Dipeptidyl aminopeptidase III of guinea-pig brain: specificity for short oligopeptide sequences.
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.
Smyth
M
M
Department of Biochemistry, University College, Galway, Ireland.
O'Cuinn
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000302:isolation & purification / Q000378:metabolism*; D017886:Durapatite; D006168:Guinea Pigs; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D009842:Oligopeptides; D011480:Protease Inhibitors; D017886:Durapatite; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III