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Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro
Author(s) -
Zhang Jianshe,
Masui Yoshio
Publication year - 1997
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/(sici)1098-2795(199707)47:3<302::aid-mrd9>3.0.co;2-p
Subject(s) - tissue transglutaminase , biology , biochemistry , polyclonal antibodies , in vitro , rana , immunoprecipitation , microbiology and biotechnology , enzyme , endocrinology , antibody , immunology , gene
Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca 2+ , and is designated primary CSF (1°CSF). During storage of Ca 2+ ‐containing cytosols at 2°C, stable CSF activity appears, designated secondary CSF (2°CSF). In Rana pipiens egg cytosols, the development of 2°CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2°CSF activity when purified (Shibuya and Masui, 1989: Development 106:799–808). The present study shows that both the formation of 2°CSF protein complex and the development of its activity are inhibited by ethylamine and glycine‐ethyl‐ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity‐purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68‐kDa protein in fresh egg cytosols, as well as with the 2°CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2°CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible for the formation of 2°CSF, and that transglutaminase itself is incorporated into 2°CSF molecules. Mol. Reprod. Dev. 47:302–311, 1997. © 1997 Wiley‐Liss, Inc.