Fractionation and identification of metaphase-specific phosphorylated forms of high-mobility-group proteins.
In the present work chromatography on phosphocellulose and blue Sepharose have been used to fractionate the different phosphorylated forms of the low-molecular-mass high-mobility-group (HMG) proteins from metaphase arrested HeLa cells. The proteins in the different fractions from the blue Sepharose column were analysed by acetic acid/urea gel electrophoresis. Aliquots from the same fractions were also treated with alkaline phosphatase and the dephosphorylated and phosphorylated proteins were then compared by electrophoresis to identify the phosphorylated proteins. It was found that HMG 14 consisted of a mixture of an unphosphorylated and two phosphorylated forms, while HMG Y existed as one homogeneous superphosphorylated form. These findings remove previous uncertainty about phosphorylation of HMG Y and HMG 14. The presence of HMG M and phosphorylated forms of HMG 17 was confirmed. Peptide mapping of HMG I and HMG M gave further evidence that HMG M is a superphosphorylated form of HMG I, and it is suggested that the term HMG Im be used instead of HMG M. The results suggested that HMG I and Y from HeLa cells contained at least three and two metaphase-specific phosphate groups respectively, while HMG 14 and 17 both consisted of an unphosphorylated form and two phosphorylated forms. A protein corresponding to HMG Im from HeLa cells was also found to be present in metaphase-arrested human lymphocytes, while HMG I and from two different rodent species seemed to be less phosphorylated then their counterparts from HeLa metaphase cells.
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006609:High Mobility Group Proteins / Q000302:isolation & purification*; D006801:Humans; D008214:Lymphocytes / Q000032:analysis; D008677:Metaphase; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000302:isolation & purification*; D014508:Urea
D000085:Acetates; D006609:High Mobility Group Proteins; D010750:Phosphoproteins; D014508:Urea; D019342:Acetic Acid