[64] Lactate-oxaloacetate transhydrogenase from Veillonella alcalescens

The lactate-oxaloacetate transhydrogenase or malatelactate transhydrogenase is found in only one genus, Veillonella. This chapter describes the assay method and properties of lactate–oxaloacetate transhydrogenase isolated from Veillonella alcalescens. The lactate–oxaloacetate transhydrogenase functions to oxidize lactate to pyruvate probably by coupling with an L-malate dehydrogenase present in Veillonella. Lactate entering the cell is converted to pyruvate, which is metabolized either to acetic or to propionic acids. The transhydrogenase is readily reversible and enzyme activity is assayed in either direction. The assay is performed with L-malate and pyruvatc as substrates. Two assay procedures are used. The direct assay depends upon the increase in absorbance at 258 nm because of oxaloacetate formation. The indirect assay, employing nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidation, which is approximately seven times more sensitive than the direct assay, measures the formation of oxaloacctate from L-malate and pyruvate by coupling the transhydrogenase with the malate dehydrogenase. Purification to homogeneity requires only a two-step procedure, using diethylaminoethyl (DEAE)-cellulose batch treatment, followed by QAE-Sephadex chromatography.