NADP-MALIC ENZYME FROM MAIZE LEAF - PURIFICATION AND PROPERTIES

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C4 pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s20,w) of 9.7 and molecular weight of 2.27 × 105 in sucrose density gradient centrifugation, and molecular weight of 2.26 × 105 calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 104 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO3- proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: Km (malate), 0.4 mm; Km (NADP), 17.6 μm; Km (Mg2+), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol. © 1979.