title: Effects of vitamin C on phosphoenolpyruvate carboxykinase from rat liver.
abstract: Phosphoenolpyruvate carboxykinase (GTP) (PEPCK) is one of the key enzymes of gluconeogenesis. Mechanisms responsible for rapid regulation of enzyme activity include activation by bivalent cations such as Mn2+ and Fe2+ and/or alterations of the oxidation state of the enzyme's SH groups. A cytosolic cell free system prepared from rat liver was used to study the effects of the thiol reagents GSH and dithiothreitol (DTT) and particularly of vitamin C on PEPCK activity. (1) Basal activity and Mn(2+)-stimulated activity were not affected by variations in the concentrations of GSH and DTT, indicating that some components of the cell free system provided sufficient protection against PEPCK-inactivation due to disulfide bond formation. The latter phenomenon is known to occur with purified PEPCK in the absence of added thiols. Only in the presence of 2 microM Fe2+, GSH/DTT addition increased PEPCK activity. (2) Addition of vitamin C in the range of 0.6-1.2 mM resulted in a marked stimulation of the PEPCK reaction, ranging from 1.5-fold (with 2 microM Mn2+) to 4-7-fold (for basal activity and with 2 microM Fe2+). (3) When 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) was used to induce disulfide bond formation and subsequent inactivation of PEPCK, reactivation experiments with GSH/DTT but not with vitamin C restored full enzyme activity. It is concluded that vitamin C activates PEPCK or protects it from inactivation caused by oxidants by a mechanism that does not involve the reduction of the enzyme's thiols.
authors.affiliation: Department of Biochemistry, University of Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Maggini
authors.forename: P
authors.initials: P
authors.lastname: Walter
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002474:Cell-Free System; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004793:Enzyme Reactivators; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005978:Glutathione; D001205:Ascorbic Acid; D004229:Dithiothreitol