adenosine-5--o-(3-thiotriphosphate) and 8-azidoadenosine-5--triphosphate

Adenosine tri- and diphosphate (ATP and ADP) and their structural analogues stimulate insulin secretion from the isolated perfused rat pancreas, an effect mediated by P2Y-purinoceptor activation. Concerning the base moiety of the nucleotide, it was previously shown that purine but not pyrimidine nucleoside triphosphates were active and that substitution on purine C2 with the 2-methylthio group greatly enhanced the potency. In this study, we further analyze the consequences of ribose and polyphosphate chain modifications. Modifications in 2' and 3' position on the ribose led to a decrease in insulin response when bulky substitutions were made: indeed, 2'-deoxy ATP was similar in activity to ATP, whereas arylazido-aminopropionyl ATP (ANAPP3) was weakly effective and trinitrophenyl ATP (TNP-ATP) was inactive. Substitution on the gamma phosphorus of the triphosphate chain led to a decrease (gamma-anilide ATP) or no change (gamma-azido ATP) in potency; the replacement of the bridging oxygen between beta and gamma phosphorus by a peroxide group did not significantly change the activity, whereas substitution by a methylene group completely abolished stimulation of insulin secretion. As for the phosphorothioate analogues, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) induced an insulin response similar to that produced by ATP, whereas adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) was about 100-fold more potent than ATP, as previously shown. In conclusion, two structural features seem to have a strategic importance for increasing the insulin secretory activity of ATP analogues: substitution at the C2 position on the adenine ring of ATP and modifications of the polyphosphate chain at the level of the beta phosphorus.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Area Under Curve; Azides; Deoxyadenine Nucleotides; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Pancreas; Polyphosphates; Rats; Receptors, Purinergic P2; Ribose; Structure-Activity Relationship; Thionucleotides

The beta- and delta-subunits of the nicotinic acetylcholine receptor from Torpedo californica were covalently photolabeled at the synaptic surface with the ATP photoaffinity analogue [alpha-32P]-8-azido-ATP. The specificity of labeling for nucleotide binding sites was demonstrated by the saturation of labeling with increasing concentration of 8-azido-ATP and the inhibition of photolabeling by ATP. Protection studies suggest that the binding sites for the photolabel are unique and are not associated with the cholinergic ligand binding sites.

Topics: Adenosine Triphosphate; Affinity Labels; Animals; Azides; Binding Sites; Photochemistry; Receptors, Nicotinic; Sodium-Potassium-Exchanging ATPase; Synapses; Torpedo

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.

Topics: Adenosine Triphosphate; Affinity Labels; Azides; Bacterial Proteins; Binding, Competitive; Cross-Linking Reagents; DNA-Binding Proteins; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Ultraviolet Rays

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Azides; Kinesins; Microscopy, Electron; Microtubules; Movement; Nerve Tissue Proteins; Sea Urchins