AFFINITY LABELING OF C-H-RAS P21 CONSENSUS ELEMENTS WITH PERIODATE-OXIDIZED GDP AND GTP

The amino acid sequence motifs of human c-H-ras p21 involved in the in teraction with guanosine nucleotides were cross-linked to in situ peri odate-oxidized [alpha-P-32]GDP or [alpha-P-32]GTP. Site-specific react ion was achieved by cross-linking conserved lysine residues close to t he G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivati ves of GDP or GTP under kinetically controlled conditions. After endop roteinase Asp-N digestion, HPLC separation of P-32-labeled peptides an d N-terminal microsequence analysis, two single lysine residues, namel y, K117 and K147, which are parts of the N-K-X-D and S-A-K/L consensus elements of ras proteins, respectively, were identified. No significa nt divergences in the position and extent of covalent modification cou ld be detected between p21 . GDP and p21 . GTP. This is in contrast to Thermus thermophilus EF-Tu . GDP and EF-Tu . GTP, which were investig ated with the same technique [Peter, M. E., Wittmann-Liebold, B. & Spr inzl, M. (1988) Biochemistry 27, 9132-9139] and which exhibited consid erable differences in cross-linking efficiency in the GTP form as comp ared to the GDP form of the protein. The described affinity labeling t echnique of cross-linking [alpha-P-32]GTP with GTP-binding proteins ca n be used as a general analytical method for the detection and identif ication of consensus elements in GTPases from different organisms.