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 interaction with guanosine nucleotides were cross-linked to in situ periodate-oxidized [alpha-32P]GDP or [alpha-32P]GTP. Site-specific reaction was achieved by cross-linking conserved lysine residues close to the G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivatives of GDP or GTP under kinetically controlled conditions. After endoproteinase Asp-N digestion, HPLC separation of 32P-labeled peptides and N-terminal microsequence analysis, two single lysine residues, namely, 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 significant divergences in the position and extent of covalent modification could be detected between p21.GDP and p21.GTP. This is in contrast to Thermus thermophilus EF-Tu.GDP and EF-Tu.GTP, which were investigated with the same technique [Peter, M. E., Wittmann-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] and which exhibited considerable differences in cross-linking efficiency in the GTP form as compared to the GDP form of the protein. The described affinity labeling technique of cross-linking [alpha-32P]GTP with GTP-binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.
Laboratorium für Biochemie, Universität Bayreuth, Germany.
A
A
Löw
M
M
Sprinzl
H G
HG
Faulhammer
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D006153:Guanosine Diphosphate / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000737:chemistry; D017433:Protein Structure, Secondary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010504:Periodic Acid; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C009288:metaperiodate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)