[Purification and some properties of isozymes 1 and 5 of lactate dehydrogenase from fox heart and skeletal muscles]

Abstract

An improved method is described for the isolation of isozymes 1 and 5 of lactate dehydrogenase (LDH) from heart and skeletal muscles of foxes. The method includes salt fractionation with ammonium sulphate, chromatography on DEAE- and CM-celluloses and affinity chromatography on AMP-Sepharose. The preparations of LDH isozymes 1 and 5 turned out to be homogeneous both in 7,5% polyacrylamide gel electrophoresis and under immunodiffusion analysis. It is shown that the pH optimum for LDH-1 is 10.2-10.4 for LDH-5 it is 9.5-9.6 in the case of the direct reaction, and the pH optimum is 7.6-7.8 for LDH-1 and 7.3-7.4 for LDH-5 in the case of reverse reaction. The values of Mikhaelis constants were determined for substrates and coenzymes in direct and reverse reactions. It is found that the excess of lactate and pyruvate causes substrate inhibition of both LDH-1 and LDH-5. The activities of LDH-1 and LDH-5 showed an unexpected similar sensitivity to the inhibitory effect of high pyruvate concentrations.