title: Deoxyguanosine nucleotide analogues: potent stimulators of microtubule nucleation with reduced affinity for the exchangeable nucleotide site of tubulin.
abstract: Four analogues of guanosine 5'-triphosphate (GTP) (dGTP, 3'-deoxy-GTP, arabinosyl-GTP, and 2',3'-dideoxy-GTP), which support more rapid and extensive microtubule assembly than GTP, were hydrolyzed more rapidly than GTP in reaction mixtures containing tubulin plus microtubule-associated proteins (MAPs). As with GTP, hydrolysis of the four analogues was initially closely coupled to the onset of polymerization and continued at a slower rate at the turbidity plateau. Relative to GTP, however, these analogues (and the cognate GDP analogues), particularly 3'-deoxy-GTP and 2',3'-dideoxy-GTP, bound poorly to tubulin and had a reduced ability to displace bound radiolabeled GDP under nonpolymerizing reaction conditions. Despite their reduced binding to the tubulin dimer, if polymerization occurred, all four analogues were incorporated into microtubules (as the diphosphates) in stoichiometric amounts comparable to the incorporation of GTP (in the form of GDP) with displacement of the GDP initially present in the exchangeable site. Microtubule nucleation was specifically enhanced in the presence of the analogues. With MAPs the analogues initiated microtubule assembly at temperatures 10-15 degrees C below that required by the GTP-supported reaction, and the average microtubule length was significantly reduced. In addition, MAP-independent polymerization occurred only with 2',3'-dideoxy-GTP with tubulin at 1.0 mg/mL, with the other three analogues at 2.0 mg/mL, and with GTP at 5.0 mg/mL. GTP inhibited analogue-supported polymerization at 20 degrees C with MAPs and at 37 degrees C without MAPs (tubulin, 3.5 mg/mL). Both 3'-deoxy-GTP and 2',3'-dideoxy-GTP were poor inhibitors of GTP binding and hydrolysis, but GTP potently inhibited the more vigorous hydrolysis of these analogues. We conclude that alteration of the ribose moiety reduces the affinity of a guanine nucleotide for the exchangeable site of tubulin but that a nucleotide's affinity for this site is not the major factor in its ability to support the nucleation of tubulin polymerization.
mesh_terms: D000818:Animals; D002417:Cattle; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
chemical_list: D008869:Microtubule-Associated Proteins; D011108:Polymers; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate