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 GT Pase activity was induced by al colchicine analogues that contain ring s A and C (or C'), the strength varying in a manner parallel to the fr ee energy of binding of the ligand. The specific GTPase activity was f ound to be independent of the tubulin-colchicine complex concentration over the range of 2-20 mu M. Sedimentation velocity examination of th e product of the reaction showed that GDP-tubulin-colchicine generated by hydrolysis of the E-site GTP was indistinguishable from that produ ced by nucleotide exchange at the site, the protein assuming the ''cur ved'' conformation in both cases. Steady-state kinetic analysis in the presence of high concentrations of microtubule-inducing cosolvent add itives revealed an increase in k(cat)/K-m of up to 1 order of magnitud e that followed the order poly(ethylene glycol) 6000 (PEG-6000 > PEG-1 000 = 2-methyl-2,4-pentanediol > sucrose > L-glutamate > glycerol = PE G-200 > betaine, with no apparent change in K-m. This enhancement of t he GTPase activity was shown to be due neither to cosolvent-induced pr otein self-association nor to an effect of the additives on the soluti on viscosity that would affect substrate diffusion. Initiation of the GTPase reaction (in the presence of PEG-6000) by the addition of the s lowly binding colchicine analogue allocolchicine or the rapidly bindin g 2,3,4-trimethoxy-4'-(carbomethoxy)-1,1'-biphenyl (TCB) resulted in s imilar lags in inorganic phosphate release. Since the rate of binding of allocolchicine and TCB to tubulin was found not to be affected by t he cosolvent, this lag has been attributed to a slow step in the activ ation of the GTPase activity and the presence of an intermediate speci es in the GTPase pathway, not detected by spectroscopic techniques, wh ose concentration is modulated by the presence of cosolvent.