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

Jeffrey S. Rush, Jurgen Klein, Paolo Fanti, Narayan R. Bhat, Charles J. Waechter

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)304-310
Number of pages7
JournalAnalytical Biochemistry
Volume207
Issue number2
DOIs
StatePublished - Dec 1992

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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