title: The effects of various GTP analogues on microtubule assembly.
abstract: We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.
authors.affiliation: Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Tokyo.
authors.forename: M
authors.initials: M
authors.lastname: Muraoka
authors.forename: H
authors.initials: H
authors.lastname: Fukuzawa
authors.forename: A
authors.initials: A
authors.lastname: Nishida
authors.forename: K
authors.initials: K
authors.lastname: Okano
authors.forename: T
authors.initials: T
authors.lastname: Tsuchihara
authors.forename: A
authors.initials: A
authors.lastname: Shimoda
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Sato
authors.forename: M
authors.initials: M
authors.lastname: Osumi
authors.forename: H
authors.initials: H
authors.lastname: Sakai
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D001921:Brain; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D019281:Dimerization; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006860:Hydrogen Bonding; D007293:Inosine Triphosphate / Q000494:pharmacology; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011687:Purines / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D012266:Ribose / Q000378:metabolism; D013552:Swine; D014404:Tubulin / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000227:Adenine Nucleotides; D003848:Deoxyguanine Nucleotides; D008869:Microtubule-Associated Proteins; D011108:Polymers; D011687:Purines; D012265:Ribonucleotides; D014404:Tubulin; D007293:Inosine Triphosphate; D006153:Guanosine Diphosphate; C065326:xanthosine 5'-triphosphate; D012266:Ribose; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; C030985:purine