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.
ASJC Scopus subject areas
- Molecular Biology