Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: Evidence for an inhibitor of vincristine binding
✍ Scribed by Laura C. Bowman; Janet A. Houghton; Peter J. Houghton
- Publisher
- John Wiley and Sons
- Year
- 1990
- Tongue
- English
- Weight
- 754 KB
- Volume
- 144
- Category
- Article
- ISSN
- 0021-9541
No coin nor oath required. For personal study only.
✦ Synopsis
In cytosols from human rhabdomyosarcoma xenografts, the formation of vincristine (VCR)-tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Kiophys. Res. Commun., 735:695-700, 1986). Wc 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 GTPdepleted cytosols from tumor xenografts HxRH18 and HxRhl2, VCR binding activity was stable for at least 2 hours at 37"C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 n M GTP increased complex formation slightly, with ccrnplex formation increasing a5 GTP concentrations were increased to 5 pM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation threefold, while GMP, GMP-I'NP, and ITP increased formation 1.5-fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific fur the guanine nucleotide5 GDP and GTP. Microtubule protein, purified from Rh18 and RhlZ tumors by cycles ot polymerization-depolynierization, bound VCK 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 m w 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 i 5 modulated by GTP. Two potential models for VCR binding are proposed.