NADP-MALIC ENZYME ISOFORMS IN SOYBEAN. PURIFICATION, CHARACTERIZATION AND DEVELOPMENTAL PROFILE OF NADP-ME FROM GERMINATING COTYLEDONS

FALCONE FERREYRA, M.LORENA; ANDREO, CARLOS S.; PODESTA, FLORENCIO E.

Carlos Paz, Córdoba

Congreso; XXXVIII Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2002

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

NADP-malic enzyme catalyses the oxidative decarboxylation of l-malate to yield pyruvate, CO2, and NADPH. It is widely distributed in higher plants as photosynthetic (some C4 and CAM plants) and non-photosynthetic (different tissues of C3, C4 and CAM plants) isoforms that either serve an anapleurotic role, providing NADPH and pyruvate to biosynthetic pathways  or participate in the respiratory and carbon asssimilation pathways. The purpose of this work was to study the presence of NADP-ME isoforms in different tissues of soybean, evaluate enzyme levels during germination and kinetically and structurally characterize NADP-ME from cotyledons. Western blots and native IEF showed that the same NADP-ME isoform is present in roots, leaves and cotyledons with a subunit molecular mass of 72 kDa and pI 5.9. The developmental profile showed a peak in activity (1.53 U/g FW and 0.8U) around day 7 postimbibition, declining markedly thereupon. In contrast, immunoreactive protein increased up to day 7 but remained relatively constant afterwards. The enzyme was purified (180-fold) with a yield of 4% and a specific activity of 3.77 U/mg, one of the highest values reported for NADP-ME from C3 plants. The optimal pH was 7.2, similar to NADP-ME from C3 plants. Hyperbolic kinetics was obtained for NADP and l-malate with Km values of 12.6 mM and 0.52 mM, respectively. Both the high specific activity and levels of the enzyme in soybean cotyledons suggest a crucial role for NADP-ME in this tissue during the heterotrophic period