[61] Lactate Dehydrogenase Isozymes from Mouse

This chapter describes the assay method and properties of lactate dehydrogenase isozymes isolated from the mouse. Three homotetrameric isozymes of lactate dehydrogenase, designated as A₄, B₄, and C₄ or X, are found in mice. Each isozyme is expressed by a separate structural gene. All the lactate dehydrogenase isozymes require nicotinamide adenine dinucleotide kinase (NAD⁺) or nicotinamide adenine dinucleotide dehydrogenase (NADH) as the coenzyme and catalyze the same chemical reactions. The purification of these enzymes is achieved by general ligand affinity chromatography in combination with ion-exchange chromatography to separate the multiple forms of lactate dehydrogenase. The activity of lactate dehydrogenase isozymes is determined at 25°C spectrophotometrically by following the changes in absorbance at 340 nm. The reaction mixture in a total volume of 1 ml contains 0.1 M Tris-HCl, pH 8.0, 1.0 mM pyruvate, 0.15 mM NADH, and a suitable amount of enzyme to produce a decrease in the absorbance of 0.05–0.l per minute at 340 nm. Lactate dehydrogenase isozymes are virtually inactive with α-ketoglutarate as the substrate. Thin-layer chromatography of the product is performed on cellulose plates with a fluorescent indicator.