Tubulin-colchicine complexes differentially poison opposite microtubule ends.
The kinetics of radiolabeled guanosine 5'-triphosphate-tubulin dimer addition to preformed microtubule copolymers, containing large numbers of tubulin-colchicine complexes (TCs), were examined at apparent equilibrium. The results indicated that radiolabeled dimer addition to copolymers occurs predominantly by a "treadmilling" reaction, analogous to that described for unpoisoned microtubules, and that some labeled dimer uptake also occurs by equilibrium exchange. The data further showed that TCs decrease the steady-state treadmilling reaction in a concentration-dependent manner. Since microtubule copolymers exhibited a treadmilling reaction, it was possible to differentially radiolabel opposite copolymer ends with [3H]- and [14C]guanine nucleotides and thus to measure the effects of TCs on dimer loss from opposite copolymer ends upon copolymer dilution. Dimer loss from both copolymer ends was inhibited in a concentration-dependent manner, but dimer loss from copolymer net assembly (A) ends (defined under steady-state conditions) was inhibited to a far greater extent than that from the opposite, net disassembly (D) copolymer ends. TCs therefore exhibited a graded, polar poisoning action, with copolymer A-end association and dissociation rate constants being far more susceptible to TC inhibition than those at the opposite copolymer D ends. The potential significance of this TC effect for regulating microtubule spatial orientation in vivo is discussed.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003078:Colchicine / Q000378:metabolism / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D014316:Tritium; D014404:Tubulin / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D014404:Tubulin; D014316:Tritium; D006160:Guanosine Triphosphate; D003078:Colchicine