Purification and properties of prolylcarboxypeptidase (angiotensinase C) from human kidney.

Abstract

Prolylcarboxypeptidase was purified from human kidney 1200-fold with 18% yield. The enzyme had no cathepsin A activity and appeared to be homogeneous in gel electrophoresis. The molecular weight of prolylcarboxypeptidase was estimated to be 115,000 by gel filtration. Under denaturing conditions the enzyme dissociated into subunits of 45,000 and 66,500 molecular weight. The enzyme cleaved benzyloxycarbonyl (Cbz)-Pro-Phe, representing the COOH-terminal end of angiotensin II and des-Asp1-angiotensin II (angiotensin III), at a rate of 31 micronmol/h/mg of protein. The rate of hydrolysis increased when phenylalanine in the N-protected dipeptide was replaced with alanine, valine, or leucine or when the octapeptide angiotensin II or the heptapeptide angiotensin III were the substrates. The enzyme also cleaved the angiotensin II antagonist saralasin (Sar1-Ala8-angiotensin II). The Km values were 1 mM, 2mM, and 0.77 mM with Cbz-Pro-Phe, angiotensin II, and angiotensin III, respectively. The enzyme had an acid pH optimum (4.5 to 5.5), but hydrolyzed angiotensin III at pH 7 at 50% of the optimal rate. Prolylcarboxypeptidase was inhibited by diisopropyl phosphorofluoridate and pepstatin, but not by sequestering agents or -SH reagents.