IDENTIFICATION, PURIFICATION AND SEPARATION OF DIFFERENT ISOZYMES OF NADP-SPECIFIC MALIC ENZYME FROM TRITRICHOMONAS-FETUS

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. K(m) 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.