Purification and Characterization of Cysteine Conjugate Transaminases from Rat Liver

Abstract

1. Soluble cysteine-conjugate α-ketoglutarate transaminase (CAT-I) was purified about 670-fold from rat liver cytosol using s-(p-bromophenyl)-L-cysteine as amino acid substrate. The enzyme preparation of the final step of purification showed a single band in polyacrylamide gel electrophoresis. CAT-I accounted for 64% of the transaminase activity in cytosol.

2. The mol. wt of the enzyme was about 64000 as determined by gel filtration. Respective K_m values for s-(p-bromophenyl)-L-cysteine and α-ketoglutaric acid were 1.0 and 1.3 mM in Tris-acetate buffer (pH 7.0). Aminooxyacetic acid, hydroxylamine, and KCN inhibited the enzyme activity.

3. In addition to CAT-I, two isozymes (CAT-IIA and CAT-IIB) were partially purified from rat liver cytosol. In respect of mol. wt, substrate specificity towards cysteine conjugates, and several other properties, CAT-IIA and CAT-IIB were very similar to CAT-I. However, differences were observed for these enzymes in the rate of reverse reaction (formation reaction of cysteine conjugates and α-ketoglutaric acid) and substrate specificity towards L-aspartic acid and L-cysteinesulphinic acid.