Quantification and properties of tubulin polymerization in crude brain extracts and preparations of microtubular and purified tubulin

Abstract

Removal of assembled tubulin by centrifugation, followed by measurement in the supernatant of the residual colchicine binding capacity of the non-polymerized, non-precipitable tubulin, is a sensitive and reliable method of measuring tubulin polymerization. This method can be used in both crude and purified preparations of brain tubulin and allows the molar quantification of the total, polymerized and non-polymerized tubulin species in each sample. Only 40--50% of the total tubulin present in crude adult brain extracts is capable of polymerizing when incubated with GTP. The percentage of tubulin polymerizing with GTP is slightly higher in crude foetal brain extracts than in the adult. Incubation of first polymerization supernatants, containing exclusively the GTP-insensitive tubulin, with guanosine 5'-[alpha, beta-methylene]triphosphate (GuoP[CH2]PP) but not with 2.4 M glycerol results in tubulin polymerization. High concentrations of glycerol (2.4 M) promote the polymerization of tubulin in adult but not in crude foetal brain extracts. Both ATP and adenosine 5'-[alpha, beta-methylene]triphosphate (AdoP[CH2]PP) are effective in promoting the polymerization of GDP-free (90%) microtubular protein. The microtubular protein assembled with GuoP[CH2]PP or AdoP[CH2]PP has the unique character of being resistant to calcium concentrations (2mM), which cause complete depolymerization of the tubulin assembled with GTP or ATP. Phosphocellulose-purified tubulin significantly assembles when incubated with GuoP[CH2]PP or AdoP[CH2]PP at protein concentrations at which GTP or ATP do not promote polymerization. 2 micrometer tubulin is the minimal concentration required for polymerization in both crude and purified preparations of adult rat brain tubulin.