Isolation and characterization of a post-proline cleaving enzyme and its inhibitor from sperm of the ascidian, Halocynthia roretzi.
A post-proline cleaving enzyme and its endogenous inhibitor have been demonstrated to be present in sperm of the ascidian, Halocynthia roretzi. The enzyme was extracted with artificial sea water from frozen and thawed sperm and isolated from accompanying acrosin-like and chymotrypsin-like enzymes by DEAE-cellulose chromatography. It was then separated from the endogenous inhibitor by ammonium sulfate fractionation and DEAE-Sephacel chromatography. Three subsequent chromatographic operations using hydroxylapatite, Sephadex G-150 and Z-Gly-Pro-Leu-Gly-aminohexyl-Sepharose yielded the highly purified enzyme. The molecular weight and isoelectric point of the enzyme were estimated to be 66,000 and 5.5, respectively. The pH optimum of the activity was 7.0. The enzyme was inactivated with diisopropylphosphorofluoridate, phenylmethylsulfonyl fluoride, Z-Gly-Pro-chloromethyl ketone and sulfhydryl-directed reagents; these inhibitor susceptibilities were similar to those reported for the enzymes of mammalian origins. The ascidian enzyme hydrolyzed oxytocin, angiotensin II, luteinizing hormone releasing hormone and neurotensin at the carboxyl side of proline residues. The endogenous inhibitor was heat stable. The molecular weight of its main component was estimated to be about 8,000. The presence of salt at high concentrations weakened the enzyme-inhibitor interaction. Z-Gly-Pro-chloromethyl ketone inhibited fertilization of the ascidian, suggesting possible involvement of the post-proline cleaving enzyme in fertilization.
D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification*; D005260:Female; D005306:Fertilization; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity; D014561:Urochordata / Q000201:enzymology*
D011480:Protease Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases