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 isolated from hydrogenosomes of Trichomonas vaginalis. A positively charged isoform at pH 7 was obtained in a single purification step using cation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchange and affinity chromatography. Both isoforms displayed similar physical and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits. The enzyme utilized NAD+ (Km, 6-6.3 microM) preferentially to NADP+ (Km, 125-145 microM). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be present inside hydrogenosomes. Immunocytochemical studies using a polyclonal 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 sensitive to non-enzymatic cleavage by an iron-ascorbate system resulting in two enzymatically inactive fragments of about 31 kDa. Microsequencing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp279-Ile280. The enzyme inactivation was inhibited by an excess of manganese. Iron-dependent posttranslational modification might contribute to the regulation of malic enzyme activity in vivo.
Drmota
T
T
Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.
Proost
P
P
Van Ranst
M
M
Weyda
F
F
Kulda
J
J
Tachezy
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002458:Cell Fractionation; D002712:Chlorides / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017895:Manganese Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D015388:Organelles / Q000201:enzymology; D017421:Sequence Analysis; D014246:Trichomonas vaginalis / Q000201:enzymology*
D002712:Chlorides; D005296:Ferrous Compounds; D007527:Isoenzymes; D017895:Manganese Compounds; D009243:NAD; C020748:ferrous sulfate; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating); D001205:Ascorbic Acid; C025340:manganese chloride