Purification of NADP malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A.A. IGLESIAS, C.S. ANDREO

PLANT AND CELL PHYSIOLOGY

Año: 1989 vol. 30 p. 399 - 406

0032-0781

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves Alberto A. Iglesias and Carlos S. Andreo Centra de Estudios Fotosinte'ticos y Bioquimicos, CEFOBI, (CONICET, F. M. Lillo and U. N. Rosario) Suipacha 531, 2000 Rosario, Argentina A procedure is described for the purification of phosphoenolpyruvate carboxylase (EC 4.1.1.31 [EC]) and NADP-dependent malic enzyme (EC 1.1.1.40 [EC]) from sugar cane leaves. Each enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, with about 30% yield. Phosphoenolpyruvate carboxylase was purified 54-fold. A molecular weight of 400,000 and a homotetrameric structure were determined for the native enzyme. The purified carboxylase had a specific activity of 20.0 {diaeresis}mol (mg protein)–1 min–1, and was activated by glucose-6-phosphate and inhibited by L-malate. Km values at pH 8.0 for phosphoenolpyruvate and bicarbonate were 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-fold purified, exhibited a specific activity of 71.2 {diaeresis}mol (mg protein)–1 min–1 and was characterized as a homotetramer with native molecular weight of 250,000. Purified malic enzyme showed an absolute specificity for NADP+ and required a divalent metal ion for activity. Km values of 0.33 and 0.008 mM for L-malate and NADP+, respectively, were determined. This enzyme was inhibited by several organic acids, including keto and amino acids; while succinate and citrate increased the enzyme activity when assayed with 10{diaeresis}M L-malate. The effects shown by amino acids and by citrate were dependent on pH, being higher at pH 8.0 than at pH 7.0.