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., & Pantaloni, D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5372-5376; Andreu, J. M., & 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 Km for GTP of 10 microM. 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 all 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 microM. 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 kcat/Km 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 Km.(ABSTRACT TRUNCATED AT 250 WORDS)
Perez-Ramirez
B
B
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Shearwin
K E
KE
Timasheff
S N
SN
D000818:Animals; D001713:Biphenyl Compounds / Q000378:metabolism / Q000494:pharmacology; D001923:Brain Chemistry; D002417:Cattle; D003078:Colchicine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008870:Microtubules / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D012997:Solvents; D014404:Tubulin / Q000378:metabolism*
D001713:Biphenyl Compounds; D012997:Solvents; D014404:Tubulin; C087411:2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl; D006153:Guanosine Diphosphate; D011092:Polyethylene Glycols; C032782:allocolchicine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D008274:Magnesium; D003078:Colchicine