Regulation of ncd by the oligomeric state of tubulin

We have compared the interaction of ncd (non-claret disjunctional), a kines in related protein, with microtubules and tubulin heterodimer. Ultracentrif ugation experiments revealed that the ncd motor domain, residues 335-700 (n cd335), does not induce tubulin polymerization but stabilizes pre-formed mi crotubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 bind ing to tubulin or microtubules was estimated by following the change in flu orescence polarization of an exogenous dye attached to Cys670 of ncd335. Nc d335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06-0.08 sec(-1) and 1.8-2.0 sec(-1) for the ncd335- tubulin and ncd335-microtubules complexes, respectively. Only the polymeriz ed complex is fully functional, suggesting the presence of additional conta cts between adjacent protofilaments. Moreover, the data reveal that the oli gomeric state of microtubules is a potent regulator for the activity of kin esin related proteins. (C) 2000 Academic Press.