Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

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Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

Biochemical Journal ◽

10.1042/bj2070133 ◽

1982 ◽

Vol 207 (1) ◽

pp. 133-138 ◽

Cited By ~ 20

Author(s):

M G Battelli ◽

E Lorenzoni

Keyword(s):

Rat Liver ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Xanthine Dehydrogenase ◽

Enzymic Activity ◽

Oxidized Glutathione ◽

Adult Rats ◽

Purification And Properties ◽

Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

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Dephosphorylation of 1d-myo-inositol 1,4-bisphosphate in rat liver

Biochemical Journal ◽

10.1042/bj2540655 ◽

1988 ◽

Vol 254 (3) ◽

pp. 655-660 ◽

Cited By ~ 8

Author(s):

A J Morris ◽

D J Storey ◽

C P Downes ◽

R H Michell

Keyword(s):

Phosphatase Activity ◽

Rat Liver ◽

Substrate Concentration ◽

Gel Filtration ◽

Fold Increase ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Inositol Monophosphatase ◽

Liver Cytosol ◽

Rat Liver Cytosol

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.

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Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol

Canadian Journal of Biochemistry ◽

10.1139/o79-076 ◽

1979 ◽

Vol 57 (6) ◽

pp. 605-612 ◽

Cited By ~ 40

Author(s):

Patrick C. Choy ◽

S. Blake Farren ◽

Dennis E. Vance

Keyword(s):

Molecular Weight ◽

Rat Liver ◽

Neutral Lipids ◽

Gel Filtration ◽

Fold Increase ◽

High Cholesterol Diet ◽

Liver Cytosol ◽

Ctp:Phosphocholine Cytidylyltransferase ◽

Molecular Weight Form ◽

Rat Liver Cytosol

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 °C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.

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Identification and partial purification of a unique phenolic steroid sulphotransferase in rat liver cytosol

Biochemical Journal ◽

10.1042/bj2240947 ◽

1984 ◽

Vol 224 (3) ◽

pp. 947-953 ◽

Cited By ~ 11

Author(s):

Y Sugiyama ◽

A Stolz ◽

M Sugimoto ◽

J Kuhlenkamp ◽

T Yamada ◽

...

Keyword(s):

Rat Liver ◽

Organic Anion ◽

Substrate Inhibition ◽

Gel Filtration ◽

Male Rat ◽

Partial Purification ◽

Ph Optimum ◽

Liver Cytosol ◽

Bile Acid Binding ◽

Rat Liver Cytosol

Phenolic steroid sulphotransferase activity for both oestradiol and oestrone was identified in male rat liver cytosol in the 30 000-40 000 Mr fractions on gel filtration when activity was assayed at pH 5.5 (pH optimum 5.5-6.0). Activity for oestradiol but not oestrone was found in the 60 000-70 000-Mr range when assayed at pH 8.0 (pH optimum biphasic, 5.5-6.0 and 7.0-8.0). Km for oestradiol (1.3 microM) was lower than published values for hydroxysteroid sulphotransferases (15-35 microM) and previously reported oestradiol sulphotransferases (71-85 microM). At above 2 microM-oestradiol phenolic sulphotransferase activity exhibited substrate inhibition. The phenolic steroid sulphotransferase activity was found to be distinct in chromatofocusing from organic-anion-binding and bile acid-binding proteins previously identified in this Mr range. Further purification on hydroxyapatite yielded a 44-fold enriched fraction that contained two monomeric bands, Mr 32 500 and 29 500.

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Mechanism of action of β-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA

Biochemical Journal ◽

10.1042/bj2130153 ◽

1983 ◽

Vol 213 (1) ◽

pp. 153-158 ◽

Cited By ~ 7

Author(s):

P M Jordan ◽

P N B Gibbs

Keyword(s):

Rat Liver ◽

Direct Evidence ◽

Gel Filtration ◽

Acetyl Coa ◽

Liver Cytosol ◽

Enzyme Reactions ◽

Single Turnover ◽

The One ◽

Enzyme Intermediate ◽

Rat Liver Cytosol

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and −4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.

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