Partial purification and characterization of mitotic factors from HeLa cells.
Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006133:Growth Substances / Q000302:isolation & purification* / Q000494:pharmacology; D006367:HeLa Cells / Q000032:analysis*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009865:Oocytes / Q000166:cytology; D011374:Progesterone / Q000494:pharmacology; D014462:Ultrafiltration; D014982:Xenopus laevis
D002868:Chromosomal Proteins, Non-Histone; D006133:Growth Substances; D011374:Progesterone; D000469:Alkaline Phosphatase; D008274:Magnesium; D002118:Calcium