This paper describes a simple and direct procedure for assaying Ca2+-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 [γ-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.
|Number of pages||7|
|State||Published - Dec 1992|
Bibliographical noteFunding Information:
i This work was supported by Grant BC-583 from the American Cancer Society and Grant GM 36065 from the National Institutes Health awarded to CJW. ’ Present address: Department of Neurology, South Carolina, Charleston, SC 29425. 3 To whom correspondence should be addressed. ’ Abbreviations used PMA, phorboll2-myristate 13-acetate; PKC, phospholipid/calcium-dependent protein kinase; PS, phosphatidylserine; FCS, fetal calf serum; PBS, Dulbecco’s phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; EGTA, ethylene glycol bis(&aminoethyl ether) NJ’-tetraacetic acid TCA, trichloroacetic acid SDS, sodium dodecyl Chaps, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology