Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

Abstract

NAD malic enzyme (EC 1.1.1.39), which is involved in C(4) photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C(4) species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M(r) of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C(4) species is an octamer of identical subunits. The purified NAD malic enzyme from each C(4) species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn(2+) and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn(2+) was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K(0.5) (the concentration giving half-maximal activity) for Mn(2+). The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn(2+). In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn(2+). Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C(4) dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.