Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine
- PMID: 2285146
- DOI: 10.1016/0003-2697(90)90133-t
Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine
Abstract
5'-p-Fluorosulfonylbenzoyladenosine (FSBA) is a useful reagent for the affinity labeling of adenine nucleotide binding proteins. We have developed an immunochemical approach to the detection of proteins that have been covalently modified with FSBA, which provides an alternative to the use of a radiolabeled ligand. Antibodies have been prepared against FSBA-modified glutamate dehydrogenase and purified by chromatography on ATP-agarose. The resulting affinity-purified antibodies react on Western blots only with proteins that have been labeled previously with the affinity reagent. The degree of immunoreactivity on Western blots correlates well with the extent of covalent modification as shown by studies on the modification and inhibition of the catalytic subunit of cAMP-dependent protein kinase. In crude cellular extracts, numerous proteins can be labeled with FSBA and then detected by using this approach. The labeling and subsequent detection of these proteins can be blocked by including an excess of MgATP, which competes with FSBA for nucleotide-binding sites. The labeling of specific proteins in crude mixtures is saturable, as shown by labeling studies of p56lck, a protein-tyrosine kinase that is abundantly expressed in membranes from the T lymphoma cell line LSTRA.
Similar articles
-
Affinity labeling of a tyrosine residue in the ATP binding site of the recA protein from Escherichia coli with 5'-p-fluorosulfonylbenzoyladenosine.J Biol Chem. 1985 Aug 25;260(18):10177-84. J Biol Chem. 1985. PMID: 3894365
-
Partial purification and characterization of the lck protein-tyrosine kinase from bovine thymus.Biochem J. 1991 Oct 15;279 ( Pt 2)(Pt 2):567-74. doi: 10.1042/bj2790567. Biochem J. 1991. PMID: 1953650 Free PMC article.
-
FSBA modifies both alpha- and beta-subunits of F1 specifically and can be bound together with AXP at the same alpha-subunit.Biochim Biophys Acta. 1997 Jan 16;1318(1-2):107-22. doi: 10.1016/s0005-2728(96)00110-7. Biochim Biophys Acta. 1997. PMID: 9030259
-
Antibodies to fluorylsulfonylbenzoyladenosine permit identification of protein kinases.FEBS Lett. 1993 Nov 22;334(3):347-50. doi: 10.1016/0014-5793(93)80709-4. FEBS Lett. 1993. PMID: 8243645
-
Identification of the catalytic subunit of an oligomeric casein kinase (G type). Affinity labeling of the nucleotide site using 5'-[p-(fluorosulfonyl)benzoyl]adenosine.Biochemistry. 1983 Mar 15;22(6):1452-9. doi: 10.1021/bi00275a020. Biochemistry. 1983. PMID: 6573204
Cited by
-
MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1.EMBO J. 2003 Oct 1;22(19):5090-101. doi: 10.1093/emboj/cdg447. EMBO J. 2003. PMID: 14517247 Free PMC article.
-
Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L.Plant Physiol. 1993 Aug;102(4):1211-1218. doi: 10.1104/pp.102.4.1211. Plant Physiol. 1993. PMID: 12231896 Free PMC article.
-
Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5'-p-fluorosulphonylbenzoyladenosine ATP analogue.Biochem J. 1995 Dec 1;312 ( Pt 2)(Pt 2):351-6. doi: 10.1042/bj3120351. Biochem J. 1995. PMID: 8526842 Free PMC article.
-
Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.Mol Cell Biol. 1995 Dec;15(12):7043-9. doi: 10.1128/MCB.15.12.7043. Mol Cell Biol. 1995. PMID: 8524271 Free PMC article.
-
Mechanisms of p34cdc2 regulation.Mol Cell Biol. 1993 Mar;13(3):1675-85. doi: 10.1128/mcb.13.3.1675-1685.1993. Mol Cell Biol. 1993. PMID: 8441405 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources