|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥2,000,000 (Direct Cost : ¥2,000,000)
The fragile envelope of an unfertilized egg changes into hard envelope after fertilization and/or activation of the egg. We analyzed such the egg envelope (chorion) hardening of rainbow trout, Onco.rhynchus mykiss. The hardening was caused by two transglutaminases, P1 (76 and 82kDa) and P2 (76kDa), catalyzing epsilon-(gamma-glutamyl) lysine cross-link formation between chorion subunit proteins (49, 56 and 65kDa). The cross-linking resulted in polymerization of all the subunits. The enzymes were localized exclusively in unfertilized egg chorion. Activity of the P1 enzyme was apparent in a medium of low ionic strength such as fresh water. After egg activation, the P1 changed into a 48kDa protein. Such the proteolytic processing resulted in augmentation of its activity, suggesting that the egg activation-associated conversion of P1 to 48kDa enzyme promotes the efficient cross-linking at the restricted sites of chorion proteins. On the other hand, the P2 was released from chorion into peri
vitelline space. Such the release probably implies the cross-linking of universal sites in chorion. Both the changes of transglutaminases were inhibited by leupeptin and EDTA.
In addition to polymerization of chorion proteins by transglutaminase, we found post-activation occurrence of limited hydrolysis of a subunit. The 49kDa subunit changed into a 42kDa protein. This proteolysis was also inhibited by leupeptin and EDTA.The EDTA-sensitive proteinase was a metalloproteinase directly hydrolyzing the subunit. Some preliminary studies suggested that the leupeptin-sensitive proteinase activated the metalloproteinase to indirectly participate in the limited hydrolysis of the subunit.
Such chorion hardening process in fish egg was triggered by Ca^2 ions released from egg after activation. Thus, the chorion hardening reaction is a cascade" comprised by transglutaminases, and EDTA-sensitive and leupeptin-sensitive proteinases. A mode of chorion hardening in medaka (Oryzias 1atipes) eggs was essentially consistent with that in rainbow trout, except that transglutaminases were not extracted from unfertilized egg chorions, . Less