Linked Life Data

8387733

Source:http://linkedlifedata.com/resource/pubmed/id/8387733

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1993-6-10
pubmed:abstractText
A novel approach for identification of adenine and guanine nucleotide-binding proteins in permeabilized cells is described. Cells were incubated for various periods with alpha-32P-labeled nucleotides and then briefly treated with periodate. Condensation products formed in situ between the protein bound alpha-32P-labeled oxidized nucleotide (NTPoxi) and a lysine residue near the nucleotide-binding sites were rapidly stabilized by the addition of cyanoborohydride. Analysis by two-dimensional isoelectric focusing/sodium dodecylsulfate-poly-acrylamide gel electrophoresis showed that in the human leukemic T-cell line Jurkat a number of distinct intracellular proteins could be labeled with ATPoxi (M(r) 40,000-200,000) or GTPoxi (M(r) 19,000-80,000). Competition with deoxyribonucleotides confirmed the selectivity of these affinity labeling reactions. To test this method two classical GTP-binding proteins were further examined. First the alpha-subunits of the Gs and Gi-2 proteins were specifically labeled with [alpha-32P]GTPoxi but not with [alpha-32P]ATPoxi. Second, p21ras was crosslinked specifically to [alpha-32P]GTPoxi or to its bound endogenous ligand. Surprisingly, under optimized conditions 60% of the ras protein was specifically modified, demonstrating the high efficiency and sensitivity of the method. As a first step toward isolation of hitherto unidentified nucleotide-binding proteins, rabbit antisera specific for the modified amino acid residues were raised. The presented labeling method can be applied for identification of nucleotide-binding proteins in all eukaryotic cells.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:volume
210
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
77-85
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1993
pubmed:articleTitle
Labeling of adenine and guanine nucleotide-binding proteins in permeabilized cells with in situ periodate-oxidized nucleotides.
pubmed:affiliation
Division of Immunology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't