DIPEPTIDYL AMINOPEPTIDASE-III OF GUINEA-PIG BRAIN - SPECIFICITY FOR SHORT OLIGOPEPTIDE SEQUENCES

A dipeptidyl aminopeptidase III-type activity has been purified from t he cytoplasm of guinea-pig brain using arginyl-arginyl-7-amido-4 methy lcoumarin as substrate. The enzyme was purified 754-fold relative to t he crude homogenate and with a 12.7% recovery. The purified enzyme was found to have a relative molecular weight of 85,000 and consists of o ne polypeptide chain of relative molecular weight 80,000, on the basis of its migration on calibrated sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. It is highly sensitive to the presence of ch elating agents, sulphydryl reactive agents, and the dipeptide Tyr-Tyr. Dithiothreitol (1 mM) reduced activity by 28%, and 36 and 65% inhibit ion was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 mM), respectively. Little or no inhibition was observed with best atin, bacitracin, captopril, amastatin, and arphamenine B. The purifie d enzyme released dipeptide moieties from a wide range of peptides inc luding enkephalin sequences and also angiotensin sequences up to the o ctapeptide angiotensin II, These sequences inhibited the hydrolysis of arginyl-arginyl-7-amido-4-methylcoumarin by dipeptidyl aminopeptidase III with Ki values in the micromolar range. No hydrolysis was observe d with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences c ontaining a Pro residue on either side of the sissile bond. Peptides c ontaining less than four amino acids were not hydrolysed.