Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source.
The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.
Caplow
Michael
M
Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA. caplow@med.unc.edu
Fee
Lanette
L
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D019281:Dimerization; D004355:Drug Stability; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D021122:Protein Subunits; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate