guanosine-triphosphate and 8-azidoadenosine-5--triphosphate

The 8-azidopurine analogs of adenosine and guanine nucleotides have proved to be very useful probes for nucleotide-binding sites. In most systems they have proved to be effective mimics of the natural compounds with 1) both 8-azidoadenosine-3',5'-monophosphate and 8-azidoguanosine-3',5'-monophosphate activating their respective kinases, 2) 8-azidoguanosine-5'-triphosphate effecting tubulin polymerization and activation of adenylate cyclase, and 3) 8-azidoadenosine-5'-triphosphate appearing to be a substrate for a large number of ATPases and several kinases. As photoprobes they have been used to 1) isolate and study active site peptides; 2) determine the membrane sidedness and cellular location of binding sites; 3) detect the availability of various nucleotide-binding sites as cells progress through development, maturation, infectious stages, etc.; 4) study membrane-soluble partitioning of binding sites relative to nucleotide regulation of a biochemical process; 5) detect nucleotide-binding sites exposed by small molecules such as Ca2+ and calmodulin; and 6) detect potential catalytic and regulatory subunits of protein kinases found in preparations that actively phosphorylate endogenous substrates. The difference between the gamma-32P-labeled 8-azidopurine nucleotide triphosphate and the alpha-32P-labeled species has been used to study the in situ hydrolysis of the nucleotides on specific protein receptors and determine the fate of the produced nucleotide diphosphate. Such factors are important in studying the molecular dynamics of such systems as tubulin polymerization, G-actin to F-actin conversions, and GTP activation of adenylate cyclase. A review of techniques used and data obtained with these probes is presented.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Affinity Labels; Animals; Azides; Calcium; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Nucleotides; Phosphorylation; Photochemistry; Receptors, Cyclic AMP

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Azides; Baculoviridae; Binding Sites; Calcium-Binding Proteins; Cation Transport Proteins; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; Hydrolysis; Magnesium; Photoaffinity Labels; Protein Binding; Protein Interaction Mapping; Proteolysis; Sf9 Cells; Sodium Radioisotopes; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Transfection; Ultraviolet Rays

Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties.. We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[gamma] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9-12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A.. SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately.

Topics: Adenosine Triphosphate; Animals; Azides; Binding Sites; Cells, Cultured; Guanosine Triphosphate; Humans; NAD; Nucleotides; Protein Structure, Tertiary; Rats; Vesicular Transport Proteins

The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.

Topics: Adenosine Triphosphate; Affinity Labels; ATP-Binding Cassette Transporters; Azides; Chloramphenicol; Clotrimazole; GTP Phosphohydrolases; Guanosine Triphosphate; Imidazoles; Kinetics; Saccharomyces cerevisiae Proteins; Tritium

In this work, we demonstrate that the phosphatidylinositol 3,4,5-trisphosphate-binding protein JFC1 is an ATP-binding protein with magnesium-dependent ATPase activity. We show that JFC1 specifically binds to the ATP analog 8-azido-[alpha-(32)P]ATP. The affinity of JFC1 for [alpha-(32)P]ATP was 10x greater than its affinity for [alpha-(32)P]ADP; the protein did not appear to bind to [alpha-(32)P]GTP. JFC1 hydrolyzed [alpha-(32)P]ATP in a Mg(2+)-dependent manner. JFC1, which also hydrolyzed dATP, has a relatively high affinity for ATP, with a K(M) value of 58 microM, and a k(cat) value of 2.27 per min. The predicted amino acid sequence of JFC1 denotes a putative nucleotide-binding site similar to those in the GHKL ATPase/kinase superfamily. However, a truncation of JFC1 that contains boxes G2 and G3 but not boxes N and G1 of the Bergerat-binding site showed residual ATPase activity. Secondly, the antitumor ATP-mimetic agent geldanamycin, which inhibits the ATPase activity of Hsp-90, did not affect JFC1 ATPase. Therefore, the characteristics of the ATP-binding site of JFC1 are unique. Phosphatidylinositol 3,4,5-trisphosphate, a high-affinity ligand of JFC1 did not affect its ATPase kinetics parameters, suggesting that the phosphoinositide have a different role in JFC1 function.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Affinity Labels; Amino Acid Sequence; Animals; Azides; Benzoquinones; Binding Sites; Cross-Linking Reagents; Deoxyadenine Nucleotides; Glutathione Transferase; Guanosine Triphosphate; Kinetics; Lactams, Macrocyclic; Magnesium; Membrane Proteins; Molecular Sequence Data; Phosphatidylinositol Phosphates; Quinones; Recombinant Fusion Proteins; Sequence Deletion; Substrate Specificity

Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, succinyl-CoA, and Pi with purine nucleoside triphosphate, succinate, and CoA via a phosphorylated histidine (H246alpha) intermediate. Two potential nucleotide-binding sites were predicted in the beta-subunit, and have been differentiated by photoaffinity labeling with 8-N3-ATP and by site-directed mutagenesis. It was demonstrated that 8-N3-ATP is a suitable analogue for probing the nucleotide-binding site of SCS. Two tryptic peptides from the N-terminal domain of the beta-subunit were labeled with 8-N3-ATP. These corresponded to residues 107-119beta and 121-146beta, two regions lying along one side of an ATP-grasp fold. A mutant protein with changes on the opposite side of the fold (G53betaV/R54betaE) was unable to be phosphorylated using ATP or GTP, but could be phosphorylated by succinyl-CoA and Pi. A mutant protein designed to probe nucleotide specificity (P20betaQ) had a Km(app) for GTP that was more than 5 times lower than that of wild-type SCS, whereas parameters for the other substrates remained unchanged. Mutations of residues in the C-terminal domain of the beta-subunit designed to distrupt one loop of the Rossmann fold (I322betaA, and R324betaN/D326betaA) had the greatest effect on the binding of succinate and CoA. They did not disrupt the phosphorylation of SCS with nucleotides. It was concluded that the nucleotide-binding site is located in the N-terminal domain of the beta-subunit. This implies that there are two active sites approximately 35 A apart, and that the H246alpha loop moves between them during catalysis.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Azides; Binding Sites; Conserved Sequence; Enzyme Activation; Escherichia coli; Guanosine Triphosphate; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Phosphorylation; Photoaffinity Labels; Purine Nucleotides; Substrate Specificity; Succinate-CoA Ligases

Elongation factor 2 (eEF-2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A.M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J.P. (1993) Biochemistry 32, 1976-1980]. Here we studied sites of these interactions by using photoactivable 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF-2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH,poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non-competitive inhibitors of the photoincorporation of 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP, respectively. eEF-2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N-terminal sequencing. An octapeptide, Y264FDPANGK271, was the only peptide photolabeled with 8-azido-[gamma-32P]GTP whereas a N-terminal fragment of about 7 kDa was the only one photolabeled with 8-azido-[gamma-32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF-2.

Topics: Adenosine Triphosphate; Affinity Labels; Amino Acid Sequence; Azides; Binding Sites; Binding, Competitive; Guanosine Triphosphate; Light; Molecular Sequence Data; Peptide Elongation Factor 2; Peptide Elongation Factors; Peptide Fragments

Using gamma-32P-labeled 8-azidopurine nucleotide photoaffinity probes of GTP and ATP, the respective purine ring binding domain peptides of tubulin have been identified. First, the location of the GTP-specific binding site was shown to be on the beta-subunit, whereas the major ATP-specific binding site was on the alpha-subunit. Using a combination of anion-exclusion and immobilized Al3+ column chromatography, the respective photolabeled tryptic peptides of both nucleotide binding sites were isolated, further purified by reverse phase high performance liquid chromatography (HPLC) and sequenced. Chymotryptic peptides were also generated for the GTP binding site. High retention of the photoinserted radiolabel was observed with many of the peptides on reverse phase HPLC at low flow rates. The stability of the photoinserted radiolabel to HPLC varied with different peptides. However, certain peptides were easily distinguished as being within the base binding domains of the GTP and ATP binding sites of tubulin. Two beta-tubulin peptides containing the majority of photoinserted [gamma-32P]8-azidoguanosine 5'-triphosphate corresponded to N-terminal beta-tubulin amino acid residues 3EIVHIQAGQCGNQIGAK19 and 20FWEVISDEHGIDPTGS35. The peptide containing the majority of photoinserted [gamma-32P]8-azidoadenosine 5'-triphosphate corresponded to the C-terminal alpha-tubulin sequence 431DYEEVGVDSVEGEGEEEGEE450.

Topics: Adenosine Triphosphate; Affinity Labels; Amino Acid Sequence; Animals; Azides; Binding Sites; Brain; Cattle; Guanosine Triphosphate; Kinetics; Microtubule Proteins; Molecular Sequence Data; Peptide Fragments; Peptide Mapping; Trypsin; Tubulin

A protein of M(r) 59,000 (p59) was recently cloned and identified as a Heat shock protein Binding Immunophilin (p59/HBI). It participates to the heterooligomeric, non-DNA binding form of steroid receptors, in association with the heat shock protein of M(r) 90,000 (hsp90). It binds the immunosuppressants FK506 and rapamycin and possesses three FKBP-12 (FK506 binding protein of M(r) 12,000)--like domains (I to III), plus a tail containing a putative calmodulin binding site (domain IV). Following expression in E. Coli and purification on Glutathione-Sepharose of either the full-length recombinant p59/HBI, or the recombinant FKBP-like domains, we demonstrate by autoradiography of [alpha 32P]-8-azido ATP and of [alpha 32P]-8-azido GTP photoaffinity labeled complexes, that an ATP (GTP) binding site is located in the domain II. This nucleotide binding property is also found with the highly purified rabbit uterus p59/HBI. The latter, but not the recombinant protein, can be phosphorylated in vitro in the presence of Mn++ and/or of Ca++/Calmodulin in an ATP but not GTP dependent manner, suggesting copurification of a CaM kinase II-like enzyme. Thus it appears that p59/HBI is a multifunctional immunophilin which may be at the crossroad of the endocrine and immunological systems.

Topics: Adenosine Triphosphate; Animals; Autoradiography; Azides; Carrier Proteins; Cloning, Molecular; Cytosol; Electrophoresis, Polyacrylamide Gel; Female; GTP-Binding Proteins; Guanosine Triphosphate; Heat-Shock Proteins; Kinetics; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Rabbits; Recombinant Fusion Proteins; Restriction Mapping; Tacrolimus Binding Proteins; Uterus

An ATPase was isolated from synaptosomal plasma membranes derived from bovine cerebral cortex. The protein has an apparent molecular mass of 50 kDa and a pI of 5.3 to 5.9. It can be labelled by incubation of intact synaptosomes with azido-GTP or azido-ATP. The isolated ATPase can be activated to a similar extent in the presence of millimolar concentrations of Mg2+ or Ca2+. It does not hydrolyze ADP. Maximal activity is obtained between pH 7.5 and 8.5. Typical inhibitors of cytoplasmic ATPases do not affect enzyme activity. The enzyme is specifically inhibited after previous incubation of intact synaptosomes in the presence of the slowly membrane-permeable enzyme inhibitor diazotized sulfanilic acid. Incubation of intact synaptosomes with diazotized sulfanilic acid results in a small increase in the apparent molecular mass of the enzyme. Our results suggest that the active site of the membrane bound enzyme faces the extracellular medium. It thus would represent an ecto-ATPase.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Affinity Labels; Animals; Azides; Brain; Ca(2+) Mg(2+)-ATPase; Cattle; Diazonium Compounds; Guanosine Triphosphate; Hydrogen-Ion Concentration; Substrate Specificity; Sulfanilic Acids; Synaptic Membranes; Synaptosomes

We have previously shown that ATP interacts with an intracellular, stereoselective, regulatory site(s) on the human erythrocyte sugar transport system to modify transport function in a hydrolysis-independent manner. This present study examines the nucleotide binding properties of the human erythrocyte sugar transport system. We demonstrate by transport studies in ghosts, by nucleotide binding studies with purified transport protein by measurements of nucleotide inhibition of 8-azidoadenosine 5'-[gamma-32P]triphosphate (azido-ATP) photoincorporation into purified carrier, and by analysis of nucleotide inhibition of carboxyl-terminal peptide antisera binding to purified glucose carrier than the glucose transport protein binds (with increasing order of affinity) AMP, ADP, ATP, 5'-adenylyl imidodiphosphate (AMP-PNP), and 1,N6-ethenoadenosine 5'-triphosphate (EATP) at a single site. The carrier lacks detectable ATPase activity and GTP binding capacity. While AMP and ADP bind to the carrier protein and act as competitive inhibitors of ATP binding, these nucleotides are unable to mimic the ability of ATP, AMP-PNP, and EATP to modify the catalytic properties of the sugar transport system. Limited tryptic digestion of azido-ATP-photolabeled carrier suggests that the region of the glucose transport protein containing the intracellular cytochalasin B binding and extracellular bis(mannose) binding domains [residues 270-456; Holman, G. D., & Rees, W. D. (1987) Biochim. Biophys. Acta 897, 395-405] may also contain the intracellular ATP binding site.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Azides; Biological Transport, Active; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Glucose; Guanosine Triphosphate; Humans; Models, Biological; Monosaccharide Transport Proteins

We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.

Topics: Adenosine Triphosphate; Affinity Labels; Animals; Azides; Binding Sites; Eukaryotic Initiation Factor-2; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Kinetics; Proteins; Rabbits; Reticulocytes

The rod photoreceptor outer segment maintains a remarkable morphology. Two of the proteins which have been implicated in the maintenance of this structure are the 240 kDa spectrin-like protein, and the 220 kDa glycoprotein often referred to as the rim protein. We have probed rat rod outer segment proteins with light-activated (azido-labeled) radioactive nucleotides and found a nucleotide binding site(s) on the rim protein which has a preference for guanine nucleotides. Binding to this site is stimulated by the divalent cations zinc, manganese and magnesium, but not calcium. This site is under investigation and may play a role in stabilizing protein structure.

Topics: Adenosine Triphosphate; Animals; ATP-Binding Cassette Transporters; Azides; Binding Sites; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Guanosine Triphosphate; Magnesium; Manganese; Membrane Proteins; Molecular Weight; Photoreceptor Cells; Rats; Rod Cell Outer Segment; Zinc

The photoaffinity reagent 8-azido-alpha-[32P]ATP was used to label a protein of 170 kDa in membrane vesicle preparations from a highly multidrug-resistant cell line, KB-V1, but not from the drug-sensitive parental cell line KB-3-1. The 170-kDa labeled protein was immunoprecipitated with a monoclonal antibody (MRK-16) to P glycoprotein. Both ATP and GTP inhibited labeling by 8-azido-alpha-[32P]ATP. Labeling of P170 was not inhibited by 5 mM ADP, 5 mM ribose-5-phosphate, or 100 microM vinblastine. These data directly demonstrate that P glycoprotein has a nucleotide-binding site that could supply energy for drug transport.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Cell Membrane; Drug Resistance; Glycoproteins; Guanosine Triphosphate; Humans; KB Cells; Molecular Weight; Photochemistry; Protein Binding; Ribosemonophosphates

RNA polymerase type II from human term placenta has been isolated and characterized with respect to its template, ammonium sulfate, divalent cation, and buffer preferences. In addition, the apparent Michaelis constants for AMP and UMP incorporation have been determined. The enzyme was also analyzed by native and denaturing polyacrylamide gel electrophoresis, and evidence is presented that a single polypeptide is radiolabeled with azido purine nucleoside triphosphate photoprobes.

Topics: Adenosine Triphosphate; Azides; Electrophoresis, Polyacrylamide Gel; Female; Guanosine Triphosphate; Humans; Kinetics; Macromolecular Substances; Molecular Weight; Placenta; Pregnancy; RNA Polymerase II; Uridine Triphosphate

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.

Topics: Adenosine Triphosphate; Affinity Labels; Azides; Guanosine Triphosphate; Kinetics; Molecular Weight; Nicotiana; Plants; Plants, Toxic; Ribonucleotides; Tobacco Mosaic Virus; Viral Proteins

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Affinity Labels; Animals; Azides; Binding Sites; Cyclic AMP; Guanosine Triphosphate; Hormones; In Vitro Techniques; Nucleotides; Photochemistry; Protein Kinases