The co-polymerization of tubulin and tubulin chochicine complex in the absence and presence of associated proteins.
Tubulin x colchicine complex (TC) is a potent inhibitor of microtubule assembly whereas microtubule-associated proteins (MAPs) facilitate assembly. Previous studies done under MAP-depleted conditions (Sternlicht, H., and Ringel, I. (1979) J. Biol. Chem. 254, 10540) suggested that tubulin binds to the ends of the microtubule with an apparent dissociation constant (affinity T-1) which increases as the TC/tubulin ratio in the microtubule increases. Affinity T-1 was identified with the critical, i.e. minimum tubulin concentrations required for assembly. In this study, we examined the TC-tubulin co-polymerization reaction as a function of MAP availability. Critical tubulin concentrations, which increases in the presence of TC and decreases in the presence of MAP, could be approximated as a sum of contributions from MAPs and TC. An expression was derived which successfully predicted the per cent inhibition observed for a variety of TC, tubulin, and MAP concentrations. In all cases, increases in critical tubulin concentrations correlated with increases in the TC/tubulin ratio in the microtubules. Our data suggest that substoichiometric inhibition by TC is not a consequence of impaired MAP function, nor is it a consequence of a marked increase in the apparent free energy of assembly in the presence of TC. Rather, the large per cent inhibition values observed at low TC concentrations (TC less than or equal to 5 microM) appear to be a consequence of the small concentrations of tubulin typically used in assembly studies (less than or equal to 2 to 4 mg/ml active tubulin) and the constraint that assembly in the presence of TC requires a minimum concentration of tubulin equal to affinity T-1. The molecular processes by means of which TC and MAPs affect affinity T-1 remain to be established.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003078:Colchicine / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D011506:Proteins; D014404:Tubulin; D003078:Colchicine