Affinity labeling of GTP‐binding proteins in cellular extracts

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Abstract
GTP‐binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate‐oxidized [α‐32P]GTP as affinity label. Site‐specific reaction of individual GTP‐binding proteins was achieved by cross‐linking the protein‐bound 2′,3′‐dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP‐binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross‐linking of [α‐32P]GTP with GTP‐binding proteins was demonstrated in model systems using different purified GTPases, human c‐H‐ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF‐Tu) from T. themophilus and initiation factor 2 (1F2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras‐proteins, elongation and initiation factors, and heterotrimeric signal transducing G‐proteins.