65] d(−)-Lactate dehydrogenases from fungi

Abstract

Publisher Summary This chapter discusses the assay method, purification procedure, and properties of D (–)-lactate dehydrogenases from fungi. Lactate dehydrogenases are key enzymes in anaerobic energy metabolism. Two activities are determined in cell-free extracts and at various stages of purification by following the rate of NADH oxidation at 25° spectrophotometrically, at 340 nm. The purification procedure includes steps, such as preparation of cell-free extracts, protamine sulfate precipitation, DEAE-cellulose chromatography, ammonium sulfate fractionation, sephadex gel filtration, ammonium sulfate fractionation, and desalting. Polyacrylamide gel electrophoresis of the purified enzyme preparations is carried out at pH 8, using Tris-barbital buffer (1 g of Tris, and 5.52 g of barbital diethylbarbituric acid in 1 liter of H20) system. The enzymes are generally stable at 4° for several weeks, and at –20 ° for at least 2 years. Occasional species (example, Pythium butleri subramaniam , CBS 164.68) may have an enzyme that loses about 50% of its activity in crude extracts when stored frozen or kept on ice overnight. Oomycetes apparently contain the D(–)- LDH isozyme, which utilizes NAD + as coenzyme. GTP has been found to be an allosteric inhibitor of all D(–)-LDH's of the Oomycetes, increasing the cooperative binding of NADH and D (–)-lactate, but not of pyruvate and NAD + . ATP and ITP act as competitive inhibitors of NADH binding but are not allosterie modulators.