Labeling of Adenine and Guanine Nucleotide-Binding Proteins in Permeabilized Cells with in Situ Periodate-Oxidized Nucleotides

✍ Scribed by M.E. Peter; J. She; L.A. Huber; C. Terhorst

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 1019 KB
• Volume: 210
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1153

✦ Synopsis

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-{ }^{32} \mathrm{P})-labeled nucleotides and then briefly treated with periodate. Condensation products formed in situ between the protein bound (\alpha-{ }^{32}) P-labeled oxidized nucleotide (NTP ({ }{o x i}) ) 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 ATP ({\text {oxi }}\left(M_{\mathrm{r}}\right.) 40,000-200,000) or GTP ({ }{\text {oxi }}\left(M{\mathrm{r}} 19,000-80,000\right)). 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 (G_{n}) and (\mathrm{G}{1-2}) proteins were specifically labeled with (\left[\alpha{-3}{ }^{32} \mathrm{P}\right]) GTP ({\text {oxi }}) but not with (\left[\alpha^{-32}\right.) P (]) ATP (P{o x i}). Second, (\mathbf{p} 21^{\text {ras }}) was

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. c 1993 Academic Press, Inc.