Isolated rat brain tubulin can be repolymerized in vitro in solutions containing adenosine triphosphate or guanosine triphosphate, magnesium ions, and a good calcium chelator. The extreme sensitivity of tubulin to calcium ions explains the failure of previous efforts to obtain polymerization and suggests a possible mechanism for regulation of microtubule polymerization in vivo.

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The interactions of nucleotides and their role in the polymerization of tubulin have been studied in detail. GTP promotes polymerization by binding to the exchangeable site (E site) of tubulin. The microtubules formed contain only GDP at the E site, indicating that hydrolysis of E site GTP occurs during or shortly after polymerization. Tubulin prepared by several cycles of polymerization and depolymerization will polymerize in the presence of ATP as well as GTP. Polymerization in ATP is preceded by a distinct lag period which is shorter at higher concentrations of ATP. As reported by others ATP will transphosphorylate bound GDP to GTP. Under polymerizing conditions the maximum level of GTP formation occurs at about the same time as the onset of polymerization, and the lag probably reflects the time necessary to transphosphorylate a critical concentration of tubulin. The transphosphorylated protein can be isolated and will polymerize without further addition of nucleotide. The transphosphorylated GTP is hydrolyzed and the phosphate released during polymerization. About 25% of the phosphate transferred from ATP is noncovalently bound to the subunit as inorganic phosphate and this fraction is also released during polymerization. The nonhydrolyzable analogue of GTP, GMPPNP, will promote microtubule assembly at high concentration. GMPPNP assembled microtubules do not depolymerize in Ca concentrations several fold greater than that which will completely depolymerize GTP assembled tubules; however, addition of Ca prior to inducing polymerization in GMPPNP prevents the formation of microtubules. Thus GTP hydrolysis appears to promote depolymerization rather than polymerization. GDP does not promote microtubule assembly but can inhibit GTP binding and GTP induced polymerization. GDP does not, however, induce the depolymerization of formed microtubules. These experiments demonstrate that tubulin polymerization can not be treated as a thermodynamically reversible process, but must involve one or more irreversible steps. Exchange experiments with [3H]GTP indicate that the "E" site on both microtubules and ring aggregates of tubulin is blocked and does not exchange rapidly. However, during polymerization and depolymerization induced by raising or lowering the temperature, respectively, all the E sites become transiently available and will exchange their nucleotide. This observation does not suggest a direct morphological transition between rings and microtubules. The presence of a blocked E site on the rings explains the apparent transphosphorylation and hydrolysis of "N" site nucleotide reported by others.

The self-association of calf brain microtubule subunit protein (tubulin) has been studied. Divalent cations (Mg or Ca) induce the reversible aggregation of tubulin.Depending upon the divalent ion concentration, aggregation may result in an increase in sedimentation rate, formation of a 30S peak, or formation of a fibrous precipitate. Divalent cation induced precipitation is specific for the active, colchicine binding protein and is a useful step in the purification of tubulin. Colchicine alone has no apparent effect on the

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1 mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28~