Labeling of adenine and guanine nucleotide-binding proteins in permeabilized cells with in Situ periodate-oxidized nucleotides

M. E. Peter*, J. She, L. A. Huber, C. Terhorst

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

A novel approach for identification of adenine and guanine nucleotide-binding proteins in permeabilized cells is described. Cells were incubated for various periods with α-32P-labeled nucleotides and then briefly treated with periodate. Condensation products formed in situ between the protein bound α-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 dodecyl sulfate-polyacrylamide gel electrophoresis showed that in the human leukemic T-cell line Jurkat a number of distinct intracellular proteins could be labeled with ATPoxi (Mr 40,000-200,000) or GTPoxi (Mr 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 α-subunits of the Gs and Gi-2 proteins were specifically labeled with [α-32P]-GTPoxi but not with [α-32P]ATPoxi. Second, p21ras was crosslinked specifically to [α-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.

Original languageEnglish (US)
Pages (from-to)77-85
Number of pages9
JournalAnalytical Biochemistry
Volume210
Issue number1
DOIs
StatePublished - Apr 1993

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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