Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.     

Inhibition of adenylate kinase by P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate and P1-(lin-benzo-5'-adenosyl)-P5-(5'-adenosyl pentaphosphate.

P1-(lin-Benzo-5'-adenosyl)-P5-(5'-adenosyl) penraphosphate and P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate have been synthesized from lin-benzoadenosine 5'-monophosphoromorpholidate and adenosine 5'-tetraphosphate and adenosine 5'-triphosphate. These mixed dinucleoside polyphosphates are potent inhibitors of porcine muscle adenylate kinase, with association constants of 2 x 10(5) M-1 for the pentaphosphate and 2 x 10(6) M-1 for the tetraphosphate, respectively, as determined by kinetics and fluorescence experiments. The increase in fluorescence intensities and fluorescence lifetimes of both inhibitors upon binding to adenylate kinase results from a breaking of the intramolecular stacking interaction observed when these ligands are free in solution and implicates their binding to the enzyme in an "open" or "extended" form. These results and the dimensional requirements of these inhibitors are discussed in relation to our current knowledge of the active site of adenylate kinase and to the known inhibitors of adenylate kinase, P1,P5-bis(5'-adenosyl) pentaphosphate and P1,P4-bis-(5'-adenosyl) tetraphosphate.