Purification and properties of porcine brain glutathione S-transferases.

Abstract

Glutathione S-transferase was purified from porcine brain. Four enzyme fractions were obtained by column chromatography on CM-cellulose and the main enzyme fraction was purified to apparent homogeneity as judged by disc gel electrophoresis. The action spectra of these enzyme fractions toward some typical substrates were roughly similar. The optimum pH range of the purified enzyme was from 6.5 to 7.5 with o-dinitrobenzene as a substrate. The main enzyme showed a molecular weight of about 43,000 on Sephadex G-150 chromatography, and about 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was composed of two subunits of apparently identical molecular weight bound to each other noncovalently. The amino acid composition showed characteristic high contents of leucine and glutamic acid residues. A notable difference was observed in the lysine content as compared with that of the liver enzyme. The brain enzyme bound bilirubin less strongly than the monkey liver enzyme and human albumin. In addition, the regional and subcellular distributions of the enzyme in porcine brain was investigated with o-dinitrobenzene as a substrate. The enzyme activity was found to be distributed fairly evenly in various regions of the brain, and was especially abundant in the cytosol fraction. However, the enzyme activity was also detected considerably in the microsomal and mitochondrial fractions but not in the synaptosomal fraction.