THE COLCHICINE-INDUCED GTPASE ACTIVITY OF TUBULIN - STATE OF THE PRODUCT - ACTIVATION BY MICROTUBULE-PROMOTING COSOLVENTS

Colchicine induces a weak assembly-independent GTPase activity in calf brain tubulin [David-Pfeuty, T., Erickson, H. P., and Pantaloni, D. (1977) Proc. Nail. Acad. Sci. U.S.A. 74, 5372-5376; Andreu, J. M., and Timasheff, S. N. (1981) Arch. Biochem. Biophys. 211, 151-157]. Kinetic analysis shows a turnover number of 2 X 10(-4) s(-1) in 0.01 M sodium phosphate and 4 mM MgCl2, pH 7.0, with an apparent K-m for GTP of 10 mu M This activity, which requires Mg2+ ions and attains a plateau at 4 mM MgCl2, is independent of pH over the pH range of 6.6-7.4. This GTPase activity was induced by al colchicine analogues that contain rings A and C (or C'), the strength varying in a manner parallel to the free energy of binding of the ligand. The specific GTPase activity was found to be independent of the tubulin-colchicine complex concentration over the range of 2-20 mu M. Sedimentation velocity examination of the product of the reaction showed that GDP-tubulin-colchicine generated by hydrolysis of the E-site GTP was indistinguishable from that produced by nucleotide exchange at the site, the protein assuming the ''curved'' conformation in both cases. Steady-state kinetic analysis in the presence of high concentrations of microtubule-inducing cosolvent additives revealed an increase in k(cat)/K-m of up to 1 order of magnitude that followed the order poly(ethylene glycol) 6000 (PEG-6000 > PEG-1000 = 2-methyl-2,4-pentanediol > sucrose > L-glutamate > glycerol = PEG-200 > betaine, with no apparent change in K-m. This enhancement of the GTPase activity was shown to be due neither to cosolvent-induced protein self-association nor to an effect of the additives on the solution viscosity that would affect substrate diffusion. Initiation of the GTPase reaction (in the presence of PEG-6000) by the addition of the slowly binding colchicine analogue allocolchicine or the rapidly binding 2,3,4-trimethoxy-4'-(carbomethoxy)-1,1'-biphenyl (TCB) resulted in similar lags in inorganic phosphate release. Since the rate of binding of allocolchicine and TCB to tubulin was found not to be affected by the cosolvent, this lag has been attributed to a slow step in the activation of the GTPase activity and the presence of an intermediate species in the GTPase pathway, not detected by spectroscopic techniques, whose concentration is modulated by the presence of cosolvent.