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

doi:10.1016/0003-2697(92)90016-Z

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

Molecular and Cellular Biochemistry

Research output: Contribution to journal › Article › peer-review

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 language	English
Pages (from-to)	304-310
Number of pages	7
Journal	Analytical Biochemistry
Volume	207
Issue number	2
DOIs	https://doi.org/10.1016/0003-2697(92)90016-Z
State	Published - Dec 1992

Bibliographical note

Funding 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

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/0003-2697(92)90016-Z

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@article{b706e568aaca452d877fad84f1751bf9,

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

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.",

author = "Rush, {Jeffrey S.} and Jurgen Klein and Paolo Fanti and Bhat, {Narayan R.} and Waechter, {Charles J.}",

note = "Funding 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. {\textquoteright} Present address: Department of Neurology, South Carolina, Charleston, SC 29425. 3 To whom correspondence should be addressed. {\textquoteright} Abbreviations used PMA, phorboll2-myristate 13-acetate; PKC, phospholipid/calcium-dependent protein kinase; PS, phosphatidylserine; FCS, fetal calf serum; PBS, Dulbecco{\textquoteright}s phosphate-buffered saline; DMEM, Dulbecco{\textquoteright}s modified Eagle{\textquoteright}s medium; EGTA, ethylene glycol bis(&aminoethyl ether) NJ{\textquoteright}-tetraacetic acid TCA, trichloroacetic acid SDS, sodium dodecyl Chaps, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate.",

year = "1992",

month = dec,

doi = "10.1016/0003-2697(92)90016-Z",

language = "English",

volume = "207",

pages = "304--310",

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AU - Rush, Jeffrey S.

AU - Klein, Jurgen

AU - Fanti, Paolo

AU - Bhat, Narayan R.

AU - Waechter, Charles J.

N1 - Funding 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.

PY - 1992/12

Y1 - 1992/12

N2 - 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.

AB - 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.

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

Abstract

Bibliographical note

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