Dipeptidyl Aminopeptidase III of Guinea‐Pig Brain: Specificity for Short Oligopeptide Sequences

Abstract

A dipeptidyl aminopeptidase III‐type activity has been purified from the cytoplasm of guinea‐pig brain using arginyl‐arginyl‐7‐amido‐4 methylcoumarin as substrate. The enzyme was purified 754‐fold relative to the 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 one 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 chelating agents, sulphydryl reactive agents, and the dipeptide Tyr‐Tyr. Dithiothreitol (1 mM) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 mM), respectively. Little or no inhibition was observed with bestatin, bacitracin, captopril, amastatin, and arphamenine B. The purified enzyme released dipeptide moieties from a wide range of peptides including enkephalin sequences and also angiotensin sequences up to the octapeptide 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 observed with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences containing a Pro residue on either side of the sissile bond. Peptides containing less than four amino acids were not hydrolysed.

Activity Staining and Inhibition Characterization of Dipeptidylpeptidase-III Enzyme from Goat Brain

“…Our results are in agreement with human placental DPP-III [9], whereas Co 2+ was most effective in restoring the activity of EDTA pretreated DPP-III from human RBCs [10]. Ca 2+ and Co 2+ were most effective in restoring the activity of EDTA pretreated guinea pig brain DPP-III [11]. On the other hand, untreated DPP-III was activated by Co 2+ and inhibited by Zn 2+ , Ni 2+ , and Cu 2+ .…”

Dipeptidyl peptidase III: a multifaceted oligopeptide N‐end cutter

“…By virtue of its affinity for different types of bioactive peptides and synthetic substrates, it was named enkephalinase B [18], red cell angiotensinase [19], dipeptidyl aminopeptidase III [18] and dipeptidyl arylamidase III [17]. Nevertheless, the exact relationship of DPP III with enkephalinase B is still unclear [20,21].…”

Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins

“…Previous findings excluded Pro from positions P 1 and P 1 9 in the peptide sequence of DPP III substrates (Abramić et al, 1988;Smyth and O'Cuinn, 1994). Appropriate standard (di)peptides were co-chromatographed.…”