MONOCLONAL-ANTIBODIES KI-S3 AND KI-S5 YIELD NEW DATA ON THE KI-67 PROTEINS

The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new ma bs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-e mbedded tissues. They recognize proteins with the same molecular mass as Ki-67 in western blot and for the first time also in immunoprecipit ation experiments. With these mabs we were able to enrich and purify t he ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the ki-67 proteins. Since we were able to immunoprecipi tate the Ki-67 proteins, we performed various immunoprecipitation expe riments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [S-35]-methionine we were able t o immunoprecipitate the Ki-67 proteins after only 5 minutes of labelli ng time. In turnover experiments the Ki-67 proteins could not be detec ted 3 hours after the end of labelling. These data indicate a half-lif e of the Ki-67 proteins of about 90 minutes. Labelling experiments wit h [P-32]-orthophosphate revealed that the Ki-67 proteins are phosphory lated. After dephosphorylation was blocked with okadaic acid or cell g rowth was arrested by means of Colcemid, the phosphorylation of the Ki -67 proteins was greatly increased, indicating that the ki-67 proteins are phosphorylated via serine and threonine, and that the phosphoryla tion of the Ki-67 proteins increases in cycling cells. Labelling exper iments with [H-3]-mannose and [H-3]-glucose revealed that the protein is weakly N-glycosylated.