Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves |
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Authors: | Iglesias, Alberto A. Andreo, Carlos S. |
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Affiliation: | Centra de Estudios Fotosinte'ticos y Bioquimicos, CEFOBI, (CONICET, F. M. Lillo and U. N. Rosario) Suipacha 531, 2000 Rosario, Argentina |
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Abstract: | A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31[EC]) and NADP-dependent malic enzyme (EC1.1.1.40[EC]) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)1 min1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.Km values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)1 min1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP+ and requireda divalent metal ion for activity. Km values of 0.33 and 0.008mM for L-malate and NADP+, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989) |
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