M‐phase‐specific histone H1 kinase in fish oocytes: Purification, components and biochemical properties

We demonstrate, for the first time in fish, that a Ca²⁺‐independent and cyclic‐nucleotide‐independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them. The kinase, M‐phase‐specific histone H1 kinase (M‐H1K), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPKKAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serine/threonine‐specific protein kinase encoded by the fission yeast cdc2⁺ gene (cdc2 kinase). The M‐H1K and maturation‐promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M‐H1K and MPF. The final preparation was purified 5000‐fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10000‐fold with a recovery of 7% when SP peptide was used. The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33,34,46 and 48 kDa. Anti‐PSTAIR antibody recognizing cdc2 kinase cross‐reacted with the 33‐kDa and 34‐kDa proteins, while the 46‐kDa and 48‐kDa bands cross‐reacted with monoclonal antibodies raised against cyclin B. The 33‐kDa protein was also recognized by an antibody against a goldfish cdk2 (Eg1) kinase, a cdc2‐related kinase which has the PSTAIR sequence and binds to p13^suc1 but does not form a complex with cyclin B. M‐H1K activity corresponded well to the 34‐kDa, 46‐kDa and 48‐kDa proteins but not to the 33‐kDa protein. These results strongly suggest that M‐H1K consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M‐H1K, although it is found in the highly purified M‐H1K. The purified M‐H1K utilized Mg²⁺, Mn²⁺, ATP and GTP, and had a wide pH optimum ranging over 8.0–10.5. The kinase was thermolabile and sensitive to freezing/thawing.