Purification and partial characterization of cytosolic malate dehydrogenase from Tritrichomonas foetus.

A cytosolic malate dehydrogenase from Tritrichomonas foetus (Riedmüller) was purified to apparent homogeneity by a combination of differential centrifugation, affinity, hydrophobic interaction and ion-exchange chromatography. The purest preparation had specific activity of 309 mumoles.(mg protein)-1.min-1, which corresponded to 50-fold purification. The enzyme representing almost 2% of total cytosolic protein displayed hyperbolic affinity curves for substrate and coenzyme. Km for oxaloacetate and NADH was 17.5 microM and 13 microM, respectively. Subunit size determined by SDS-PAGE and by laser desorption mass spectroscopy was approximately 37 kDa, which corresponds to values reported for most malate dehydrogenases. The native molecular weight determined by gel filtration was 244 kDa, indicating six subunit structure of holoenzyme; this is unusual in comparison with other malate dehydrogenases, which are usually dimers or tetramers.