Differential radiolabeling of opposite microtubule ends: methodology, equilibrium exchange-flux analysis, and drug poisoning.
We describe a method which allows opposite microtubule ends to be distinguished by differentially labeling the microtubules with [3H]- and [14C]guanine nucleotides. Assembly-disassembly reactions at opposite microtubule ends can therefore be measured simultaneously and without modification of the tubulin dimers or microtubules. The method is predicated on experimental observations which demonstrate that net dimer addition to steady-state microtubules must be predominantly unidirectional. This does not preclude, however, some bidirectional dimer addition to steady-state microtubules by an equilibrium-exchange mechanism. We therefore calculated the relative contribution to dimer incorporation of bidirectional equilibrium exchange in a unidirectional microtubule system (s = 0.06). Under our conditions bidirectional dimer incorporation is negligible; net dimer addition to steady-state microtubules is overwhelmingly unidirectional. We used this method to study the effects of colchicine and podophyllotoxin on assembly-disassembly at opposite microtubule ends. Both drugs inhibit substoichiometrically net dimer addition to one microtubule end and, to a lesser extent, net dimer loss from the opposite end.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003078:Colchicine / Q000494:pharmacology*; D007553:Isotope Labeling; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008956:Models, Chemical; D011034:Podophyllotoxin / Q000494:pharmacology*; D014404:Tubulin / Q000302:isolation & purification*
D014404:Tubulin; D011034:Podophyllotoxin; D003078:Colchicine