyrins. Coproporphyrin III does not inhibit the haematin-dependent reconstitution, mesoporphyrin IX and protoporphyrin IX apparently compete with haematin for a binding site on the cytochrome apoprotein(s) and deuteroporphyrin IX binds to cytochrome apoprotein(s) and cannot be subsequently replaced by haematin. 4. The properties of electron-transport particles from cell-free extracts of the mutant strain, grown aerobically in the presence or absence of 5-aminolaevulinic acid, are described. In the absence of 5-aminolaevulinic acid no detectable cytochromes are produced, and oxidase activities are lowered but there is no apparent effect on the activities of the NADH dehydrogenase and d-lactate dehydrogenase. 5. The reconstitution of oxidase activity by electron-transport particles from cells grown in the absence of 5-aminolaevulinic acid requires ATP and haematin, and the product of the reaction was identified as NADH-reducible cytochrome b. 6. It is concluded that the cytochrome apoproteins are synthesized and incorporated into the cytoplasmic membrane of E. coli in the absence of haem synthesis. The subsequent reconstitution of functional cytochrome(s) requires protohaem, but the nature of the side chain on the 2 and 4 positions of the porphyrin appears to be important.
Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from lysine
Authors: Cox Richard A. , Hoppel Charles L.
Published: August 2015

The conversion of l-[U-14C]lysine into carnitine was demonstrated in normal, choline-deficient and lysine-deficient rats. In other experiments in vivo radioactivity from l-[4,5-3H]lysine and dl-[6-14C]lysine was incorporated into carnitine; however, radioactivity from dl-[1-14C]lysine and dl-[2-14C]lysine was not incorporated. Administered l-[Me-14C]methionine labelled only the 4-N-methyl groups whereas lysine did not label these groups. Therefore lysine must be incorporated into the main carbon chain of carnitine. The methylation of lysine by a methionine source to form 6-N-trimethyl-lysine is postulated as an intermediate step in the biosynthesis of carnitine. Radioactive 4-N-trimethylaminobutyrate (butyrobetaine) was recovered from the urine of lysine-deficient rats injected with [U-14C]lysine. This lysine-derived label was incorporated only into the butyrate carbon chain. The specific radioactivity of the trimethylaminobutyrate was 12 times that of carnitine isolated from the urine or carcasses of the same animals. These data further support the idea that the last step in the formation of carnitine from lysine was the hydroxylation of trimethylaminobutyric acid, and are consistent with the following sequence: lysine+methionine → 6-N-trimethyl-lysine → → 4-N-trimethylaminobutyrate → carnitine.
The evolutionary stability of cytochrome c-551 in Pseudomonas aeruginosa and Pseudomonas fluorescens biotype C
Authors: Ambler R. P.
Published: August 2015

Cytochrome c-551 was prepared from nine different strains of Pseudomonas aeruginosa and six of Pseudomonas fluorescens biotype C, and their amino acid sequences were compared with the sequences previously determined for the cytochromes of type strains of each species. The standard of sequence examination was such that all single amino acid substitutions, delections or insertions ought to have been detected. Balanced double changes in sites in the same part of the sequence might have escaped detection. The standard of some of the quantitative amino acid analyses was not as high as would be required for the investigation of completely unknown sequences. Eight of the Ps. aeruginosa sequences could not be distinguished from the type sequence, whereas the ninth had a single amino acid substitution. The sequences from Ps. fluorescens biotype C were more varied, differing in from zero to four substitutions from the type sequence, with the most diverse sequences differing in seven positions. The results for Ps. aeruginosa are interpreted as evidence that neutral mutations are not responsible for much molecular evolution. The superficially paradoxical differences in the results for the two species are discussed.
Further investigation of the increased transfer ribonucleic acid methylase activity in tumours of the mouse colon
Authors: Pegg Anthony E. , Hawks Andrew M.
Published: August 2015

1. Extracts prepared from tumours of the mouse colon induced by 1,2-dimethylhydrazine were considerably more active in catalysing the methylation of tRNA than were extracts from normal colon. The enhanced activity was observed when both unfractionated ‘methyl-deficient’ tRNA and purified tRNA preparations from yeast and bacteria were used as substrates for methylation. 2. The methylated bases produced in these reactions were identified. There were no differences between the products of the reaction catalysed by extracts of tumour and normal colon. 3. The increased activity of tRNA methylases was not due to the presence in the extracts of stimulatory or inhibitory molecules of low molecular weight such as polyamines or S-adenosylhomocysteine. 4. Other enzymes concerned with tRNA metabolism (RNA polymerase, ATP–tRNA adenylyltransferase, aminoacyl-tRNA ligases) were also increased in activity in the tumour tissue. 5. The extent of methylation of a limiting amount of tRNA was greater when tumour extracts were compared with controls, but in no case was it possible to achieve a stoicheiometric methylation of the purified tRNA preparations used as substrates, and the tumour extracts were not able to methylate tRNA obtained from normal mouse colon. We conclude that the tumours contained greater activities of tRNA methylases but that there was no evidence for changes in the specificity of these enzymes during neoplastic growth.
Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins
Authors: Suria D. , Liew C. C.
Published: August 2015

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [3H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110°C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [3H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N2-acetylserine and N2-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.
Inhibition of translation in liver polyribosomes by a new substituted thiopseudourea with antitumour action
Authors: González-Cadavid Néstor F. , Quijada Flor Herrera
Published: August 2015

A new thiopseudourea, S-(10-undecen-1-yl)isothiouronium iodide (compound AHR-1911), was tested for antitumour action and shown to inhibit considerably the growth of the Walker carcinoma in rats. The mechanism of its effect on protein and nucleic acid synthesis was then studied with systems in vitro from rat liver. In incubations of liver slices, 1.4mm-compound AHR-1911 decreased by 96% the incorporation of [14C]leucine into microsomal proteins, and mitochondrial protein synthesis measured in the presence of cycloheximide was decreased by 44%. At lower doses, translation, as well as the incorporation of [3H]uridine into RNA, was also considerably impaired, compound AHR-1911 being the most active of all the thiopseudoureas tested whereas undecylenic acid and thiourea by themselves showed practically no inhibition. Protein synthesis by cytoplasmic ribosomes (microsomes and C-polyribosomes) was inhibited by compound AHR-1911 at different concentrations (72% at 0.42mm), and again the other related compounds were much less effective, with the exception of one antileukaemic thiopseudourea. The same occurred with the poly(U)-stimulated incorporation of phenylalanine. The puromycin reaction with pulse-labelled C-ribosomes was strongly inhibited, particularly when preincubation with compound AHR-1911 preceded the addition of puromycin, with no release of nascent chains by the thiopseudourea alone. In the presence of GTP and pH5 fraction, to induce translocation and transform all the ribosomes to the donor state, the percentage inhibition remained the same. The ribosomes incubated with the drug are aggregated, as shown by the polyribosome profile, but, when excess of inhibitor was removed, the activity in protein synthesis and the puromycin reaction was restored, indicating that the inhibition is not due to the polyribosomal aggregation. These results suggest that the effect on translation with both 55S and 80S ribosomes is derived from inhibition of chain elongation at the level of transpeptidation and not translocation, probably together with the interference with transcription playing a role in the antitumour activity.