Partial purification of maturation-promoting factor from catfish, Clarias batrachus: identification as the histone H1 kinase and its periodic activation

Abstract

Maturation-promoting factor (MPF) has been demonstrated in the 100 000 g supernatant of 17 α,20 β-dihydroxy-4-pregnen-3-one (17 α,20 β-DP)-induced catfish, Clarias batrachus oocytes using DEAE-cellulose and sephadex G-200 chromatography. Partially purified MPF molecule eluted as a single peak on sephadex G-200 with molecular mass of ∼200 kDa in native PAGE. SDS-PAGE analysis showed the presence of five proteins of 32, 34, 45, 46 and 48 kDa. Antibody against the PSTAIR sequence of p34 cdc2 recognized 32 and 34 kDa proteins, whereas rabbit anti-cyclin B1 and B2 crossreacted with 46 and 48 kDa proteins, respectively. Cyclin B was absent in immature oocytes and appeared after 7 h of 17 α,20 β-DP stimulation, coinciding with the histone H1 kinase (HH1K) activity and start of germinal vesicle breakdown (GVBD). Our data indicate that C. batrachus MPF is a complex of cdc2 kinase and cyclin B molecules. A close relationship between HH1K activity and catfish oocyte maturation has been demonstrated using cycloheximide, cytochalasin B and colchicine. HH1K activation was inhibited by cycloheximide, while cytochalasin B and colchicine were ineffective. These finding suggests that the activation of HH1K depends on protein synthesis, whereas disruption of microfilaments influences only nucleus migration without effect on GVBD or HH1K activation. An increase of phosphorylated proteins after activation of catfish oocytes with 17 α,20 β-DP has also been observed.