title: Purification and regulatory properties of the NADP-linked malic enzyme for Crithidia fasciculata.
abstract: The NADP-linked malic enzyme (EC 1.1.1.40) from the insect flagellate Crithidia fasciculata has been purified to electrophoretic homogeneity by a procedure involving ammonium sulphate fractionation, gel filtration on Sephadex G-200, and column chromatography on DEAE-cellulose and hydroxylapatite. The regulatory properties of the purified enzyme have been studied, and compared with those of the two forms malic enzyme (I and II) present in Trypanosoma cruzi. The enzyme from C. fasciculata, like malic enzyme II from T. cruzi was activated by L-aspartate and succinate, which decreased the apparent Km values for both substrates, L-malate and NADP; L-aspartate in addition increased the apparent Vmax. The enzyme from C. fasciculata was inhibited by oxaloacetate, which was strictly competitive towards L-malate, with an apparent Ki (26 microM) intermediate between those reported for the two enzyme forms from T. cruzi. The C. fasciculata enzyme, like malic enzyme II from T. cruzi, was inhibited by adenine nucleotides, which were competitive towards both substrates; in addition, it was inhibited by acetyl-CoA, glyoxylate and NADH, which affected very little the activity of both enzyme froms forms T. cruzi. Thus the malic enzyme from C. fasciculata showed a regulatory pattern even more complex than that of the same enzyme from T. cruzi, despite the fact that there seems to be only one enzyme, present in the cytosol, in the insect trypanosomatid.
mesh_terms: D000818:Animals; D003421:Crithidia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010071:Oxaloacetates / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000201:enzymology
chemical_list: D008293:Malates; D010071:Oxaloacetates; D009249:NADP; C030298:malic acid; D008291:Malate Dehydrogenase