PURIFICATION AND PROPERTIES OF NAD-DEPENDENT AND NADP-DEPENDENT MALIC ENZYMES FROM BRADYRHIZOBIUM-JAPONICUM BACTEROIDS

Bacteroids were isolated from 6 weeks-old soybean nodules and the NAD- and NADP-dependent malic enzymes purified by ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, Bio-Gel chromatography and fast flow Q-Sepharose ion-exchange chromatography. NADP-ME (E.C. 1.1.1.40) was purified 40-fold and NAD-ME (E.C. 1.1.1.38) 14-fold. Native PAGE demonstrated that NAD-ME and NADP-ME from bacteroids are two distinct enzymes. The molecular weight of the native enzymes was estimated by means of gel filtration and SDS-PAGE. It was shown that NADP-ME is a 60kD monomer and NAD-ME is a heterodimer with subunits of 60 and 80 kD. NADP-ME (K-M: 0.18 mM) exhibited much higher affinity for malate than NAD-ME (K-M: 5 mM). The role of both enzymes in bacteroid metabolism is discussed.