bluuebunny/crossref_metadata_2025 huggingface dataset:
Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver
Authors: Mapes James P. , Krebs Hans A.
Published: August 2015

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH4Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH4Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH4Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH4Cl. 7. NH4Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH4Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.
An energy requirement for the degradation of intravenously injected125I-labeled albumin in mouse liver and kidney slices
Authors: Mego J L , Farb R M
Published: August 2015

Liver and kidney slices prepared 30min after intravenous injections of formaldehyde-treated 125I-labelled bovine serum albumin into mice degrade approx. 25-40% of the protein to a trichloroacetic acid-soluble form during 60min incubation at 37 degrees C. The presence of bicarbonate in Krebs-Ringer phosphate medium inhibited intracellular proteolysis, and similar results were obtained at pH5 or pH7 in kidney or liver slices. Cellular integrity was required to obtain substantial rates of proteolysis. This intralysosomal intracellular degradation of an exogenous protein was partially inhibited by inhibitors of oxidative ATP formation, such as cyanide, azide, 2,4-dinitrophenol and absence of oxygen. Arsenite and iodoacetamide were also effective inhibitors, but the effects of fluoride were variable. These results suggest that an energy requirement exists for intralysosomal proteolysis in intact cells and are consistent with the hypothesis that energy may be required to maintain intralysosomal acidity.
The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro
Authors: Skaper Stephen D. , O'Brien William E. , Schafer Irwin A.
Published: August 2015

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH14CO3 into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5–4-fold on incubation with 10mm-NH4Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH14CO3 into orotic acid was increased 3–4-fold in liver homogenate with NH4Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH4Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40–50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH4Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH4Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.
Ligand-binding propeties of the muscarinic acetylcholine receptor in mouse neuroblastoma cells
Authors: Strange P G , Birdsall N J M , Burgen A S V
Published: August 2015

1. Muscarinic acetylcholine receptors in a plasma-membrane fraction derived from mouse neuroblastoma clone NIE-115 bind [3-3H]quinuclidinyl benzilate according to the Law of Mass Action (Kdissociation 40 pM, h0.96). 2. Antagonist and agonist binding to the receptor was studied by displacement of [3-3H]quinuclidinyl benzilate with non-radioactive ligands. The data show good agreement with similar data obtained on rat brain and ideal smooth muscle [Birdsall & Hulme (1976) J. Neurochem. 27, 7-16] indicating that the receptor is very similar in these three tissues.
The role of phospholipids in the reduction of ubiquinone analogues by the mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase complex
Authors: Ragan C I
Published: August 2015

The isolated NADH-ubiquinone oxidoreductase complex of bovine heart mitochondria reduces ubiquinone analogues by two pathways. One pathway is inhibited by rotenone, and reduction of quinones takes place in the lipid phase of the system. The other pathway is insensitive to rotenone and reduction takes place in the aqueous phase. The variation of rates of electron transpport with the chemical nature of the quinone analogue and the concentrations of both quinone and phospholipid can be rationalized in terms of partition of the quinone between the aqueous and lipid phases of the system. Thus one function of phospholipid associated with the enzyme appears to be to act as solvent for ubiquinone reduced by the rotenone-sensitive pathway. This proposal is supported by the kinetic behaviour of enzyme whose endogenous lipids have been replaced by (1,2)-dimyristoylsn-glycero-3-phosphocholine. Thus, under certain circumstances, the rotenone-sensitive reduction of ubiquinone-1 exhibited a substantial increase in activation energy below the phase-transition temperature of the synthetic lipid, whereas the reduction of other acceptors was unaffected.
The uptake of silicic acid by rat liver mitochondria
Authors: Johnson R N , Volcani B E
Published: August 2015

1. To gain insight into a putative role for mitochondria in silicon metabolism, mitochondrial uptake (by which it is meant the removal from the medium) of silicic acid [Si(OH)4] was studied under conditions minimizing SI(OH)4 polymerization. 2. Measurements of mitochondrial respiration and swelling indicated indirectly a significant uptake of Si(OH)4 as a weak acid, but this was not confirmed when 31Si(OH)4 was used as a tracer. 31Si(OH)4 occupied a mitochondrial volume similar to that of 3H2O and was relatively unaffected by mitochondrial energy status and by the pH gradient across the mitochondrial inner membrane. 3. Uptake was directly proportional to Si(OH)4 concentration in the range 0-3 mM. 4. The uptake consisted of two components: under all conditions examined, the greater quantity, amounting to 1-2nmol of Si(OH)4/mg of mitochondrial protein, was bound, a major portion of it external to the inner membrane, with the lesser quantity free within the matrix space. 5. Equilibration of 31Si(OH)4 between medium and matrix was a slow process, having a half-time of approx. 10 min at 22 degrees C. 6. Mersalyl and N-ethylmaleimide inhibited the uptake by preferentially lowering the amount of Si(OH)4 bound. Their action was somewhat variable, depending on the precise nature of the suspending medium, and suggesting that the bound material may represent polymerized forms of Si(OH)4. 7. It is concluded that Si(OH)4 may penetrate the mitochondrial inner membrane by a simple diffusion mechanism.
Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5′-phosphate
Authors: Hemmilä I A , Mäntsälä P I
Published: August 2015

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5′-phosphate and this inactivation increased 3–4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5′-phosphate.
Purification and immunochemical characterization of malate synthase from Euglena gracilis
Authors: Woodcock E , Merrett M J
Published: August 2015

Malate synthase (EC 4.1.3.2) from dark-grown Euglena gracilis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis. The enzyme was released from acetate-grown cells by treatment with ultrasonic waves and purified from broken-cell suspensions by high-speed centrifugation and (NH4)2SO4 fractionation, followed by gel-filtration on Sepharose 6B. The final enzyme preparation was purified 190-fold compared with the crude extract. The mol.wt. of the enzyme was about 350000 as determined by gel filtration on Sepharose 6B. Treatment with sodium dodecyl sulphate and urea dissociated the enzyme into subunits of mol.wt. 175000. The pH optimum for the enzyme was 8.0 and the Km values for glyoxylate and acetyl-CoA were 50 and 80 micron respectively. Antibodies raised to the purified enzyme were shown to be monospecific by radiochemical immunoassay. Euglena anti-(malate synthase) tested on Ouchterlony double-diffusion gels gave a sharp precipitation band against acetate-grown Escherichia coli, but no immunological correspondence was observed with acetate-grown Chlorella fusca, Zea mays (maize) scutella or purified malate synthase from Ricinus communis.
A defined molecular-weight distribution of deoxyribonucleic acid after extensive sonication
Authors: Davis A W , Phillips D R
Published: August 2015

Concentrated solutions of low-molecular-weight DNA (Mw=35000) with a known molecular-weight distribution can be prepared in several hours, and require no additional fractionation procedures. This is achieved by sonication of the DNA in 1.0 M-NaCl at high power at 0–2 degrees C. No denaturation of the DNA is detectable, even after 8h of continuous sonication. After 2h, the molecular-weight distribution of the total DNA sample is that of the most probably Schulz distribution, described by-Mn:-Mw:-Mz …=1:2:3 …etc. Such a molecular-weight distribution is expected for a random break-up of indefinitely long macromolecules and indicates that the sonication process is essentially by random double-strand scission. DNA was also sonicated in the presence of ligands capable of modifying the DNA tertiary structure. The results support the idea that inflexibility of the DNA is required for efficient sonic degradation.
Amino acid sequences of α-helical segments from S-carboxymethylkerateine-A. Complete sequence of a type-II segment
Authors: Crewther W G , Inglis A S , McKern N M
Published: August 2015

1. The helical fragments obtained by partial chymotryptic digestion of S-carboxymethylkeratine-A, the low-sulphur fraction from wool, were fractionated into type-I and type-II helical segments in aqueous urea under conditions limiting carbamoylation. 2. The amino acid sequence of a 109-residue type-II segment was completed by using the sequenator. 3. When the data were incorporated into a helical model of 3.6 residues per turn the hydrophobic residues generated a band aligned at a slight angle to the helical axis. This result is in accord with the postulated coiled-coil structure of the crystalline regions of alpha-keratin.
Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite
Authors: Kelly J M , Goddard J P , Maden B E H
Published: August 2015

The reaction of HeLa-cell 5.8S rRNA with NaHSO3 under conditions in which exposed cytidine residues are deaminated to uridine was studied. It was possible to estimate the reactivities of most of the 46 cytidine residues in the nucleotide sequence by comparing ‘fingerprints’ of the bisulphite-treated RNA with those of untreated RNA. The findings were consistent with the main features of the secondary-structure model for mammalian 5.85S rRNA proposed by Nazar, Sitz, & Busch [J. Biol. Chem (1975) 250, 8591–8597]. Five out of six regions that are depicted in the model as single-stranded loops contain cytidine residues that are reactive towards bisulphite at 25 degrees C (the other loop contains no cytidine). The cytidine residue nearest to the 3′-terminus is also reactive. Several cytidines residues that are internally located within proposed double-helical regions show little or no reactivity towards bisulphite, but the cytidine residues of several C.G pairs at the ends of helical regions show some reactivity, and one of the proposed loops appears to contain six nucleotides, rather than the minimum of four suggested by the primary structure. Two cytidine residues that are thought to be ‘looped out’ by small helix imperfections also show some reactivity.
Hyaluronic acid in the pulmonary secretions of patients with asthma
Authors: Sahu S , Lynn W S
Published: August 2015

Hyaluronic acid was the only glycosaminoglycan found in the pulmonary secretions of patients with asthma. The compound had a hexuronate/hexosamine molar ratio of about 1:1. Glucosamine constituted over 98% of the hexosamines, the remaining being galactosamine. The compound moved as a single spot with the mobility of standard hyaluronic acid on cellulose acetate electrophoresis, and this spot disappeared after digestion with testicular hyaluronidase. Even after extensive proteolysis and purification, the compound was associated with small amounts of protein, the major amino acids of which were aspartic acid, threonine, serine, glutamic acid, glycine and valine.
Occurrence of glycosphingolipids in chicken egg yolk
Authors: Li S C , Chien J L , Wan C C , Li Y T
Published: August 2015

Chicken egg yolk was found to contain a unique glycosphingolipid pattern not seen in other types of tissue or cell. These glycosphingolipids were isolated in pure form and their structures established by sequential enzymic hydrolysis and permethylation analysis. The major gangliosides in chicken egg yolk are N-acetylneuraminosylgalactosylceramide, N-acetylneuraminosyl-lactosylceramide and di-N-acetylneuraminosyl-lactosylceramide. The only neutral glycosphingolipid found in chicken egg yolk is galactosylceramide.
The mechanism of reaction of fully reduced membrane-bound cytochrome oxidase with oxygen at 176K
Authors: Clore G M , Chance E M
Published: August 2015

1. The results of non-linear optimization studies on the mechanism of reaction of fully reduced cytochrome oxidase with O2 at 176K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at three wavelength pairs (604-630, 608-630 and 830-940 nm) and at three O2 concentrations (60, 200 and 1180 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, good determination of the optimized parameters and a random distribution of residuals is a three-species sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from low-temperature trapping, e.p.r. and magnetic-susceptibility studies, the possible valence states of the metal centres in each of the intermediates are discussed.
Solubilization of isolated central-nervous-system myelin preparations by the amniotic detergent sodium dodecyl sulphate
Authors: Crang A J , Rumsby M G
Published: August 2015

The mechanism for the solubilization of isolated central-nervous-system myelin by sodium dodecyl sulphate was studied in detail. The release of protein and phospholipid to the 100000 g x 1 h supernatant fraction is dependent on the total amount of detergent relative to the amount of membrane present and on the ionic strength of the solubilization system. Gel-filtration analysis of supernatant fractions indicate that at suboptimal concentrations of detergent these contain lipid-protein complexes. The complete dissociation of the individual protein components from lipid is dependent on the total amount of sodium dodecyl sulphate present in the system. The results indicate that for the analysis of membrane components in sodium dodecyl sulphate it is essential that sufficient detergent is present.
Use of the sulphite adduct of nicotinamide–adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase
Authors: Parker David M. , Lodola Alberto , Holbrook J. John
Published: August 2015

1. The formation of the non-enzymic adduct of NAD+ and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD+. 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD+ and sulphite was investigated. The u.v. spectrum of the NAD–sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD–sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO3−=SO32−+H+), the enzyme-bound adduct showed a bell-shaped pH–stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD+ and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD+ binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD+ by rapid reaction with sulphite. The rate of NAD+ dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward Vmax.. No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD+.
Regulation of pyruvate dehydrogenase activity in rat epididymal fat-pads and isolated adipocytes by adrenaline
Authors: Smith S J , Saggerson E D
Published: August 2015

1. Dose-dependent effects of adrenaline on PDHa activity were investigated with both incubated rat epidiymal fat-pads and isolated adipocytes. 2. Adrenaline (10nM- 5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]glucose + insulin (20 munits/ml). Changes in [U-14C]glucose incorporation into fatty acids in these tissues correlated only loosely with changes in PDHa activity. There was a good inverse relationship between adrenaline-induced changes in PDHa activity and increases in lipolysis (glycerol release). 3. Adrenaline (10nM - 0.5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]pyruvate + insulin (20 munits/ml), whereas 1 micrometer- and 5 micrometer-adrenaline slightly increased PDHa activity. All concentrations of adrenaline tested decreased [U-14C]pyruvate incorporation into fatty acids. Between 10nM- and 0.5 micrometer-adrenaline percentage decreases in PDHa activity paralleled decreases in faty acid synthesis. 4. Effects of adrenaline on PDHa activity and fatty acid synthesis in fat-pads incubated with 5mM-[U-14C]pyruvate + insulin (20 munits/ml) could not be mimicked by addition of albumin-bound palmitate. 5. The response of PDHa activity to adrenaline (0.1 nM - 1 micrometer) in isolated adipocytes differed with the carbohydrate substrate used in the incubations. With 5 mM-glucose + insulin (20 munits/ml), PDHa activity was significantly increased by 10 nM-adrenaline, but not by 1 micrometer-adrenaline, the response to adrenaline being biphasic. There was some correlation between PDHa activity and accumulation of non-esterified fatty acids. With 5 mM-glucose alone adrenaline (0.1 nM - 1 micrometer) had no effect on PDHa activity even though lipolysis was increased by adrenaline (0.1 micrometer - 1 micrometer). With 5mM-fructose in the presence and absence of insulin, lipolytic doses of adrenaline decreased PDHa activity. No tested concentrations of adrenaline increased PDHa with this substrate. 6. In the presence of 5 mM-fructose, palmitate was significantly more effective than adrenaline with respect to the maximum decrease in PDHa activity that could be elicited. 4. The relationship of changes in PDHa activity to changes in lipogenesis and the likelihood of adrenaline-induced changes in PDHa activity being secondary to changes in non-esterified fatty acid metabolism are discussed.
Superoxide dismutase and catalase in the protection of the proton-donating systems of nitrogen fixation in the blue-green alga Anabaena cylindrica
Authors: Henry L E A , Gogotov I N , Hall D O
Published: August 2015

1. Superoxide dismutase activity was present in the heterocysts and vegetative cells of Anabaena cylindrica, but was always lower in the heterocysts. 2. No qualitative differences were found in the superoxide dismutase from the two cellular types. 3. Catalase activity was also present in both cellular types. 4. Most of the NADP reductase activity, as assayed with menadione or ferredoxin as electron acceptor, was localized within the heterocysts. 5. Studies on H2 consumption showed that most of the hydrogenase activity was associated with the heterocysts. 6. The results are discussed in terms of the postulate that superoxide dismutase and catalase are involved in the protection of the proton-donating systems participating in N2 fixation and H2 metabolism of heterocysts.
Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes
Authors: Seglen P O
Published: August 2015

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.
Incorporation of ribonucleic acid in vitro into dense ribonucleoprotein-like materials by isolated rat liver nuclei
Authors: Suzuka I
Published: August 2015

A cell-free system of isolated rat liver nuclei is described which permits an active incorporation of newly synthesized RNA into ‘dense’ ribonucleoprotein-like materials. The reaction is stimulated with increasing amounts of cytosol protein isolated from rat liver. This indicates that cytosol protein plays an important role in the formation of such material.
Aerobic glycolysis and lymphocyte transformation
Authors: Hume David A. , Radik Judith L. , Ferber Ernst , Weidemann Maurice J.
Published: August 2015

1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U-14C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U-14C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [3H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25μm) stimulated DNA synthesis half-maximally, but maximum [3H]thymidine incorporation was observed only when the glucose concentration was raised to 1mm. Lactate addition did not alter the sensitivity of [3H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis.
The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens
Authors: Hume D A , Vijayakumar E K , Schweinberger F , Russell L M , Weidemann M J
Published: August 2015

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN′N′-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.
Activity and androgenic control of enzymes associated with the tricarboxylic acid cycle, lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat
Authors: Brooks D E
Published: August 2015

1. Enzyme activities (units/g wet wt.) were determined in the caput and cauda epididymidis and in epididymal spermatozoa of the rat. 2. The activity of most enzymes in the cauda was between 50 and 100% of that in the caput, except that ATP citrate lyase was barely detectable in the cauda. 3. Spermatozoa, unlike epididymal tissue, contained sorbitol dehydrogenase but lacked ATP citrate lyase. NADP+-malate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, succinate dehydrogenase, carnitine acetyltransferase and citrate synthase were 5 to 400 times as active in spermatozoa as in epididymal tissue. 4. 2-Oxoglutarate dehydrogenase was the least active member of the tricarboxylic acid cycle in all tissues and most closely matched the measured flux through the cycle. 5. The concentrations of hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase were equivalent to the more active enzymes of the tricarboxylic acid cycle, indicating the capacity for extensive lipid oxidation, and the presence of 3-hydroxybutyrate dehydrogenase suggests that these tissues can also oxidize ketone bodies. 6. Transfer of reducing equivalents from cytoplasm to mitochondrion is unlikely to occur by means of the glycerol phosphate cycle because mitochondrial glycerol 3-phosphate dehydrogenase is relatively inactive in epididymal tissue, whereas the cytoplasmic enzyme has little activity in spermatozoa, but transfer may be accomplished by the malate-aspartate shuttle. 7. Transfer of acetyl units from mitochondrion to cytoplasm could be effected by the pyruvate-malate cycle in the caput of androgen-maintained rats, but not in the other tissues because of the low activity of ATP citrate lyase. Acetyl unit transfer could take place via acetylcarnitine, mediated by carnitine acetyltransferase. 8. Castration resulted in a decrease in the concentration of nearly all enzymes, although subsequent administration of testosterone restored concentrations to values similar to those in animals maintained by endogenous androgen. The extent to which enzyme concentration was changed by an alteration in androgen status was highly variable, but was most marked in the case of pyruvate carboxylase.
Subcellular structure of bovine thyroid gland. The localization of the peroxidase activity in bovine thyroid
Authors: De Wolf M J S , Lagrou A R , Hilderson H J J
Published: August 2015

1. After differential pelleting of bovine thyroid tissue the highest relative specific activities for plasma membrane markers are found in the L fraction whereas those for peroxidase activities (p-phenylenediamine, guaiacol and 3,3′-diaminobenizidine tetrachloride peroxidases) are found in the M fraction. 2. When M + L fractions were subjected to buoyant-density equilibration in a HS zonal rotor all peroxidases show different profiles. The guaiacol peroxidase activity always follows the distribution of glucose 6-phosphatase. 3. When a Sb fraction is subjected to Sepharose 2B chromatography three major peaks are obtained. The first, eluted at the void volume, consists of membranous material and contains most of the guaiacol peroxidase activity. Most of the protein (probably thyroglobulin) is eluted with the second peak. Solubilized enzymes are recovered in the third peak. 4. p-Phenylenediamine peroxidase activity penetrates into the gel on polyacrylamidegel electrophoresis, whereas guaiacol peroxidase activity remains at the sample zone. 5. DEAE-Sephadex A-50 chromatography resolves the peroxidase activities into two peaks, displaying different relative amounts of the different enzymic activities in each peak. 6. The peroxidase activities may be due to the presence of different proteins. A localization of guaiacol peroxidase in rough-endoplasmic-reticulum membranes (or in membranes related to them) seems very likely.
Activities and some properties of 5′-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine
Authors: Arch J R S , Newsholme E A
Published: August 2015

1. The maximal activities of 5′-nucleotidase, adenosine kinase and adenosine deaminase together with the Km values for their respective substrates were measured in muscle, nervous tissue and liver from a large range of animals to provide information on the mechanism of control of adenosine concentration in the tissues. 2. Detailed evidence that the methods used were optimal for the extraction and assay of these enzymes has been deposited as Supplementary Publication SUP 50088 (16pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K.,from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5. This evidence includes the effects of pH and temperature on the activities of the enzymes. 3. In many tissues, the activities of 5′-nucleotidase were considerably higher than the sum of the activities of adenosine kinase and deaminase, which suggests that the activity of the nucleotidase must be markedly inhibited in vivo so that adenosine does not accumulate. In the tissues in which comparison is possible, the Km of the nucleotidase is higher than the AMP content of the tissue, and since some of the latter may be bound within the cell, the low concentration of substrate may, in part, be responsible for a low activity in vivo. 4. In most tissues and animals investigated, the values of the Km of adenosine kinase for adenosine are between one and two orders of magnitude lower than those for the deaminase. It is suggested that 5′-nucleotidase and adenosine kinase are simultaneously active so that a substrate cycle between AMP and adenosine is produced: the difference in Km values between kinase and deaminase indicates that, via the cycle, small changes in activity of kinase or nucleotidase produce large changes in adenosine concentration. 5. The activities of adenosine kinase or deaminase from vertebrate muscles are inversely correlated with the activities of phosphorylase in these muscles. Since the magnitude of the latter activities are indicative of the anaerobic nature of muscles, this negative correlation supports the hypothesis that an important role of adenosine is the regulation of blood flow in the aerobic muscles.
Product inhibition of o-aminophenol glucuronidation by p-nitrophenyl glucuronide in detergent-treated microsomal fractions
Authors: Howland R D
Published: August 2015

The glucuronidation of o-aminophenol is unaffected by p-nitrophenyl gluronide when native microsomal fractions are the source of UDP-glucuronyltransferase. When microsomal fractions treated with Lubrol detergent are the source of the enzyme, however, p-nitrophenyl glucuronide exhibits competitive inhibition of o-aminophenol glucuronidation. In addition, the apparent K1 for p-nitrophenyl glucuronide is the same whether o-aminophenol or p-nitrophenol is the acceptor substrate. The data suggest that UDP-glucuronyltransferase has one binding site for the two phenols and that the absence of inhibition observed in native microsomal fractions is dependent on an intact microsomal membrane.
Sodium-dependent alanine transport in plasma-membrane vesicles from rat liver
Authors: van Amelsvoort J M M , Sips H J , van Dam K
Published: August 2015

L-Alanine transport was studied in plasma-membrane vesicles from rat liver. A gradient of NaSCN, but not of KSCN, stimulated alanine uptake. Monensin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished the observed overshoot in uptake. After equilibration of alanine, NaSCN induced uphill transport.
Modification of yeast ribosomal proteins. Phosphorylation
Authors: Kruiswijk T , de Hey J T , Planta R J
Published: August 2015

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly.
Beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions
Authors: MacDermot J , Westgaard R H , Thompson E J
Published: August 2015

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).
Chloroperoxidase-catalysed oxidation of 4-chloroaniline to 4-chloronitrosobenze
Authors: Corbett M D , Chipko B R , Baden D G
Published: August 2015

The incubation of 4-chloroaniline with chloroperoxidase and H2O2 resulted in a rapid formation of 4-chloronitrosobenzene. This enzymic oxidation displayed a pH optimum at 4.4 with a Km of 8.1×10(−4)M and catalytic-centre activity of 312. The initial rate of the reaction was strongly affected by the presence of halide ions. 4-Chlorophenylhydroxylamine was even more rapidly converted into the nitroso compound. A reaction mechanism is proposed on the basis of currently accepted theory for the catalytic action of chloroperoxidae. A noteworthy aspect of this new reaction is the difference in the products previously reported for the action of classical peroxidases on anilines and the single nitroso product resulting from chloroperoxidase oxidation.
The β-glucosidase from Botryodiplodia theobromae Pat. Kinetics of enzyme-catalysed hydrolysis of o-nitrophenyl β-d-glucopyranoside in dioxan/water
Authors: Umezurike G M
Published: August 2015

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Botryodiplodia theobromae Pat. has been studied in the presence of added dioxan. 2. At donor saturation, the maximum rate of hydrolysis in the presence of up to 50%(v/v) dioxan was pH4.3-4.5 (pH of the buffer system in water) in McIlvaine's buffer. 3. Increasing dioxan concentrations progressively decreased the maximum rate of hydrolysis. 4. The rate of enzyme-catalysed reaction was enhanced at high donor concentrations, but inhibited at low donor concentrations in the presence of glycerol, methanol, fructose of sucrose. 5. The hydrolytic reaction was found to proceed with retention of configuration at the anomeric carbon atom. 6. The kinetics of the enzyme-catalysed process in the presence of added acceptors indicated that water was necessary for the maintenance of the active enzyme conformation apart from its acceptor function.
The necessity of magnesium cation for acid assistance aglycone departure in catalysis by Escherichia coli (lacZ) β-galactosidase
Authors: Sinnott M L , Withers S G
Published: August 2015

1. Removal of Mg2+ from Escherichia coli (lacZ) beta-galactosidase slightly increases the rate of hydrolysis of galactosyl pyridinium salts, but decreases the rate of hydrolysis of arylgalactosides. 2. Fair correlation of logkcat. and log (Km) with the pKa of aglycone is now observed for arglygalactosides, as well as for glycosyl pyridinium salts. 3. Degalactosylation of Mg2+-free enzyme is the rate-limiting step in the hydrolysis of 2,4-dinitrophenyl galactoside. 4. alpha-Deuterium kinetic isotope effects for both sets of substrates are consistent with the rate-determining generation of a glycosyl cation. 5. The pH-independent, SNl hydrolysis of 3,4-dinitrophenyl galactoside has been measured: it is as fast as that of the galactosyl 3-chloropyridinium ion. 6. Hydrolysis of these two substrates by Mg2+-free enzyme proceeds at very similar rates. 7. It is concluded that loss of both types of aglycone takes place, without acid catalysis, from the first ES complex of substrate and apoenzyme. 8. Data for galactosyl azide and thiopicrate confirm that neither charge nor change of atom is the cause of the differences in behavior between aryl galactosides and galactosylpyridinium salts.
Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli
Authors: Blackwell Carol M. , Turner John M.
Published: August 2015

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, Km values for ethanolamine and coenzyme B12 were 44μm and 0.42μm respectively. The Km for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (Ki 0.86μm), dl-1-aminopropan-2-ol (Ki 2.2μm) and dl-1,3-diaminopropan-2-ol (Ki 88.0μm) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (Ki 1.4nm), hydroxocobalamin (Ki 2.1nm) and cyanocobalamin (Ki 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K+ ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s20,w of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.
A radioimmunoassay for chicken avidin. Comparison with a [14C]biotin-binding method
Authors: Kulomaa M S , Elo H A , Tuohimaa P J
Published: August 2015

A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin.
Isolation and partial characterization of the major sialoglycoprotein of human T-lymphoblastoid cells of a MOLT-4B cell line
Authors: Saito M , Toyoshima S , Osawa T
Published: August 2015

A sialoglycoprotein with an approx. mol.wt. of 95000 was isolated from human lymphoblastoid cells of a MOLT-4B cell line, which was of human T-lymphocyte origin, by ion-exchange chromatography, affinity chromatography on a column of wheat-germ agglutinin-Sepharose and preparative slab-gel electrophoresis. The localization of this glycoprotein on the cell surface was indicated by surface labelling by the periodate/NaB3H4 and lactoperoxidase-catalysed iodination methods. Carbohydrate analyses of this glycoprotein revealed that its total carbohydrate content is 28% (w/w), and it contains fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in molar proportions 1.0:4.0:3.7:3.5:1.2:2.5, suggesting that it has two types of sugar chain, i.e. sugar chains like those of serum glycoproteins and sugar chains of the type found in mucins. Actually, alkaline borohydride treatment of this glycoprotein yielded tri- and tetra-saccharide, the latter containing 1 molecule of fucose in addition to each molecule of galactose, N-acetylgalactosamine and sialic acid. This glycoprotein bound to Ricinus communis agglutinin and concanavalin A as well as to wheat-germ agglutinin.
Comparison of the molybdenum centres of native and desulpho xanthine oxidase. The nature of the cyanide-labile sulphur atom and the nature of the proton-accepting group
Authors: Gutteridge S , Tanner S J , Bray R C
Published: August 2015

The non-functional form of xanthine oxidase known as the desulpho enzyme was compared with the functional enzyme in various ways, to obtain information on the structure of the molybdenum centre and the mechanism of the catalytic reaction. The desulpho enzyme, like the functional one, possesses a site for the binding of anions, presumably as ligands of molybdenum. Evidence is presented that in the Mo(V) e.p.r. signal from the desulpho-enzyme, as in that from the functional enzyme, a weakly coupled proton, in addition to a strongly coupled proton, interacts with the metal. Measurements were carried out by e.p.r. on the rate at which the proton strongly coupled to molybdenum exchanged, on diluting enzyme samples with 2H2O. For the desulpho enzyme the exchange rate constant was 0.40s-1, at pH 8.2 and 12 degrees C, and for the functional enzyme it was 85 s-1. It is shown that the great majority of reported differences between the enzyme forms are consistent with functional enzyme containing an (Enzyme)-Mo=S grouping, replaced in the desulpho form by (Enzyme)-Mo=O. Protonation of these groups, with pK values of about 8 and 10 respectively, would give (Enzyme)-Mo-SH and (Enzyme)-Mo-OH, these being the forms observed by e.p.r. The accepting group in the functional enzyme, for the proton transferred from the substrate while molybdenum is reduced in the catalytic reaction [Gutteridge, Tanner & Bray (1978) Biochem J. 175 869-878], is thus taken to be Mo=S.