Pituitary Arylamidases and Peptidases

Abstract

Abstract The hydrolysis of lysyl-, arginyl-, leucyl-, methionyl-, and phenylalanyl-p-nitroanilides by bovine anterior pituitary glands is attributable to the action of at least two distinct enzymes, designated as lysyl and arginyl arylamidases because of their preferential action on the respective p-nitroanilides. The arginyl arylamidase is associated with a dipeptidase during fractionation with (NH4)2SO4 and chromatography on diethylaminoethyl cellulose. The purified arylamidase is principally active upon Arg- and, to a somewhat lesser extent, on Lys-p-nitroanilide; it hydrolyzes other p-nitroanilides at a very low rate. The lysyl arylamidase is distinguishable from the arginyl arylamidase by its requirement for thiol compounds for activation and by a strong sensitivity to puromycin. Although Lys-p-nitroanilide is hydrolyzed preferentially, a significant rate of hydrolysis also is seen with Arg-, Phe-, Met-, and Leu-p-nitroanilide. Purified lysyl arylamidase preparations completely hydrolyze Ala5, Ala4, Ala3, Ala2, Lys4, Lys3, and Lys2 to the free amino acids, but only the hydrolysis of Ala5,, Ala4, and Lys4 is subject to a consistent and substantial activation by mercaptoethanol and inhibition by puromycin. The total hydrolysis of the tetra- and pentapeptides is ascribed to the consecutive action of an aminopolypeptidase, a tripeptidase, and a dipeptidase. Since the aminopolypeptidase is associated with the lysyl arylamidase during purification and displays the same properties as the latter, both activities are assumed to be due to the action of a single enzyme. Both arylamidases require thiol groups and a metal for activity and are strongly inhibited by dipeptides, tripeptides, polylysine, polyarginine, protamine, and the arginine-rich calf thymus histones. The subcellular distribution, the effect of various inhibitors, and the stability of the arylamidases are noted.