guanosine-triphosphate and 2--3--dideoxyguanosine-5--triphosphate

Despite reduced affinity for the exchangeable nucleotide binding site of tubulin relative to GTP, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) and guanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pG] are highly active in promoting tubulin assembly. Like the antimitotic drug paclitaxel, which interacts with the same part of the beta-tubulin molecule as exchangeable-site GTP, both analogs enhance nucleation reactions and promote formation hyperstable polymers. These observations led us to synthesize the doubly modified analog 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pddG]. We compared the effects of pp(CH2)pddG to those of ddGTP, pp(CH2)pG, and the three-cognate diphosphates in their interactions with tubulin. We found that pp(CH2)pddG was as active as ddGTP and pp(CH2)pG in supporting formation of polymer of increased stability, but that its affinity for the exchangeable site was lower than that of both singly modified analogs [relative affinities for the exchangeable site for pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP were 1:2.8:10:273]. There were significant differences in interactions of each of the three analogs with tubulin, and the behavior of pp(CH2)pddG was intermediate between that of ddGTP and that of pp(CH2)pG. Most importantly, under the reaction conditions studied, with heat-treated microtubule-associated proteins (MAPs) ddGTP-induced polymer consisted of short microtubules, while polymer formed with both pp(CH2)pddG and pp(CH2)pG consisted of short sheets. On the other hand, assembly without MAPs had a fivefold lower critical concentration for tubulin with ddGTP and pp(CH2)pddG (0.5 mg/ml) than with pp(CH2)pG (2.5 mg/ml). De novo assembly, which occurs readily with 2',3'-dideoxyguanosine 5'-diphosphate, was not observed with either alpha, beta-methylenediphosphate GDP analog.

Topics: Antineoplastic Agents; Binding, Competitive; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; Dideoxynucleotides; Dose-Response Relationship, Drug; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Microtubule-Associated Proteins; Microtubules; Paclitaxel; Polymers; Temperature; Tubulin

The ribonucleoprotein enzyme telomerase is a specialized type of cellular reverse transcriptase which synthesizes one strand of telomeric DNA, using as the template a sequence in the RNA moiety of telomerase. We analyzed the effects of various nucleoside analogs, known to be chain-terminating inhibitors of retroviral reverse transcriptases, on Tetrahymena thermophila telomerase activity in vitro. We also analyzed the effects of such analogs on telomere length and maintenance in vivo, and on vegetative growth and mating of Tetrahymena cells. Arabinofuranyl-guanosine triphosphate (Ara-GTP) and ddGTP both efficiently inhibited telomerase activity in vitro, while azidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate (ddITP) or ddTTP were less efficient inhibitors. All of these nucleoside triphosphate analogs, however, produced analog-specific alterations of the normal banding patterns seen upon gel electrophoresis of the synthesis products of telomerase, suggesting that their chain terminating and/or competitive actions differ at different positions along the RNA template. The analogs AZT, 3'-deoxy-2',3'-didehydrothymidine (d4T) and Ara-G in nucleoside form caused consistent and rapid telomere shortening in vegetatively growing Tetrahymena. In contrast, ddG or ddI had no effect on telomere length or cell growth rates. AZT caused growth rates and viability to decrease in a fraction of cells, while Ara-G had no such effects even after several weeks in culture. Neither AZT, Ara-G, acycloguanosine (Acyclo-G), ddG nor ddI had any detectable effect on cell mating, as assayed by quantitation of the efficiency of formation of progeny from mated cells. However, AZT decreased the efficiency of programmed de novo telomere addition during macronuclear development in mating cells.

Topics: Animals; Arabinonucleotides; Base Sequence; Cell Survival; Deoxyguanine Nucleotides; Dideoxynucleotides; DNA Nucleotidylexotransferase; Guanosine Triphosphate; Molecular Sequence Data; Nucleotides; Telomere; Tetrahymena thermophila; Thymine Nucleotides; Zidovudine

Topics: Animals; Deoxyguanine Nucleotides; Dideoxynucleotides; Guanosine Triphosphate; Kinetics; Microtubules; Tubulin

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg2+, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-beta-D-arabinofuranosylguanine-5'-diphosphate (araGDP). Omission of Mg2+ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernible relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.

Topics: Animals; Cattle; Deoxyguanine Nucleotides; Dideoxynucleotides; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Macromolecular Substances; Magnesium; Microscopy, Electron; Microtubule-Associated Proteins; Nerve Tissue Proteins; Proteins; Ribose; Tubulin; Tubulin Modulators

We have examined the effects of dilution, Ca2+, reduced temperature, and triphosphate depletion on microtubules formed from purified tubulin, heat-treated microtubule-associated proteins (MAPs), and either GTP, 2',3'-dideoxyguanosine 5'-diphosphate (ddGDP), or 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP). The stability of the polymer formed with tubulin plus ddGTP without MAPs was also examined. In all cases dilution resulted in rapid depolymerization of polymer until a new turbidity plateau was established. These experiments yielded estimates of the critical concentration of tubulin of 0.09 mg/ml with GTP plus MAPs, 0.04 mg/ml with either ddGDP or ddGTP plus MAPs, and 0.07 mg/ml with ddGTP minus MAPs. Addition of CaCl2 to polymer resulted in depolymerization of microtubules formed with either GTP or ddGDP plus MAPs; but both with and without MAPs the polymer formed with ddGTP was stable to Ca2+. The polymer formed with ddGTP minus MAPs was the most cold-labile, major depolymerization occurring at 25 degrees C. With MAPs, microtubules were progressively less cold-labile when formed with GTP, ddGDP, or ddGTP. Depolymerization with GTP was virtually complete at 15 degrees C, with ddGDP at 5 degrees C, and with ddGTP at 0 degrees C. Rapid triphosphate depletion was achieved with phosphofructokinase. GTP-formed tubules were rapidly and completely depolymerized at all GTP concentrations after the enzyme was added to the reaction mixture. Both with and without MAPs polymer formed with ddGTP was progressively more stable upon enzyme addition the higher the initial ddGTP concentration. At specific ddGTP concentrations, however, less depolymerization was observed following enzyme addition if MAPs were present. Microtubules formed with ddGDP plus MAPs were unaffected by phosphofructokinase addition. This comparison of the properties of microtubules formed with MAPs and either ddGDP or ddGTP demonstrates that their stability is enhanced rather than reduced following nucleotide hydrolysis. The greater stability of microtubules formed with ddGTP plus MAPs than of the polymer formed with ddGTP minus MAPs similarly implies substantial enhancement of microtubule stability by the MAPs.

Topics: Animals; Brain; Calcium; Cattle; Deoxyguanine Nucleotides; Dideoxynucleotides; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Nerve Tissue Proteins; Proteins; Temperature; Tubulin

Interactions of tubulin with a number of guanine nucleotides modified at the 2' and 3' ribose hydroxyls were examined. Deoxy analogs of GTP were equal or superior to GTP in supporting tubulin polymerization, but analogs bearing either methyl or phosphate groups on the hydroxyls had significantly reduced ability to support polymerization. These substituted GTP analogs were hydrolyzed at the 5'-gamma-phosphate position, although less rapidly than GTP, at rates exceeding those of polymerization. GTP hydrolysis, however, was closely coupled to polymerization. Moreover, the partially active GTP analogs were not effective inhibitors of GTP-dependent polymerization. These data indicate that the substituted GTP analogs have reduced affinity for tubulin at the exchangeable site because of steric factors. No deoxy or substituted GDP analog was as effective as GDP itself in inhibiting GTP-supported tubulin polymerization. Furthermore, there was no apparent relationship between the ability of nucleoside 5'-triphosphates to support polymerization and that of nucleoside 5'-diphosphates to inhibit the reaction. These findings suggest that GTP and GDP may actually bind to different, mutually exclusive sites rather than to a single exchangeable site.

Topics: Animals; Binding Sites; Brain; Cattle; Deoxyguanine Nucleotides; Dideoxynucleotides; Glutamates; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Substrate Specificity; Tubulin; Vinblastine