Partial purification and properties of rat liver glutaminase.
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ABSTRACT:
SUBMITTER: Patel M
PROVIDER: S-EPMC1153663 | biostudies-other | 1984 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. Polynucleotide phosphorylase was partially purified from the inner membrane of rat liver mitochondria. 2. The partially purified particulate enzyme catalyses phosphorolysis of poly(A), poly(C), poly(U) and RNA to nucleoside diphosphates. 3. It is devoid of nucleoside diphosphate-polymerization activity. 4. Variable amounts of ADP/P(i)-exchange activity are associated with the polynucleotide phosphorylase and are probably due to a different enzyme. 5. ADP is the preferred substrate for exchange, and little or no reaction occurs with other nucleoside diphosphates, but ATP/P(i)-exchange takes place at one-third the rate observed with ADP. 6. The partially purified enzyme is free from the phosphatases found in the crude mitochondrial inner membrane, but is associated with an endonuclease activity and some adenylate kinase activity; no cytidylate kinase activity analogous to the latter was detectable.
| S-EPMC1174413 | biostudies-other
Project description:Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.
| S-EPMC1164719 | biostudies-other
Project description:1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg(2+), has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.
| S-EPMC1179382 | biostudies-other
Project description:Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.
| S-EPMC1178266 | biostudies-other
Project description:1. An enzyme that catalyses the transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to l-tyrosine methyl ester and tyramine was purified approx. 70-fold from female rat livers. 2. The partially purified preparation is still contaminated with adenosine 3'-phosphate 5'-sulphatophosphate-phenol sulphotransferase (EC 2.8.2.1), but a partial separation of the two enzymes can be achieved by chromatography on columns of Sephadex G-200 and DEAE-Sephadex. 3. The enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is activated by dithiothreitol, 2-mercaptoethanol and GSH, the degree of activation being more marked with preparations previously stored at 0 or -10 degrees C. In contrast, the enzymic sulphation of p-nitrophenol is inhibited by all three thiols. Again, there is a quantitative difference in the degree of inhibition of the two enzymes by o-iodosobenzoate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate. 4. Mixed-substrate experiments support the hypothesis that the enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is separate from that responsible for the sulphation of p-nitrophenol. However, p-nitrophenol is a potent inhibitor of the sulphation of both tyrosyl derivatives whereas these latter compounds have no effect on the sulphation of p-nitrophenol.
| S-EPMC1185501 | biostudies-other
Project description:A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.
| S-EPMC1153702 | biostudies-other
Project description:Background & aimsParacrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study.MethodsHepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic acid), PPADS), and liver regeneration after hepatectomy was analyzed.ResultsA robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction.ConclusionExtracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.
| S-EPMC3625734 | biostudies-literature
Project description:An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.
| S-EPMC1148977 | biostudies-other
Project description:1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.
| S-EPMC1184023 | biostudies-other
Project description:1. Glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9-7.6 and at temperatures below 29 degrees C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000-400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.
| S-EPMC1176648 | biostudies-other