A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely.
A cytosolic cell-free system prepared from rat liver was used to study the effect of bivalent cations on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Steady-state concentrations of oxaloacetate in the range 5-50 microM were generated from increasing concentrations of malate+fumarate (10:1); 2 mM ITP and 3 mM Mg2+ were added as cofactors. Micromolar concentrations of Mn2+, Fe2+ and, to a lesser extent, of Zn2+ and Co2+ were shown to stimulate PEPCK activity. Vmax. (mumol/min per g of liver) increased from 0.67 to 1.68 on addition of 5 microM Fe2+ and to 2.34 with 2 microM Mn2+, whereas no significant effect on the Km for oxaloacetate was observed. The apparent K(a) values (total) were 0.62 microM for Mn2+, 1.48 microM for Zn2+, 1.92 microM for Co2+ and 3.37 microM for Fe2+, being 2-8-fold lower than the corresponding published values. Variations of the free Mn2+ concentration were obtained (a) by increasing the Mn2+ concentration (i.e. activation curve) and (b) by simultaneous addition of Mn2+ and increasing concentrations of the chelating agent EGTA (i.e. inactivation curve). Different results were obtained for the activation and inactivation curves. The inactivation curve showed that PEPCK activity was almost unaffected by variations of the free Mn2+ concentration over the range 0.05-0.15 microM. Under comparable experimental conditions, rat liver arginase (another Mn(2+)-dependent enzyme) was completely inactivated. From kinetic evidence, the existence of two distinct molecular forms of cytosolic rat liver PEPCK with different Mn2+ affinities is postulated. Considering the high affinity of PEPCK for Mn2+ and its relative insensitivity to changes in the free Mn2+ concentration, it seems rather unlikely that changes in the free cation concentration play a major role in regulating PEPCK activity in vivo.
Department of Biochemistry, University of Basel, Switzerland.
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Maggini
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Stoecklin-Tschan
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Mörikofer-Zwez
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P
Walter
D000818:Animals; D003035:Cobalt / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008345:Manganese / Q000502:physiology*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D003035:Cobalt; D008345:Manganese; D007501:Iron; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D015032:Zinc