2 NOVEL ENDOPEPTIDASES RELEASED INTO THE MEDIUM DURING MATING OF GAMETES OF CHLAMYDOMONAS-REINHARDTII

During the mating reaction between mt(+) and mt(-) gametes of Chlamydo monas reinhardtii, two novel endopeptidases, each of which was able to digest the B chain of insulin, were released into the culture medium, together with a gamete lytic enzyme (GLE) which is responsible for di gestion of the gametic cell walls. Both endopeptidases and GLE were co purified from the mating medium by column chromatography on DEAE-cellu lose and concanavalin A. Gel filtration separated the peptidases, whic h were unable to digest gametic cell walls, into two fractions, endope ptidase-1 and endopeptidase-2. These enzymes were also separated from GLE, which was unable to digest the B chain of insulin. Endopeptidase- 1, with a molecular mass of about 200 kDa, cleaved the B chain of insu lin at the Ala(14)-Leu(15) peptide bond, and this activity was inhibit ed by EDTA. Endopeptidase-2, with a molecular mass of about 110 kDa, d igested the peptide at the Leu(15)-Tyr(16) peptide bond and was sensit ive to iodoacetate and chymostatin. When the cell walls of gametes of either mating-type were digested prior to mating with exogenously adde d GLE, the two endopeptidases were released into the medium, a result that suggests that they are stored, like GLE, outside the plasmalemma.