Presence and characteristics of nadp-malic enzyme in soybean nodule bacteroids

Abstract

The localization and characteristics of NADP-malic enzyme [EC 1.1.1.40], a key enzyme for succinate oxidation in nodule bacteroids, were surveyed in soybean nodules and bacteroids. Protoplasts prepared from nodules were separated into cortex cell, infected cell, and uninfected cell groups following the determination of NADP-malic enzyme activity in those cells. The enzyme activity was localized in the cortex cells and the infected cells. In the infected cells the malic enzyme activity was not localized in the cytosol but in the bacteroids. The uninfected cells showed no detectable enzyme activity. The NADP-malic enzyme activity in the bacteroids prepared by Percoll density gradient centrifugation or by differential centrifugation was almost identical with that of the infected cells (mg protein basis). These data indicate that NADP-malic enzyme in soybean nodules was localized both in the cortex cells and in the bacteroids. Staining for the detection of the NADP-malic enzyme activity after polyacrylamide gel electrophoresis and the enzyme activity assay after isoelectrofocusing revealed that the malic enzyme in the bacteroids exhibited a unique Rf value from that in the whole nodules, indicating that the NADP-malic enzyme in Bradyrhizobium japonicum J501 bacteroids was from that in the cortex cells. The NADP-malic enzyme in the bacteroids was partially purified, and the characteristics were surveyed. At least two NADP-malic enzymes were present in the nodule bacteroids, and a major malic enzyme activity in the bacteroids was purified 127 fold. The enzyme showed an optimum pH at 7.5, a K m value at 109µM for malic acid and absolute requirement of NADP for the enzyme reaction. Strong inhibition of the enzyme activity by citrate, malonate, and glyoxylate was observed, suggesting a possible regulatory role of the malic enzyme in the organic acid metabolism in bacteroids.