Identification, purification and separation of different isozymes of NADP-specific malic enzyme from Tritrichomonas foetus.
Tritrichomonas foetus was found to contain NADP-specific malic enzyme. The activity was present in the cytosolic fraction and was about 5-fold higher in extracts of a metronidazole-resistant strain (KV1-1MR-100) than of the parent strain (KVc1). Electrophoresis under non-denaturing conditions and activity staining indicated the existence of 3 isozymes termed I, II and III in order of increasing electrophoretic mobility. Isozymes I and II were much less active than isozyme III in the parent strain, whereas all three isozymes had comparable activities in the resistant strain. NADP-malic enzymes were purified from the cytosolic fraction of the resistant strain to apparent homogeneity and were identified by SDS-PAGE as polypeptides of 41.5 kDa (I), 40.5 kDa (III) and as a mixture of both in equal amounts (II). The molecular mass of the three holoenzymes was about 180 kDa, as determined by gel-filtration on Sephacryl S-300 HR, indicating a tetrameric structure. Isozyme III was also purified from parent strain and shown to consist of the 40.5-kDa polypeptide. Km values for malate were 0.31, 0.65 and 1.35 mM for isozyme I, II and III, respectively. From these results we conclude that T. foetus+, which is required for the formation of ethanol by alcohol dehydrogenase, an NADP-specific enzyme in this species. This is particularly important for the resistant strain, in which ethanol is the major end-product of glucose metabolism.
Hrdý
I
I
Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Brussels, Belgium.
Mertens
E
E
Van Schaftingen
E
E
D000818:Animals; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification*; D008795:Metronidazole / Q000494:pharmacology; D008970:Molecular Weight; D016835:Tritrichomonas foetus / Q000187:drug effects / Q000201:enzymology*
D007527:Isoenzymes; D008795:Metronidazole; D000431:Ethanol; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)