IRON-ASCORBATE CLEAVABLE MALIC ENZYME FROM HYDROGENOSOMES OF TRICHOMONAS-VAGINALIS - PURIFICATION AND CHARACTERIZATION

Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.39) were is olated from hydrogenosomes of Trichomonas vaginalis. A positively char ged isoform at pH 7 was obtained in a single purification step using c ation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchang e and affinity chromatography. Both isoforms displayed similar physica l and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits, The enzyme utilized NAD(+) (K-m, 6-6.3 mu M) preferentially to NADP() (K-m, 125-145 mu M). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be pr esent inside hydrogenosomes. Immunocytochemical studies using a polycl onal rabbit antibody raised against purified T. vaginalis malic enzyme proved hydrogenosomal localization of the enzyme. Subfractionation of hydrogenosomes suggested an association of the malic enzyme with the hydrogenosomal membranes. The 60 kDa malic enzyme subunit was highly s ensitive to non-enzymatic cleavage by an iron-ascorbate system resulti ng in two enzymatically inactive fragments of about 31 kDa. Microseque ncing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp(279)-Ile(280). The enzyme inactiva tion was inhibited by an excess of manganese. Iron-dependent posttrans lational modification might contribute to the regulation of malic enzy me activity in vivo. Copyright (C) 1996 Elsevier Science B.V.