PURIFICATION AND PARTIAL CHARACTERIZATION OF NAD-DEPENDENT MALIC ENZYME ISOLATED FROM VIGNA-UNGUICULATA HYPOCOTYLS

NAD-malic enzyme (L-malate: NAD oxidoreductase (decarboxylating); EC 1 .1.1.39 (NAD-ME)) was purified to homogeneity from etiolated hypocotyl s of Vigna unguiculata (L.) Walp (cowpea). Purification sequentially i nvolved mitochondria purification, anion-exchange, dye-ligand and affi nity chromatographies. The NAD-ME was predominantly present in a dimer ic form in native gradient gel stained for activity. Two subunits of t he NAD-ME were isolated with M(r) of 61,000 and 64,000 respectively, i n SDS-PAGE (10% resolving). Immunoprecipitation and immunoblot analysi s confirmed the existence of these two different subunits. The purifie d enzyme showed a sequential kinetic mechanism with each substrate bou nd randomly to the NAD-ME. The K-m value for each substrate (malate an d NAD), in the presence of Mn2+, decreased when the other was linked t o the NAD-ME. The NAD-ME also used Mg2+ as a metal cofactor, but in a minor extent compared to Mn2+. Polyclonal antibodies produced against the NAD-ME from etiolated hypocotyls were reactive against the isoform of V. unguiculata and Eucalyptus citriodora leaves.