Purification of maturation-promoting factor, an intracellular regulator of early mitotic events.

Abstract

Maturation-promoting factor causes germinal vesicle breakdown when injected into Xenopus oocytes and can induce metaphase in a cell-free system. The cell-free assay was used to monitor maturation-promoting factor during its purification from unfertilized Xenopus eggs. Ammonium sulfate precipitation and six chromatographic procedures resulted in a preparation purified >3000-fold that could induce germinal vesicle breakdown within 2 hr when injected into cycloheximide-treated oocytes. Proteins of 45 kDa and 32 kDa were correlated with fractions of highest activity in both assays. These fractions contained a protein kinase activity able to phosphorylate the endogenous 45-kDa protein, as well as histone Hi, phosphatase inhibitor 1, and casein. The highly purified preparations described here should help to identify the mechanism of action of maturation-promoting factor and to elucidate the role of protein kinases in the induction of metaphase.

A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF‐1γ and EF‐1β

“…Cell division is controlled by a cytoplasmic factor, MPF, which has been recently purified [1]. It is constituted in part by a protein of 34 kDa, which shares H1 kinase activity and is related to the product of the gene cdc2 controlling the cell division in the yeast, Schizosaccharomyces pombe [2][3][4][5].…”

Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast.

“…Homologues of both types of these proteins have been found in many eukaryotes, suggesting that mitotic controls are highly conserved. Such proteins are components of the maturation promoting activity (MPF) which upon injection causes immature Xenopus eggs or invertebrate oocytes to enter meiotic metaphase (Lohka et al, 1988). Moreover, deregulation of the kinase through mutation causes premature entry into mitosis in the fission yeast Schizosaccharomyces pombe (Nurse and Thuriaux, 1980).…”

Expression of an EF-1α-like Rat cDNA, S1, in Escherichia coli and Production of a Rabbit Polyclonal Antiserum to the Recombinant Protein

“…Inttially MPF was purified from Xellopus, and the fraction with the highcst MPF activity was found to be associated with two proteins of about 32 kDa and 45 kDa (Lohka et al, 1988). This fraction contained a protein kinase activity ablc to phosphorylate the 45 kDa protein.…”