Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of Brij 58.
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.
Rush
J S
JS
Department of Biochemistry, University of Kentucky College of Medicine, Lexington 40536.
Klein
J
J
Fanti
P
P
Bhat
N R
NR
Waechter
C J
CJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002592:Cetomacrogol / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D005260:Female; D005910:Glioma; D007700:Kinetics; D051379:Mice; D008811:Mice, Inbred DBA; D010006:Osteoblasts / Q000201:enzymology; D010761:Phosphorus Radioisotopes; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D003902:Detergents; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D002592:Cetomacrogol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate