Affinity labeling of GTP-binding proteins in cellular extracts.
GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-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 [alpha-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. thermophilus and initiation factor 2 (IF2) 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.
Löw
A
A
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Faulhammer
H G
HG
Sprinzl
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013824:Thermus / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010761:Phosphorus Radioisotopes; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins