Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: evidence for an inhibitor of vincristine binding.
In cytosols from human rhabdomyosarcoma xenografts, the formation of vincristine (VCR)-tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Biophys. Res. Commun., 135:695-700, 1986). We have further examined this modulation to determine whether a) GTP was protecting the VCR binding site from denaturation, b) the enhancement of complex formation was guanosine specific, and c) whether this influence was a direct interaction between GTP, VCR, and tubulin, or was mediated through another factor. In GTP-depleted cytosols from tumor xenografts HxRH18 and HxRh12, VCR binding activity was stable for at least 2 hours at 37 degrees C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 nM GTP increased complex formation slightly, with complex formation increasing as GTP concentrations were increased to 5 microM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation three-fold, while GMP, GMP-PNP, and ITP increased formation 1.5-fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific for the guanine nucleotides GDP and GTP. Microtubule protein, purified from Rh18 and Rh12 tumors by cycles of polymerization-depolymerization, bound VCR rapidly and binding was not influenced by GTP. This suggested that GTP modulation of VCR binding in cytosols was through a soluble factor lost in tubulin purification. In experiments with cytosol fractionated by molecular weight, there was inhibition of VCR binding activity by fractions with an mw range 20-50 kD. This inhibition was decreased by 25% by the addition of GTP. These data suggest that in tumor cytosols there may be competition between VCR and a natural ligand that is modulated by GTP. Two potential models for VCR binding are proposed.
Bowman
L C
LC
Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Houghton
J A
JA
Houghton
P J
PJ
D000818:Animals; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000502:physiology; D006801:Humans; D051379:Mice; D011485:Protein Binding / Q000187:drug effects; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D014750:Vincristine / Q000378:metabolism*
D006150:Guanine Nucleotides; D014404:Tubulin; D014750:Vincristine; D006160:Guanosine Triphosphate