Trypsin-like enzyme from eggs of the ascidian (protochordate), Halocynthia roretzi. Purification, properties, and physiological role.
A trypsin-like enzyme has been purified to apparent homogeneity from eggs of the ascidian, Halocynthia roretzi, by a procedure including column chromatography on diethylaminoethyl-cellulose, phenyl-Sepharose, and soybean trypsin inhibitor-immobilized Sepharose 4B. The molecular weight of the enzyme was estimated to be 31,000 and 33,000 by gel electrophoresis in sodium dodecyl sulfate under the reducing and the nonreducing conditions, respectively. The isoelectric point of the enzyme was 4.8. The pH optimum of the activity was 8.4. The enzyme was stable between pH 6 and 9 in the presence of 0.005% Brij 35 as a stabilizer. Substrate specificity of the purified enzyme was broad toward various peptidyl-arginine (or -lysine) 4-methylcoumaryl-7-amides and was similar to that of a trypsin-like enzyme found in the fertilization product. The purified enzyme was inhibited by diisopropyl fluorophosphate and a variety of trypsin inhibitors including leupeptin, but not, or scarcely, inhibited by p-chloromercuribenzoic acid, pepstatin, chymostatin, bestatin, elastatinal, and tosyl-phenylalanyl-chloromethane. The rankings in the potencies of leupeptin and its six analogs as the inhibitors of the purified enzyme were well correlated with those found in their inhibitory effects on the expansion of perivitelline space. Thus, the trypsin-like enzyme possibly present in the fertilization product participates in the expansion of perivitelline space of the egg during fertilization of the ascidian.
D000818:Animals; D005306:Fertilization; D007526:Isoelectric Point; D007976:Leupeptins / Q000494:pharmacology; D008970:Molecular Weight; D010063:Ovum / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014357:Trypsin / Q000302:isolation & purification*; D014561:Urochordata / Q000201:enzymology*
D007976:Leupeptins; D011480:Protease Inhibitors; D014357:Trypsin; C032854:leupeptin