bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyruvate metabolism by aminopterin-inhibited Aerobacter aerogenes
Authors: Webb M.
Published: August 2015

1. The synthesis and utilization of both alanine (by reductive amination, oxidative deamination and transamination) and valine (by transamination only) in Aerobacter aerogenes are unaffected by aminopterin. These amino acids, which accumulate in aminopterin-treated cultures of this organism, are therefore considered to be formed as secondary products from the excess of pyruvate that also accumulates. 2. Oxidative metabolism of pyruvate and the synthesis of acetylmethylcarbinol by A. aerogenes cells are unaltered by growth in the presence of aminopterin. 3. Cells from static and anaerobic cultures that have been treated with the folic acid antagonist in the early exponential phase have a decreased ability to cleave pyruvate to acetate and formate, and to effect the exchange of formate with the carboxyl group of pyruvate. 4. 3-Methyl-2-oxobutanoate, the keto acid precursor of valine, cannot replace pyruvate as substrate in either the phosphoroclastic or the exchange reaction.
Some properties of adenosine kinase from Ehrlich ascites-tumour cells
Authors: Murray A. W.
Published: August 2015

1. Adenosine kinase was measured in dialysed extracts from Ehrlich ascites-tumour cells by a chromatographic procedure. 2. In the absence of added Mg2+ the Km values for ATP and adenosine were 0·22mm and 2·8μm respectively. 3. The maximum velocity of adenosine kinase with free ATP was about three times that with the Mg2+–ATP complex. Free Mg2+ was a non-competitive inhibitor of the reaction. A small amount of added Mg2+, Mn2+ or Ca2+ was required for maximum adenosine kinase activity after cation bound to the enzyme had been released by treatment with p-chloromercuribenzoate and then removed by dialysis. 4. GTP, ITP, deoxy-ATP, deoxy-GTP, CTP, xanthosine triphosphate, UTP and thymidine triphosphate could partially or completely replace ATP as a phosphate donor. 5. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, GMP, IMP, ADP, deoxy-ADP and IDP (Ki 0·2, 1·1, 5·9, 1·2, 0·5 and 0·78mm respectively). Enzymic activity was markedly affected by the relative concentrations of AMP, ADP and ATP in assay mixtures. 6. The results are discussed in terms of possible mechanisms regulating the rate of adenosine kinase in vivo.
A comparison of horseradish peroxidase and manganese ions as catalysts for the oxidation of dihydroxyfumaric acid
Authors: Hartree E F
Published: August 2015

With horseradish peroxidase as catalyst the main product was dihydroxytartrate, but small amounts of glycolaldehyde, mesoxalic semialdehyde, mesoxalate and possibly glyoxal were also formed. Mn2+ catalysis gave rise only to mesoxalate and oxalate. When oxygen uptake was followed by a manometric method the rate of the peroxidase-catalysed reaction was proportional to oxygen concentration and marked inhibition by cyanide was obtained only at low buffer concentration. The catalytic effects of peroxidase and Mn2+ were almost always additive. Chelating agents inhibited the Mn2+-catalysed reaction, but had either no effect or a slight accelerating effect on the peroxidase-catalysed reaction. It is concluded that Mn2+ does not function as cofactor in the peroxidase-catalysed oxidation.
The permeability of mitochondria to oxaloacetate and malate
Authors: Haslam J. M. , Krebs H. A.
Published: August 2015

1. A spectrophotometric assay of the rates of penetration of oxaloacetate and l-malate into mitochondria is described. The assay is based on the measurement of the oxidation of intramitochondrial NADH by oxaloacetate and of the reduction of intramitochondrial NAD+ by malate. 2. The rate of entry of both oxaloacetate and l-malate into mitochondria is restricted, as shown by the fact that disruption of the mitochondrial structure can increase the rate of interaction between the dicarboxylic acids and intramitochondrial NAD+ and NADH by between 100- and 1000-fold. 3. The rates of entry of oxaloacetate and malate into liver, kidney and heart mitochondria increased by up to 50-fold on addition of a source of energy, either ascorbate plus NNN′N′-tetramethyl-p-phenylenediamine aerobically, or ATP anaerobically. 4. In the absence of a source of energy the changes in the concentrations of intramitochondrial NAD+ and NADH brought about by the addition of l-malate or oxaloacetate were followed by parallel changes in the concentrations of NADP+ and NADPH, indicating the presence in the mitochondria of an energy-independent transhydrogenase system. 5. The results are discussed in relation to the hypothesis that malate acts as a carrier of reducing equivalents between mitochondria and cytoplasm.
Biochemical aspects of bovine ketosis
Authors: Baird G D , Hibbitt K G , Hunter G D , Lund Patricia , Stubbs Marion , Krebs H. A.
Published: August 2015

1. The concentrations of acetoacetate, β-hydroxybutyrate and metabolites related to gluconeogenesis were determined in biopsy samples of the livers of ketotic, normal lactating and normal non-lactating cows. Key enzymes of gluconeogenesis in the liver were also assayed. 2. Significant decreases were found in the ketotic liver in the concentrations of glucogenic amino acids (glutamate, glutamine, alanine) and of glucogenic oxo acids (α-oxoglutarate, pyruvate, oxaloacetate). 3. The β-hydroxybutyrate/acetoacetate concentration ratios were generally much higher than in rat liver. 4. The concentration of total fat was sevenfold higher in the ketotic liver, and that of glucose plus glycogen fourfold lower than in normal liver. 5. The blood of ketotic cows showed a marked rise in the concentration of free fatty acids. 6. The activities of pyruvate carboxylase, propionyl-CoA carboxylase, phosphopyruvate carboxylase and fructose 1,6-diphosphatase showed no clear-cut differences between normal and ketotic animals. 7. Glucose injection promptly relieved the ketotic condition with respect to both the clinical and biochemical signs. The fall in the concentrations of the ketone bodies in the blood was preceded by a fall in the concentrations of free fatty acids and glycerol. 8. The findings are taken to be consistent with the concept that an increased rate of gluconeogenesis, causing a decrease in the concentration of oxaloacetate, is a major causal factor in ketogenesis.
The comparative structure of mammalian glyceraldehyde 3-phosphate dehydrogenase
Authors: Perham R N
Published: August 2015

1. The amino acid sequences around the thiol groups of glyceraldehyde 3-phosphate dehydrogenase from badger and monkey skeletal muscle were compared with the sequences around the thiol groups in the enzyme isolated from other organisms. 2. Preliminary evidence of the existence of isoenzymes in the badger was obtained. Only the major form, however, could be purified completely. 3. The monkey enzyme contains only three cysteine residues per polypeptide chain compared with the four found in all the other mammalian enzymes so far examined, including that of badger, and the two in yeast. The missing thiol group in monkey was identified as residue 281 in the corresponding sequence of the pig enzyme. 4. These experiments rule out any essential role for cysteine-281 in the function of the mammalian enzymes. 5. Further evidence of the remarkable conservation of amino acid sequence in this enzyme during evolution is presented and discussed.
The metabolism of potassium dodecyl [35S]-sulphate in the rat
Authors: Denner W. H. B. , Olavesen A H , Powell Gillian M. , Dodgson K S
Published: August 2015

The metabolic fate of potassium dodecyl [35S]sulphate was studied in rats. Intraperitoneal and oral administration of the ester into free-ranging animals were followed by the excretion of the bulk of the radioactivity in the urine within 12hr., approximately 17% being eliminated as inorganic [35S]sulphate. Similar results were obtained in experiments in which potassium dodecyl [35S]sulphate was injected intravenously into anaesthetized rats with bile-duct and ureter cannulae. Analysis of urinary radioactivity revealed the presence of a new ester sulphate (metabolite A). This metabolite was isolated, purified and subsequently identified as the sulphate ester of 4-hydroxybutyric acid by paper, thin-layer and gas chromatography, by paper electrophoresis and by comparison of its properties with those of authentic butyric acid 4-sulphate. The identity of the metabolite was confirmed by isotope-dilution experiments. When either purified metabolite A or authentic potassium butyric acid 4[35S]-sulphate was administered to free-ranging rats the bulk of the radioactivity was eliminated unchanged in the urine within 12hr., approx. 20% of the dose appearing as inorganic [35S]sulphate. Whole-body radioautography and isolated-liver-perfusion experiments implicated the liver as the major site of metabolism of potassium dodecyl [35S]sulphate. It is suggested that butyric acid 4-sulphate probably arises by ω-oxidation of dodecyl sulphate to a fatty acid-like compound, which is then degraded by β-oxidation.
Electron-spin-resonance evidence for enzymic reduction of oxygen to a free radical, the superoxide ion
Authors: Knowles P F , Gibson J. F. , Pick F. M. , Bray R C
Published: August 2015

1. An electron-spin-resonance signal with g∥2·08 and g⊥2·00 is observed by the rapid-freezing technique during the oxidation of substrates by molecular oxygen catalysed by xanthine oxidase at pH10. 2. The intensity of this signal is shown to depend on oxygen rather than on enzyme concentration, indicating that it is due to an oxygen free radical and not to the enzyme. 3. The same species is shown to be produced in the reaction at pH10 between hydrogen peroxide and periodate ions. Studies with this system have facilitated comparison of the properties of the oxygen radical with data in the literature on the products of pulse radiolysis of oxygenated water over a wide pH range. 4. It is concluded that the species observed is the superoxide ion, O2−, and that the stability of this ion is greatly increased in alkaline solution. A mechanism explaining the alkaline stability is proposed. 5. The importance of O2− in the enzymic reaction is discussed.
Studies on metabolism of vitamin A. The effect of hormones on gestation in retinoate-fed female rats
Authors: Juneja H. S. , Moudgal N. R. , Ganguly J.
Published: August 2015

1. Female rats, raised to maturity on a vitamin A-deficient diet supplemented with retinoate as the sole source of vitamin A, conceived when allowed to mate with normal males, but the conception resulted in foetal resorption, beginning from day 14 or 15 of pregnancy. 2. Daily injections of pregnenolone or oestradiol-17β, but not of progesterone, were fully effective, and transplantation of pituitary homografts under kidney capsules was 80% effective, in preventing resorption. 3. Unilateral ovariectomy, leading to compensatory hypertrophy of the remaining ovary, resulted in significantly fewer corpora lutea and in lower weight of the hypertrophied ovary of the retinoate-treated rats, as compared with the corresponding retinyl acetate-fed ones. 4. The activity of the enzyme system Δ5-3β-hydroxy steroid dehydrogenase was significantly less in the ovaries of the retinoate-fed rats that were subjected to unilateral ovariectomy or were made pregnant, when compared with that of the corresponding controls. Also, the ovaries of the retinoate-treated rats were insensitive to both exogenous and endogenous gonadotrophin stimulus. 5. These results indicate that one of the reasons for foetal resorption in the retinoate-fed rats might be inadequate synthesis of steroid hormones such as pregnenolone and oestrogen.
Evidence for histidine in the triethyltin-binding site of rat haemoglobin
Authors: Rose M. S.
Published: August 2015

One molecule of rat haemoglobin binds two molecules of triethyltin. The binding sites are located on the globin and there is co-operativity between the sites such that the intrinsic affinity constant at pH8·0 increases from 3·5×105m−1 for the binding of the first triethyltin molecule to 5·0×105m−1 for the binding of the second. Evidence is presented, from pH studies and the kinetics of inhibition due to photo-oxidation, that each binding site contains two histidine residues.
Studies on the biosynthesis of phenols in fungi. Production of 4-methoxytoluquinol, epoxysuccinic acid and a diacetylenic alcohol by surface cultures of Lentinus degener I.M.I. 110525
Authors: Packter N. M.
Published: August 2015

1. 4-Methoxytoluquinol was secreted into the medium by surface cultures of the basidiomycete Lentinus degener Kalchbr. (approx. 100mg./l. of medium). In addition, epoxysuccinic acid (150–200mg.) and a long-chain diacetylenic alcohol (3mg.) were also secreted. Epoxysuccinic acid has previously been found in the culture medium of some Fungi Imperfecti. These metabolites were all synthesized during the early phase of growth but maximum production occurred some time later. 2. Supplementation of the medium with cycloheximide or 8-azaguanine inhibited the production of epoxysuccinic acid. 3. Sodium [1−14C]acetate and 6-methyl[14C]salicylic acid were not incorporated into 4-methoxytoluquinol, but [U−14C]tyrosine and [Me−14C]methionine were incorporated to the extent of 0·55 and 4·75% respectively (minimum values). Degradation studies established that the aromatic ring and C-methyl group were derived from the ring and β-carbon atom of tyrosine; the O-methyl group alone was formed from methionine.
The effect of starvation on the incorporation of palmitate into glycerides and phospholipids of rat liver homogenates
Authors: Vavrečka M. , Mitchell M P , Hübscher G.
Published: August 2015

1. Glyceride biosynthesis from glycerol phosphate and [1−14C]palmitate was studied in liver homogenates of rats that were fed ad libitum or starved for 36–40hr. The changes in enzyme activity were related to total DNA content or total liver homogenate as these were found to be equivalent and to be the most meaningful parameters. 2. In liver homogenates from fed rats, labelled palmitate was incorporated mainly into phosphatidate (58% of the total incorporation into lipids), diglycerides (25%) and triglycerides (16%), whereas monoglycerides, cholesterol esters and phospholipids other than phosphatidate were labelled only to a small extent. Addition of particle-free supernatant to full homogenates increased the total incorporation of palmitate by 45% and the pattern of incorporation altered to 53% incorporated into triglycerides, 24% into diglycerides and 17% into phosphatidate. This result suggested that, in liver homogenates, phosphatidate phosphohydrolase (EC 3.1.3.4) may be rate-limiting in the biosynthesis of glycerides via the glycerol phosphate pathway. 3. Upon starvation, the amount of palmitate incorporated per liver into total phospholipids plus glycerides was decreased to between 68% and 75% of that observed with fed animals. In homogenates from fed animals 41–44% of the labelled phospholipids plus glycerides was in glycerides; this value increased to between 63% and 75% with starved rats. Of the palmitate incorporated into total phospholipids, between 85% and 86% was found in phosphatidate, independent of the nutritional state of the animal. The ratio of palmitate incorporated into triglycerides/diglycerides rose from 0·7, obtained with fed rats, to 1·0 with starved animals. 4. These results indicate that starvation caused a decrease in the activity (per total liver) of acyl-CoA–glycerol phosphate acyltransferase(s) (EC 2.3.1.15) and an increase in the activity of acyl-CoA–diglyceride acyltransferase (EC 2.3.1.20). The largest change, however, seemed to be related to the increased activity of the phosphatidate phosphohydrolase in the particle-free supernatant. 5. The latter enzyme was assayed in the particle-free supernatant with membrane-bound phosphatidate as substrate. In starvation, the activity per total liver was increased to between 130% and 190% and the specific activity to between 180% and 320% of the values for fed rats.
A lipid intermediate in the synthesis of a poly-(N-acetylglucosamine 1-phosphate) from the wall of Staphylococcus lactis N.C.T.C. 2102
Authors: Brooks D. , Baddiley J
Published: August 2015

1. The enzymic synthesis of the wall polymer poly-(N-acetylglucosamine 1-phosphate) in Staphylococcus lactis N.C.T.C. 2102 was studied by using UDP-[acetyl−14C]N-acetylglucosamine and the corresponding nucleotide containing 32P. 2. Labelled material was extracted from the particulate enzyme preparation with butan-1-ol. Pulse-labelling experiments indicated that this material contained an intermediate in the biosynthesis. 3. The lipid intermediate was partially purified, and chemical and enzymic degradation showed that it was composed of N-acetylglucosamine 1-pyrophosphate in labile ester linkage to an organic-soluble alcohol, possibly a polyisoprenoid alcohol. The methanolysis of sugar 1-pyrophosphate derivatives, including nucleoside diphosphate sugars, is discussed in relation to degradation products obtained from the lipid. 4. The lipids from the particulate enzyme preparation probably contained another compound in which N-acetylglucosamine 1-phosphate is attached to an organic-soluble alcohol; this may participate in the biosynthesis of another polysaccharide. 5. The function of the lipid intermediate in polymer biosynthesis is discussed.
Acetoacetylation of insulin
Authors: Lindsay D. G. , Shall S.
Published: August 2015

Insulin was treated with diketen at pH6·9. The reaction mixture was resolved into four components by DEAE-Sephadex chromatography. The first component was unchanged insulin. The second and third components were shown by end-group analysis to be substituted on phenylalanine B-1 and glycine A-1 respectively. The fourth component was disubstituted on both phenylalanine B-1 and glycine A-1. The ∈-amino group of lysine B-29 was not involved in the reaction at low reagent concentrations. The purity of these derivatives was checked by their electrophoretic behaviour and by measurement of the rate of their reaction with trinitrobenzenesulphonic acid. The hormonal activity of the derivatives was determined. The effect of the modifications on the hormonal activity and the tertiary structure of insulin is discussed.
Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide–adenine dinucleotide in barley
Authors: Ryrie I. J. , Scott K. J.
Published: August 2015

1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD+ were measured in the first leaf of barley seedlings. 2. In small amounts, both [14C]nicotinate and [14C]quinolinate were quickly and efficiently incorporated into NAD+ and some evidence is presented suggesting that NAD+ is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [14C]Nicotinamide served equally well as a precursor of NAD+ and although significant amounts of [14C]NMN were detected, most of the [14C]NAD+ was derived from nicotinate intermediates formed by deamination of [14C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [14C]nicotinate and [14C]quinolinate but most probably it arose from the breakdown of [14C]NAD+. 5. In barley leaves where the concentration of NAD+ is markedly increased by infection with Erysiphe graminis, the pathways of NAD+ biosynthesis did not appear to be altered after infection. A comparison of the rates of [14C]NAD+ formation in infected and non-infected leaves indicated that the increase in NAD+ content was not due to an increased rate of synthesis.
Cytoplasmic effects of cortisol in liver
Authors: Cox R. F. , Mathias A P
Published: August 2015

1. The time-course of the induction by cortisol of tryptophan pyrrolase in mouse liver was established. 2. The kinetics of labelling of mouse liver polyribosomes and the distribution of radioactivity in the various sizes of polyribosomes were investigated. It is concluded that the major part of the messenger RNA is monocistronic. 3. Cortisol administered to adrenalectomized mice stimulates in parallel the ability of ribosomes and polyribosomes, both free and bound, to incorporate amino acids into protein. 4. This stimulation does not appear to affect in a selective manner particular regions of the gradients of polyribosomes. 5. Administration of cortisol enhances the ability of the denser fraction of the rough microsomes to incorporate labelled amino acids. Adrenalectomy has minor effects on the pool sizes and specific radioactivities of liver amino acids which are counteracted by the hormone. However, these appear insufficient to influence the changes observed. 6. Adrenalectomy causes an increase in the percentage of ribosomes existing as polyribosomes and a fall in the proportion bound to the membrane of the endoplasmic reticulum. After treatment of these animals with cortisol there is a marked increase in the proportion of bound ribosomes. 7. The influence of cortisol on the membranous components of liver cytoplasm is discussed.
Some properties of cytochrome b5 from liver microsomes of man, monkey, pig and chicken
Authors: Nóbrega F. G. , Araujo P. S. , Pasetto M. , Raw I.
Published: August 2015

1. Cytochrome b5 was released from liver microsomes of man, monkey, pig and chicken by incubation with a crude lipase preparation. 2. By using DEAE-cellulose chromatography, ammonium sulphate fractionation, Sephadex-gel filtration and a final gradient elution on DEAE-Sephadex A-50, cytochromes b5 were obtained from the four species studied, all possessing similar spectral properties. 3. Stokes radii of the cytochromes were measured by gel filtration. 4. N-Terminal amino acids for the different cytochromes were serine for man and monkey, alanine for pig and glycine for chicken. 5. Amino acid analyses of the cytochromes are presented. 6. Peptide ‘fingerprint’ patterns of tryptic digests of the different cytochromes are discussed and clearly show increasing similarity for more closely related species.
The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei
Authors: Haines M E , Johnston I R , Mathias A P , Ridge D.
Published: August 2015

1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed.
The solubilization of collagen and protein–polysaccharides from the developing cartilage of lathyrytic chicks
Authors: Glimcher Melvin J. , Seyer Jerome , Brickley Diane M.
Published: August 2015

1. The solubilization of collagen and protein–polysaccharides from the developing cartilage of normal and lathyritic chicks was studied by using mild extraction procedures. One-third of the protein–polysaccharides could be solubilized in salt solutions at neutral pH from normal cartilage, whereas 95–100% could be extracted from the cartilage of animals that were severely lathyritic. Likewise, whereas in normal animals the collagen of cartilage was essentially insoluble in salt solutions at neutral pH, in lathyritic animals it was almost completely soluble. 2. The increased solubility of the collagen of cartilage from lathyritic animals enabled sufficient material to be collected so that the pure α1 chains of the collagen were isolated by repeated reconstitution, precipitation and CM-cellulose column chromatography. The purified α1 component was characterized by its relatively high content of hydroxylysine (14 residues/1000 amino acids). 3. About 37% of the collagen from the cartilage of normal chick embryos could be extracted as the gelatin at pH7·4 in lithium chloride solution. This was accompanied by the extraction of approx. 14% of the protein–polysaccharide content. 4. The protein–polysaccharides and the collagen from normal animals could be extracted from the cartilage relatively independently of one another under mild conditions. These same components obtained from lathyritic animals easily separated from one another after solubilization. This provided evidence that the two components are probably not covalently cross-linked. 5. The collagen of cartilage extracted as a gelatin from normal animals contained a high proportion of α chains compared with β dimers, similar to the lathyritic collagen of cartilage and other tissues, and similar to the gelatin extracted from normal chick bone.
Identification of mycobactins by nuclear-magnetic-resonance spectroscopy
Authors: Greatbanks D. , Bedford G. R.
Published: August 2015

Details are given of the n.m.r. spectra of mycobactins F, H, M, N, P, S and T, and resonances are ascribed to all the protons in these molecules. A simplified system is described for identifying known mycobactins by the n.m.r. spectrum alone. This method will not distinguish mycobactins S and T, whose nuclei differ only in the configuration at an asymmetric centre.
Absence of a common intermediate pool among individual enzyme chains of the energy-conservation pathway in chloroplasts of Euglena gracilis
Authors: Kahn Joseph S.
Published: August 2015

Tri-n-butyltin chloride is a specific inhibitor that binds stoicheiometrically and irreversibly with the ATP-synthesizing sites of chloroplasts. Titration of Euglena chloroplasts with tri-n-butyltin chloride shows about six ATP-synthesizing sites per molecule of cytochrome c552 or per 380 molecules of chlorophyll. This system was used to study the possibility of linkage between individual enzyme chains of the energy-conservation pathway or the possible existence of a common pool of an intermediate in this pathway. The inhibition of ATP synthesis by tri-n-butyltin chloride at low rates of electron transport (low light-intensities, NADP+ or ferricyanide as electron acceptor) agrees with a kinetic model of two to four ATP-synthesizing sites per energy-conservation chain. At high rates of electron transport, however, the results occasionally agree with a model of 20 or more sites per chain. The results are interpreted as indicating the absence of a common intermediate pool, but the presence of a limited degree of linkage between individual chains. It also indicates the presence of two energy-conservation sites in these chloroplasts.
Inhibition by α-amanitin of ribonucleic acid polymerase solubilized from rat liver nuclei
Authors: Novello F. , Fiume L. , Stirpe F.
Published: August 2015

1. α-Amanitin inhibits in vitro the RNA polymerase solubilized from isolated rat liver nuclei. 2. In contrast with previous observations with whole nuclei, the inhibition occurs approximately to the same extent in the presence and in the absence of ammonium sulphate. 3. Evidence is presented that the toxin acts by interacting with the enzyme itself and not with DNA or other components.
Effect of α-p-chlorophenoxyisobutyrate on the metabolism of isoprenoid compounds in the rat
Authors: Krishnaiah K. V. , Ramasarma T.
Published: August 2015

1. Feeding of α-p-chlorophenoxyisobutyrate (CPIB) to rats increased ubiquinone concentration in the liver but not in other tissues. The increase was progressive with the time of feeding and related to the concentration of CPIB in the diet. 2. Incorporation of [1-14C]acetate, but not of [2-14C]mevalonate, into sterols in the liver in vivo or by liver slices in vitro was decreased on feeding the rats with CPIB. However, incorporation of mevalonate into ubiquinone increased. 3. CPIB, when added in low concentrations to liver slices, had no effect on isoprene synthesis from acetate; higher concentrations, however, were inhibitory. 4. No activation of ubiquinone synthesis from mevalonate was observed when CPIB was added to the liver slices synthesizing ubiquinone. 5. The increase in ubiquinone in CPIB-fed animals appears to be due to increased synthesis in the initial stages and to decreased catabolism in the later stages. 6. An inverse relationship was found between the concentration of ubiquinone in the liver and the serum sterol concentration in CPIB-fed rats.
Gluconeogenesis in the kidney cortex. Effects of d-malate and amino-oxyacetate
Authors: Rognstad R. , Katz J.
Published: August 2015

1. Rat kidney-cortex slices incubated with d-malate alone formed very little glucose. d-Malate, however, augmented gluconeogenesis from l-lactate and inhibited gluconeogenesis from pyruvate and l-malate. 2. d-Malate had little effect on the rate of the tricarboxylic acid cycle with or without other substrates added. 3. d-Malate inhibited the activity of the l-malate dehydrogenase in a high-speed-supernatant fraction from kidney cortex. 4. It was concluded that d-malate inhibited either the operation of the cytoplasmic l-malate dehydrogenase or malate outflow from the mitochondria in the intact kidney-cortex cell. This supports the hypothesis of Lardy, Paetkau & Walter (1965) and Krebs, Gascoyne & Notton (1967) on the role of malate as carrier for carbon and reducing equivalents in gluconeogenesis. 5. Gluconeogenesis from l-lactate in kidney-cortex slices was strongly inhibited by a low concentration (0.1mm) of amino-oxyacetate, whereas glucose formation from pyruvate, malate, aspartate and several other compounds was only slightly affected. 6. High concentrations of l-aspartate largely reversed the inhibition of gluconeogenesis from l-lactate caused by amino-oxyacetate. 7. Amino-oxyacetate inhibited strongly the glutamate–oxaloacetate transaminase in the 30000g supernatant fraction of a kidney-cortex homogenate. The presence of l-aspartate decreased the inhibition of the transaminase by amino-oxyacetate. 8. Detritiation of l-[2-3H]aspartate was inhibited by 90% during an incubation of kidney-cortex slices with l-lactate and amino-oxyacetate. 9. Low concentrations (10μm) of artificial electron acceptors such as Methylene Blue and phenazine methosulphate abolished most of the inhibition of gluconeogenesis from l-lactate by amino-oxyacetate. This is interpreted as an activation of net malate outflow from the mitochondria by-passing the inhibited transfer of oxaloacetate. 10. These findings support the concept that transamination to aspartate is involved in the transfer of oxaloacetate from mitochondria to cytosol required in gluconeogenesis from l-lactate.
Functional significance of isoenzymes in thermal acclimatization. Acetylcholinesterase from trout brain
Authors: Baldwin John , Hochachka P. W.
Published: August 2015

1. The effects of acclimatization temperature on the catalytic properties of acetylcholinesterase from rainbow-trout brain were examined. 2. Trout brain acetylcholinesterase occurs in two distinct forms. A single ‘warm’ variant of the enzyme is present after acclimatization to 17°C; a single ‘cold’ variant appears after acclimatization to 2°C. Both forms are present in fish after acclimatization to an intermediate temperature. 3. The Km values of the enzyme variants for acetylcholine are temperature-dependent, the lowest values coinciding with the acclimatization temperature at which each enzyme was induced. 4. It is concluded that the Km–temperature relationship is adaptive, and that the critical process during thermal acclimatization, in cases where enzymes show sharp changes in Km with temperature, is the synthesis of a new enzyme variant that is better suited for catalysis and control of catalysis under the conditions of the acclimatized state.
Purification and properties of α-d-mannosidase from the limpet, Patella vulgata
Authors: Snaith Sybil M. , Levvy G. A. , Hay A. J.
Published: August 2015

1. α-Mannosidase from the limpet, Patella vulgata, was purified nearly 150-fold, with 40% recovery. β-N-Acetylglucosaminidase was removed from the preparation by treatment with ethanol. The final product was virtually free from β-galactosidase. 2. Limpet α-mannosidase was assayed at pH3.5 and at this pH it was necessary to add Zn2+ for full activity. At pH5, the enzyme had the same activity in the presence or absence of added Zn2+. 3. On incubation at acid pH, the enzyme underwent reversible inactivation, which was prevented by adding Zn2+. 4. EDTA accelerated inactivation and the addition of Zn2+ at once restored activity. No other cation was found to reactivate the enzyme. 5. Cl- had an unspecific effect on hydrolysis by limpet α-mannosidase. It increased the rate of reaction with substrate. The anion did not prevent or reverse inactivation by EDTA. 6. It is concluded that α-mannosidase is a metalloenzyme or enzyme–metal ion complex, dissociable at the pH of activity, and that it requires Zn2+ specifically.
The effects of isoniazid on the carbohydrates of Mycobacterium tuberculosis BCG
Authors: Winder F. G. , Rooney S. A.
Published: August 2015

1. Mycobacterium tuberculosis BCG was usually grown in glycerol–asparagine–casein hydrolysate medium. A soluble fraction was obtained from the cells with aq. 50% ethanol; unbound lipids were then removed and the cells were treated with dilute alkali to give, after acidification, an alkali-extractable fraction and an insoluble fraction. On occasion, lipopolysaccharides were obtained by extracting with phenol or dimethyl sulphoxide instead of alkali. The soluble fraction contained, particularly after long extraction, polysaccharide containing mainly glucose, in addition to trehalose and monosaccharides and their derivatives. The alkali-extractable fraction contained polysaccharides containing mannose, glucose, arabinose, galactose and 6-O-methylglucose. These could be resolved into three fractions of markedly different molecular size. It is argued that the high-molecular-weight materials originated from the outside of the cell envelope and the medium-molecular-weight materials from a middle layer of the envelope. 2. Exposure of the growing cells to isoniazid, usually at 1 or 10μg/ml for 6–12h, increased the total cell carbohydrate, mainly due to an increase in trehalose and in insoluble glucan. It also facilitated the extraction of polysaccharide into the medium and the soluble fraction. This produced about a 25% decrease in the amount of carbohydrate in the alkaline-extractable fraction, mainly due to a fall in glucose, arabinose and 6-O-methylglucose. The decrease was confined to polysaccharides of large and medium molecular weight. When intact lipopolysaccharides were extracted, their amount was also decreased by isoniazid. 3. Substitution of ammonium sulphate for asparagine and casein hydrolysate in the medium, so that glycerol was the sole carbon source, decreased the carbohydrate accumulation brought about by isoniazid but did not alter its effect on polysaccharide extraction. 4. Growth with 14C-labelled substrates showed that glycerol provided two to four times as much of the cell carbon as did asparagine, when both were present. Under these conditions isoniazid inhibited the incorporation of carbon atoms from asparagine into the cells, but had little effect on the total incorporation from glycerol. These experiments also showed that the effect of isoniazid on alkali-extractable polysaccharides was due to their loss to the soluble fraction and external medium. 5. It is suggested that isoniazid inhibits a pathway (probably the synthesis of mycolic acid) involved in the formation of the cell envelope, and that this inhibition results in some re-channelling of intermediates into carbohydrate synthesis and in some loss of polysaccharides through damage to the envelope.
The control by insulin of amino acid accumulation in muscle
Authors: Manchester K. L.
Published: August 2015

1. The capacity of insulin to enhance the accumulation by muscle of several amino acids was studied. Reports that threonine uptake is enhanced by insulin were not confirmed, despite its enhanced incorporation into protein. Uptake of β-alanine and γ-aminobutyric acid also did not respond to the hormone. A stimulation of accumulation of alanine, histidine and ethionine was observed. 2. The capacity of inhibitors of protein synthesis to reveal a stimulation by insulin of accumulation of several amino acids, hitherto considered unresponsive to insulin in the absence of inhibitor, was confirmed. Cycloheximide was as effective as puromycin. However, two of these amino acids, alanine and histidine, here showed response to insulin in the absence of inhibitor. The presence of cycloheximide was found to enhance uptake of cycloleucine, ethionine and threonine; in its presence insulin enhanced uptake of β-alanine and α-methyltyrosine. 3. It is concluded that the influence of inhibitors of protein synthesis on amino acid accumulation and the response of amino acid accumulation to insulin are not adequately explained on the basis of inhibition of protein synthesis allowing amino acids to accumulate more readily. 4. The technical problems of whether linear rates of incorporation of amino acids into protein really indicate more than one cell pool are discussed and safeguards suggested. That initial rates of incorporation of label into protein are likely to be non-linear is shown.
Biosynthesis of phytoquinones. Homogentisic acid: a precursor of plastoquinones, tocopherols and α-tocopherolquinone in higher plants, green algae and blue–green algae
Authors: Whistance G. R. , Threlfall D. R.
Published: August 2015

1. By means of 14C tracer experiments and isotope competition experiments the roles of d-tyrosine, p-hydroxyphenylpyruvic acid, p-hydroxyphenylacetic acid, phenylacetic acid, homogentisic acid and homoarbutin (2-methylquinol 4-β-d-glucoside) in the biosynthesis of plastoquinones, tocopherols and α-tocopherolquinone by maize shoots was investigated. It was established that d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid can all be utilized for this purpose, whereas p-hydroxyphenylacetic acid, phenylacetic acid and homoarbutin cannot. Studies on the mode of incorporation of d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid showed that their nuclear carbon atoms and the side-chain carbon atom adjacent to the nucleus give rise (as a C6-C1 unit) to the p-benzoquinone rings and nuclear methyl groups (one in each case) of plastoquinone-9 and α-tocopherolquinone and the aromatic nuclei and nuclear methyl groups (one in each case) of γ-tocopherol and α-tocopherol. 2. By using [14C]-homogentisic acid it has been shown that homogentisic acid is also a precursor of plastoquinone, tocopherols and α-tocopherolquinone in the higher plants Lactuca sativa and Rumex sanguineus, the green algae Chlorella pyrenoidosa and Euglena gracilis and the blue–green alga Anacystis nidulans.
Evidence for the existence of a multichain proteoglycan of heparan sulphate
Authors: Jansson L. , Lindahl U.
Published: August 2015

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.
The isolation by isopycnic density-gradient centrifugation of two mitochondrial populations from livers of embryonic and fed and starved adult rats
Authors: Pollak J. K. , Munn E. A.
Published: August 2015

1. Mitochondria isolated from livers of fed adult, starved adult, and embryonic rats can be separated into three distinct bands by isopycnic density centrifugation on a sucrose density gradient. The least dense band (B1) has a mean buoyant density of 1.162 and consists mainly of disrupted mitochondria. The middle band (B2) has a mean buoyant density of 1.184. The most dense band (B3) has a mean buoyant density of 1.216. B2 and B3 consist of intact mitochondria. 2. The mitochondria in B2 and B3 have very similar protein/phospholipid ratios, virtually identical phospholipid and fatty acid compositions and similar specific activities for cytochrome oxidase, malate dehydrogenase and monoamine oxidase. Both fractions have very low glucose 6-phosphatase and acid phosphatase activities. 3. As isolated, adult rat liver mitochondria have electron-dense matrices (condensed forms); some embryonic liver mitochondria are condensed, but a significant proportion have dilated matrices. All B2 mitochondria are in the condensed form. B3 mitochondria from adult rats are condensed if fixed in their equilibrium-density sucrose, but when this is diluted rapidly to 0.25m they become swollen. Some B3 mitochondria from embryonic rats are condensed, the others have dilated matrices. They all swell if rapidly diluted to 0.25m-sucrose. B2 mitochondria retain their condensed form on dilution of the sucrose. 4. It is concluded that the matrix space of B2 mitochondria is almost totally inaccessible to sucrose, but that of B3 mitochondria is readily accessible to sucrose. 5. In liver from normally fed adult rats the B2 mitochondria predominate, whereas in starved rats B2 and B3 are present in approximately equal proportions. Mitochondrial preparations from embryonic liver consist predominantly of B3 mitochondria, but the proportion of these decreases progressively as development proceeds. 6. The B2 mitochondria from livers of fed adult rats can be converted into B3 mitochondria by incubation with 10mm-succinate and 10mm-phosphate. 7. Some B2 mitochondria are converted into B3 mitochondria by exposure to high concentrations of sucrose.
Mescaline-induced changes of brain-cortex ribosomes. Effect of mescaline on the stability of brain-cortex ribosomes
Authors: Datta R. K. , Ghosh J. J.
Published: August 2015

1. During the action of mescaline sulphate on goat brain-cortex slices the ribosomal particles become susceptible to breakdown, releasing protein, RNA, acidsoluble nucleotides and ninhydrin-positive materials, resulting in loss of ribosomal enzyme activities. 2. Ribosomes of the mescaline-treated cortex slices undergo rapid degradation in the presence of trypsin and ribonuclease. 3. Mescaline does not alter the chemical and nucleotide compositions or the u.v.-absorption characteristics of ribosomal particles, however.
Reversible inhibition in bimolecular rapid-equilibrium random-order enzyme systems. The effect of substrate–substrate and inhibitor–substrate interactions
Authors: Clark A. G.
Published: August 2015

A model is presented that accounts for all types of reversible inhibition by a single inhibitor molecule in bimolecular rapid-equilibrium random-order enzyme systems. The characterization of inhibition mechanisms by graphical methods is examined, and a system of nomenclature is suggested.
A ribonuclease from human skeletal muscle
Authors: Goldspink D. F. , Pennington R. J.
Published: August 2015

1. A ribonuclease has been prepared from human muscle by ammonium sulphate fractionation, heat treatment and ion-exchange chromatography. 2. The enzyme degrades polycytidylic acid and polyuridylic acid to the nucleoside 3′-phosphates, with nucleoside 2′:3′-cyclic phosphates as intermediates. Polyadenylic acid and polyguanylic acid are not attacked. 3. The enzyme has maximal activity at pH8.5. The molecular weight (by gel filtration) is between 11000 and 12000. It is relatively heat-stable. It exhibits optimum activity in a medium of high ionic strength, and is inhibited by several bivalent cations, particularly Zn2+.
The role of the phosphoenolpyruvate phosphotransferase system in the transport of N-acetyl-d-glucosamine by Escherichia coli
Authors: White R. J.
Published: August 2015

The properties of an N-acetyl-d-glucosamine-transport system have been studied by following the intracellular accumulation of methyl 2-acetamido-2-deoxy-α-d-[1-14C]glucoside by Escherichia coli. The same analogue was used to assay phosphoenolpyruvate phosphotransferase activity of toluene-treated cells. Transport and phosphorylation are induced by growth on d-glucosamine or N-acetyl-d-glucosamine. Mutants resistant to N-iodoacetyl-d-glucosamine are defective in the uptake and phosphorylation of the labelled glycoside.
Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate
Authors: Engel P. C. , Dalziel K.
Published: August 2015

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver–Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.
Phenothiazine-N-carbonyl chloride, a specific inactivator of chymotrypsin
Authors: Erlanger Bernard F. , Vratsanos Spyros M. , Wassermann Norbert H. , Cooper A. G.
Published: August 2015

Phenothiazine-N-carbonyl chloride inactivated chymotrypsin and trypsin by means of a 1:1 stoicheiometric reaction. Its reaction with chymotrypsin was 29 times as fast as that with trypsin and was inhibited by indole. The reaction of phenothiazine-N-carbonyl chloride with chymotrypsin resembled an enzyme–substrate reaction in which the deacylation step is rate-limiting. Slow deacylation occurred, resulting in complete regeneration of active enzyme in 15h. The pH–rate profile of the inactivation process had a maximum at pH7.8. These data and other evidence indicate that the reaction of phenothiazine-N-carbonyl chloride with chymotrypsin exhibits `kinetic specificity'. Therefore any hypothesis that attempts to describe the topography of the active site of chymotrypsin should take into account the reactivity of phenothiazine-N-carbonyl chloride. The above findings, as well as recent reports of others, are examined within the context of a hypothesis given in an earlier paper (Erlanger, 1967).
The mechanism of uptake of cobalt ions by Neurospora crassa
Authors: Venkateswerlu G. , Sastry K. Sivarama
Published: August 2015

Uptake of Co2+ by 3-day-old mycelia of Neurospora crassa involves cell-surface binding as well as transport into the intracellular space. The surface binding is rapid and accounts for 30–40% of the total Co2+ uptake. Transport of Co2+ occurs at a rate of 40μg/h per 100mg dry wt. Surface binding and overall uptake show different temperature dependence. Metabolic inhibitors such as azide, dinitrophenol and fluoride depress transport of Co2+. The overall uptake of Co2+ exhibits a high Km value and hence the concentration mechanism is one of low `affinity' for the metal. The uptake of Co2+ varies linearly with pH in the range pH3 to pH6. Mg2+ inhibits both surface binding and transport of Co2+. It is suggested that the system that transports Mg2+ is also involved in Co2+ uptake by N. crassa.