Aminopeptidase P from human leukocytes

Publisher Website
Google Scholar
Abstract
Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four‐step procedure. Buffy coats were the starting material. A Mr of 140000 was obtained by size‐exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with Mr of 71000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N‐terminal residues from peptides with N‐terminal Xaa‐Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di‐, tri‐ and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg‐Pro, Phe‐Pro > Trp‐Pro > Pro‐Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C‐terminal fragment (residues 201–206) of C‐reactive protein, oxytocin fragment Tyr‐Pro‐Leu‐Gly, morphiceptin, peptide Gly‐Pro‐Arg‐Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin‐6, was also demon‐strated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10‐phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy‐phenylalanine, as well as several N‐carbobenzoxy‐proline‐containing peptides, caused partial inhibition. The observed resistance of Gly‐Pro, Pro‐Gly, Pro‐Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline‐specific dipeptidases in the aminopeptidase‐P preparation.