Lysozyme-induced polymerization of tubulin: Burial of the colchicine-binding site as a probe

Abstract

Ample evidence has accumulated indicating that the information for forming microtubule lattice lies in the tubulin molecule. There are varieties of agents such as Mg2+ [l-3], dimethyl sulfoxide [4], polyethylene glycol, DEAE-dextran [S] and basic proteins [6-91 which could induce microtubule assembly in a purified preparation of tubulin. However, it appears that in those assembly conditions where polycations are employed, tubulin is marginally soluble and microtubule assembly occurs just before precipitation [5,10]. Polycation-induced assembly can not be reversed in all cases even by prolonged cooling [6,9], whereas, normal assembly (without adding exogenous factor) is quite susceptible to cold, Ca2+and some antimitotic drugs. Although turbidity is a reliable measure of the assembly of tubulin into larger forms, only a reversible turbidity (upon cooling the sample) could indicate the microtubule assembly [ 111. Therefore, to follow the microtubule assembly even in the presence of non-specific aggregation (which is unavoidable in the presence of lysozyme), we have explored the use of colchicine-binding as a probe. We now report experiments demonstrating the burial of the colchicinebinding site of tubulin as assembly occurs and also the sensitivity of the lysozyme-induced tubulin polymers toward Ca2+ and cold, as observed using colchitine-binding as a probe.