Separation of amino acid naphthylamidases in human plasma on sephadex G-200 and DEAE-cellulose

Abstract

Enzymatic activities for the hydrolysis of ß-naphthylamides of various amino acids (alanine, arginine, aspartic acid, glutamic acid, leucine, lysine, phenylalanine, tyrosine and valine) in human plasma were separated by column chromatography on Sephadex G-200 and DEAE-cellulose. The distribution of activities toward naphthylamides of aspartic acid and glutamic acid was identical, but different from that toward leucine naphthylamide. The distribution of activities toward naphthylamides of other aminoacids (alanine, arginine, lysine, phenylalanine, tyrosine and valine) was generally like that toward leucine naphthylamide. The molecular weight or aspartyl of glutamyl ß-naphthylamidase was 190,000, whereas that of leucyl ß-naphthylamidase was 160,000. Glutamyl ß-naphthylamidase was separated from leucyl ß-naphthylamidase by Sephadex G-200 and then DEAE-cellulose. Properties of purified ß-naphthylamidase directed toward the aspartyl or glutamyl substrates were different from those of leucyl ß-naphthylamidase. Glutamyl ß-naphthylamidase was markedly activated by CA ++, had a pH optimum at 7.5, and a K m value of 1·2 × 10 −3 M. Ca ++ had no effect on leucyl ß-naphthylamidase. Its pH optimum was 6·6 and the K m value was 3·3 × 10 −4 M. It is concluded that the enzyme in human plasma which hydrolyzes either glutamyl ß-naphthylamide or aspartyl ß-naphthylamide (aminopeptidase A) is a distinct enzyme.