Purification and characterization of maturation-promoting factor in fish

M. Yamashita, S. Fukada, M. Yoshikuni, P. Bulet, T. Hirai, A. Yamaguchi, Y. H. Lou, Z. Zhao, Y. Nagahama

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90 Citations (Scopus)

Abstract

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13sucl-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.

Original languageEnglish
Pages (from-to)8-15
Number of pages8
JournalDevelopmental Biology
Volume149
Issue number1
DOIs
Publication statusPublished - Jan 1992
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Developmental Biology
  • Cell Biology

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    Yamashita, M., Fukada, S., Yoshikuni, M., Bulet, P., Hirai, T., Yamaguchi, A., Lou, Y. H., Zhao, Z., & Nagahama, Y. (1992). Purification and characterization of maturation-promoting factor in fish. Developmental Biology, 149(1), 8-15. https://doi.org/10.1016/0012-1606(92)90259-J