Nucleosidediphosphate Kinase Associates with Rings but not with Assembled Microtubules

Abstract

Microtubules reassembled in vitro from chick brain contain significant nucleoside diphosphate (NDP) kinase activity (EC 2.7.4.6) although the specific activity decreases with successive cycles of reassembly. While the recovery of microtubule protein, as a function of initial protein concentration, exhibits a critical concentration below which there is no polymerization, the recovery of NDP kinase activity is, at low initial protein concentrations, directly proportional to the initial protein content indicating that microtubule protein and the kinase activity are independently recovered. This was confirmed by pelleting the reassembled microtubules through a sucrose cushion: the specific activity of the pelleted microtubules was reduced by .apprx. 90%. By contrast, when cold-dissociated microtubule protein, which is predominantly in the form of rings, is fractionated on a Biogel A 15 m column the microtubule protein and NDP kinase activity coeluted in the void volume and the specific activity remained constant throughout the ring fraction. Similarly, when microtubules were dissociated in the presence of NDP kinase the enzyme bound to the generated rings. Apparently, NDP kinase binds preferentially to the rings compared with the microtubules; a model is proposed to account for the presence of this enzyme in pellets of microtubule protein.