Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of Brij 58

✍ Scribed by Jeffrey S. Rush; Jurgen Klein; Paolo Fanti; Narayan R. Bhat; Charles J. Waechter

• Publisher: Elsevier Science
• Year: 1992
• Tongue: English
• Weight: 700 KB
• Volume: 207
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(92)90016-z

✦ Synopsis

This paper describes a simple and direct procedure for assaying Ca(2+)-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [gamma-32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.