guanosine-diphosphate and 8-azidoguanosine-triphosphate

Platelets have been shown to possess several, different, low-molecular-mass, guanine-nucleotide-binding proteins (G-proteins) with molecular masses about 20-30 kDa. We report here that a 25-kDa G-protein copurified with the bovine platelet actin-binding protein (ABP), a cross-linker of actin filaments which is known to generate the three-dimensional network of actin. Both the G-protein and ABP were recovered in a fraction that was insoluble in Triton X-100 and were extracted in 0.6 M NaCl. Gel-filtration chromatography of the high-salt extract and rechromatography in a low-salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G-protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G-protein and ABP was unaffected by guanosine-5'-O-[beta-thio]diphosphate and guanosine-5'-O-[gamma-thio]triphosphate, but the G-protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP-dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G-proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G-protein, rather than that the G-protein modulates the function of the membrane skeleton which comprises ABP.

Topics: Affinity Labels; Animals; Azides; Blood Platelets; Blotting, Western; Cattle; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Microfilament Proteins; Molecular Weight; Phosphorylation; Photochemistry; Precipitin Tests; Protein Kinases; Thionucleotides

Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoaffinity labeling with a GTP analog [gamma-32P]-8-AzidoGTP and a guanosine-3'-diphosphate-5'-diphosphate analog, 8-Azidoguanosine-3'-phosphate-5'-85'-32P]phosphate. The guanosine diphosphate photoprobe labeled the beta, beta', and sigma subunits with the sigma subunit being most heavily labeled. The GTP photoprobe also labeled the beta, beta', sigma subunits but the beta' subunit was most heavily labeled. In competition experiments guanosine-3'-diphosphate-5'-diphosphate decreased photolabeling by 8-Azidoguanosine-3'-phosphate-5'-[5'-32P]phosphate better than GTP, while the opposite was true for photolabeling with [gamma-32P]8- AzidoGTP. The guanosine diphosphate photoprobe inhibited transcription on E. coli DNA with Ki of ca. 150 microM. Present studies suggest a unique ppGpp binding site distinct from substrate binding site(s) and this photoprobe may be used to localize this binding site(s).

Topics: Affinity Labels; Azides; Binding Sites; Binding, Competitive; DNA-Directed RNA Polymerases; Escherichia coli; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Tetraphosphate; Guanosine Triphosphate; Photochemistry