title: Purification and characterization of maturation-promoting factor in fish.
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, p13suc1-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.
authors.affiliation: Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yamashita
authors.forename: S
authors.initials: S
authors.lastname: Fukada
authors.forename: M
authors.initials: M
authors.lastname: Yoshikuni
authors.forename: P
authors.initials: P
authors.lastname: Bulet
authors.forename: T
authors.initials: T
authors.lastname: Hirai
authors.forename: A
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Y H
authors.initials: YH
authors.lastname: Lou
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.forename: Y
authors.initials: Y
authors.lastname: Nagahama
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000737:chemistry; D002347:Carps / Q000196:embryology*; D005260:Female; D006054:Goldfish / Q000196:embryology*; D016200:Maturation-Promoting Factor / Q000737:chemistry / Q000302:isolation & purification*; D009865:Oocytes / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development; D010766:Phosphorylation; D014982:Xenopus laevis
chemical_list: D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor