Tetsuya Someno

Evidence for the participation of two sperm proteases, spermosin and acrosin, in fertilization of the ascidian, Halocynthia roretzi: Inhibitory effects of leupeptin analogs on enzyme activities and fertilization☆

Ten kinds of argininal-containing compounds were examined for their inhibitory effects on the fertilization of the solitary ascidian and on the activities of acrosin and spermosin, trypsin-like proteases isolated from spermatozoa of this animal. Benzyloxycarbonyl-Val-Pro-argininal (I) and benzyloxycarbonyl-Phe-Leu-argininal (II) showed the strongest inhibition on the fertilization. Leupeptin (acetyl-Leu-Leu-argininal, III) was ranked next (I, II greater than III). The activity of ascidian acrosin was susceptible to most of the compounds, among which II was the best inhibitor and followed with I and III (II greater than I, III). Spermosin suffered significant inhibition only with I and II (I greater than II). These results suggest that not only acrosin but also spermosin is involved in fertilization of the ascidian.

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

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.

Participation of 650-kDa protease (20 S proteasome) in starfish oocyte maturation

A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.

Inhibition of starfish oocyte maturation by leupeptin analogs, potent trypsin inhibitors☆

Inhibition of subsite-substituted leupeptin analogs, potent trypsin inhibitors, on 1-methyladenine-induced germinal vesicle breakdown was investigated in a starfish, Asterina pectinifera. Of benzyloxycarbonyl(Z)-Leu-P2-argininals, the analog with Ser at P2 residue was the strongest inhibitor, and those with Pro, Leu greater than Thr greater than Gly were followed in this order. In Z-P3-Ser-argininals, ranking of the inhibitory ability was as follows: Phe greater than Leu much greater than Pro greater than Ala at P3 residue. Among 11 analogs synthesized, Z-Phe-Ser-argininal showed the strongest inhibition. The inhibitory potency of the analog was 100-fold stronger than that of leupeptin (acetyl-Leu-Leu-argininal). Thus, trypsin-like enzyme possessing a narrow subsite specificity participates in oocyte maturation in the starfish.

Determination of urokinase activity by high-performance liquid chromatography with radioisotope detection

Abstract A specific method for the determination of two types of urokinase was developed, involving high-performance liquid chromatography (HPLC) with radioisotope detection using [3H]diisopropyl fluorophosphate (DFP) as a pre-labelling reagent. The serine moiety located in the active site of urokinase was reacted selectively with [3H]DFP to form [3H]DFP-urokinase adduct. Two types of urokinase were determined by monitoring the radioactivity of each peak separated by means of HPLC. The total radioactivity of the two peaks was in good agreement with the urokinase activity obtained by a modified fibrin plate method.

Rapid method for the determination of urokinase activity by high-performance liquid chromatography

Abstract A simple, rapid and reproducible high-performance liquid chromatographic method for the quantitation of urokinase was developed by the separation of two types of urokinase with molecular weights of 54,000 and 32,000 in its crude solutions containing various as impurities. TSK-GEL 3000 SW column chromatography with 0.2 M phosphate buffer solution of pH 3.0 as the mobile phase at room temperature was used. The ratios of the two types of urokinase determined by the method were in good agreement with those obtained by Sephadex G-100 gel chromatography followed by Waltons modified plate method.

Inhibition of Mouse Blastocyst Hatching by Subsite-Specific Trypsin Inhibitors, Peptidyl Argininals1

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P4)‐Leu(P3)‐Leu(P2)‐argininal(P1)] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P4 position inhibited the hatching more strongly than those containing acetyl group or unprotected N‐terminal amino acid. Among five Z‐Leu‐P2‐argininals, a derivative containing a P2 Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P2 position followed in this order. Then, we synthesized four Z‐P3‐Ser‐argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P3 position was the strongest inhibitor, and the Leu‐, Pro‐, and Ala‐containing derivatives were ranked in this order. Thus, among Z‐dipeptidyl‐argininals tested, Z‐Phe‐Ser‐argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P3)‐Ser(P2)‐Arg(P1) sequence as a substrate.