Comparative Studies of NAD-Malic Enzyme from Leaves of Various C4 Plants

Using butyl-TSK-gel chromatography, we purified NAD-malic enzyme(ME) (EC 1.1.1.39 EC] ), which is involved in C₄ photosynthesis,to electrophoretic homogeneity, from leaves of Amaran-thus tricolor.Molecular weights of the native and SDS-denatured enzyme fromA. tricolor were 490 kDa and 61 kDa, respectively. During assayof the enzyme there was a slow reaction transient in the formof a lag before a steady-state rate was reached. The durationof this lag was inversely proportional to the concentrationof each substrate and the activator, fructose- 1,6-bis-phosphate(FBP). The optimal pH of the reaction fell with decreasing concentrationsof either malate or FBP. High pH prolonged the lag in reaction. Double reciprocal plots of the enzymatic activity as a functionof the concentration of malate yielded straight lines and didnot show any cooperativity for binding of malate. The enzymefrom A. tricolor was not inhibited by either HCO^–₃ orCO₂. At different concentrations of malate, the nature of theactivating effect of FBP was compared among the purified enzymesfrom A. tricolor and the C₄ monocots Eleusine coracana and Panicumdichotomiflorum. At low levels of malate, FBP markedly stimulatedthe enzyme from each species. In contrast, at saturating levelsof malate, the response of enzymes to increasing concentrationsof FBP was different and depended on the source of enzyme. The immunochemical properties of the enzymes from the threespecies were compared using an enzyme-linked immunoadsorbentassay with antisera raised against the purified enzymes fromthe three species. Different cross-reactivities were observedamong the enzymes from different sources. The N-terminal aminoacid sequences of NAD-MEs from the three species were determinedand some differences were found among the three enzymes. ²Permanent address; Tohoku National Agricultural ExperimentStation, Morioka, 020-01 Japan. ³Permanent address; National Grassland Research Institute, Nishinasuno,Tochigi, 329-27 Japan. (Received December 12, 1988; Accepted February 17, 1989)