Labeling of adenine and guanine nucleotide-binding proteins in permeabilized cells with in situ periodate-oxidized nucleotides.
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.
Peter
M E
ME
Division of Immunology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215.
She
J
J
Huber
L A
LA
Terhorst
C
C
D000227:Adenine Nucleotides / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D010084:Oxidation-Reduction; D010504:Periodic Acid; D010539:Permeability
D000227:Adenine Nucleotides; D000345:Affinity Labels; D002352:Carrier Proteins; D006150:Guanine Nucleotides; D010504:Periodic Acid; D019204:GTP-Binding Proteins