Leucine Aminotransferase: III. ACTIVATION BY β-MERCAPTOETHANOL

Abstract

Abstract Highly purified leucine transaminase from pig heart was observed to undergo activation in the presence of several thiol compounds. β-Mercaptoethanol was the most effective thiol compound tested. It yielded a 3- to 4-fold increase in activity when added either directly to the standard assay system or first incubated with excess enzyme alone and diluted out just prior to assay. Leucine transaminase was inhibited markedly by low concentrations of heavy metal ions and mercaptide-forming reagents. One of our most active preparations contained eight p-chloromercuribenzoate titratable sulfhydryl groups per 75,000 g of protein; however, about 90% of the residual activity (i.e. minus β-mercaptoethanol) was lost when only four had reacted with p-chloromercuribenzoate. β-Mercaptoethanol partially reversed inhibition by excess p-chloromercuribenzoate, restoring 75% of the initial activity. Enzyme centrifuged at an initial protein concentration of 7 to 8 mg per ml gave an s20,w value near 5.0 when examined either directly in phosphate buffer or first activated and then centrifuged in phosphate-0.1 m β-mercaptoethanol. If, however, the enzyme was first activated and the β-mercaptoethanol was removed by dialysis, heavier material with an s20,w of 6.8 appeared in the schlieren pattern along with the original peak. Aggregation was correlated with a loss of the increased activity generated by prior treatment with β-mercaptoethanol.