title: Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.
abstract: It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6806-6814]. Since chemical modifications of the vinca alkaloid structure are known to modulate the overall affinity of drug binding, it is very likely that, by Wyman linkage, chemical modifications of guanine nucleotide allosteric effectors also modulate drug binding. Here we compare the effects of the GTP and GDP alpha,beta-methylene analogues GMPCPP and GMPCP on vinblastine-induced tubulin association in 10 and 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM MgSO4, and 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), pH 6. 9, at different temperatures. We found that GMPCPP perfectly mimics GTP in its effect on spiral assembly under all ionic strength and temperature conditions. However, GMPCP in 10 mM Pipes behaves not as a GDP analogue, but as a GTP analogue. In 100 mM Pipes, GMPCP has characteristics that are intermediate between GDP and GTP. These data suggest that the alpha,beta methylene group in GMPCP and GMPCPP is sufficient to produce a GTP-like effect on vinblastine-induced tubulin self-assembly. This is consistent with previous observations that GMPCP-tubulin will assemble into microtubules in a 2 M glycerol and 100 mM Pipes buffer [Vulevic & Correia (1997) Biophys. J. 72, 1357-1375]. Our results demonstrate that an alpha,beta methylene modification of the guanine nucleotide phosphate moiety can induce a salt-dependent conformational change in the tubulin heterodimer that favors the GTP-tubulin structure. This has important implications for understanding allosteric interactions that occur in the binding of guanine nucleotides to tubulin.
authors.affiliation: Department of Biochemistry and School of Nursing, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
authors.forename: B
authors.initials: B
authors.lastname: Vulevic
authors.forename: S
authors.initials: S
authors.lastname: Lobert
authors.forename: J J
authors.initials: JJ
authors.lastname: Correia
mesh_terms: D017738:Alkanesulfonic Acids; D000818:Animals; D001921:Brain; D002151:Calorimetry; D004533:Egtazic Acid; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008278:Magnesium Sulfate; D010879:Piperazines; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D014747:Vinblastine / Q000031:analogs & derivatives / Q000494:pharmacology*; D014750:Vincristine / Q000494:pharmacology; D000077235:Vinorelbine
chemical_list: D017738:Alkanesulfonic Acids; D010879:Piperazines; D014404:Tubulin; D006153:Guanosine Diphosphate; C025675:guanosine 5'-(alpha,beta-methylene)triphosphate; C004805:5'-guanylylmethylenebisphosphonate; D004533:Egtazic Acid; D014750:Vincristine; D014747:Vinblastine; D008278:Magnesium Sulfate; D006160:Guanosine Triphosphate; C008916:piperazine-N,N'-bis(2-ethanesulfonic acid); D000077235:Vinorelbine