Light/dark regulation of maize leaf phosphoenolpyruvate carboxylase by in vivo phosphorylation.
Phosphoenolpyruvate carboxylase (PEPCase) from light- and dark-adapted maize leaves was rapidly purified in the presence of L-malate and glycerol to apparent electrophoretic homogeneity by ammonium sulfate fractionation, hydroxylapatite chromatography, and fast-protein liquid chromatography on Mono Q. The resulting preparations were totally devoid of pyruvate, orthophosphate dikinase protein based on immunoblot analysis. Throughout the purification, both forms of PEPCase retained their different enzymatic properties. The specific activity of the light enzyme was consistently about twice that of the dark form when assayed at suboptimal (but physiological) pH (pH 7.0-7.3), and the former was also less sensitive to feedback inhibition by L-malate than that from darkened leaves under various conditions. Covalently bound phosphate and high-performance liquid chromatography-based phosphoamino acid analyses showed that both forms of purified PEPCase were phosphorylated exclusively on serine residues, but the degree of phosphorylation was about 50% greater in the light enzyme. Notably, incubation of purified PEPCase in vitro with exogenous alkaline phosphatase led to an increase in malate sensitivity and a decrease in specific activity of the light form enzyme to levels observed with the dark form, which was essentially not affected by phosphatase treatment. These results with the purified enzyme from light- and dark-adapted maize leaves indicate that the light-induced changes in activity and malate sensitivity of C4 PEPCase are related, at least in part, to the degree of covalent seryl phosphorylation of the protein in vivo.
Jiao
J A
JA
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Chollet
R
R
D000469:Alkaline Phosphatase / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003624:Darkness; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008027:Light; D008293:Malates / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D003313:Zea mays / Q000201:enzymology*
D008293:Malates; C030298:malic acid; D000469:Alkaline Phosphatase; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase