Substrate specificity of ascidian sperm trypsin-like proteases, spermosin and acrosin.

Abstract

In order to investigate systematically the substrate or subsite specificity of two sperm proteases, acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization.