Macrophage microtubules: an optimized method for the assay of tubulin concentration and state of polymerization in...

We describe a method for the assay of total tubulin content and the tubulin dimer/polymer ratio in cultured macrophages (MPs). The assay is based on the specific binding of [3H]colchicine to tubulin dimer units and on the separation of free and tubulin-complexed [3H]colchicine on diethylaminoethyl (DEAE)-cellulose filters. The native state of microtubules (MTs) was conserved by suspending the cells in a microtubule-stabilizing solution specifically adapted for use with MPs and by disrupting the cells under carefully chosen conditions. Intact MTs were isolated from the cell homogenate by centrifugation at 150,000 X g and were subsequently depolymerized. [3H]Colchicine binding assays were performed on both the supernatant fraction, containing the pool of soluble tubulin, and on the deploymerized MTs. By using this method, we examined the influence of low temperature and of a number of agents known to affect MP function, on the tubulin dimer/polymer ratio in guinea pig peritoneal MPs. The results of the biochemical assay correlated well with the published information, derived by morphologic and functional approaches, on the effect of cold and of these agents on MTs in phagocytic cells.