Biochemical Studies on the in Vitro Assembly and Disassembly of Microtubules*

The mechanism of assembly and disassembly of cytoplasmic microtubules in vivo is not clearly understood, Kinetic and equilibrium studies in vitro are potentially useful in suggesting reasonable models of tubulin assembly, which can then be tested in vivo.Large quantities of pure tubulin are needed for reliable kinetic studies and other solution physicochemical techniques.We have developed a purification scheme for neurotubules, based on assembly and disassembly under the conditions defined by Weisenberg,' modified by the addition of glycerol as a stabilizing agent.'Several minor components present in these tubulin preparations have been characterized.3One has similar physi- cal properties to the ATPase known as dynein, found in the microtubules of the axonemes of flagella and cilia;'.except that the brain dynein has very low ATPase activity.The kinetics and equilibria of tubule assembly have been studied mainly by quantitative turbidimetry.The results of these studies are insufficient to establish a unique model for tubulin assembly, but they do place strong constraints on possible models of assembly in vitro. PURIFICATION AND ANALYSIS OF NEUROTUBULESFIGURE 1 summarizes the procedure we use to purify tubulin from porcine, guinea pig, and calf brains.About one-half of the total soluble tubulin in the brain is recovered after one cycle of assembly.Tubulin stored at -20" C, in running buffer, or RB (0.1 M Mes, 1 mM EGTA, and 0.5 mM MgCL, pH 6.6) that contains 4 M glycerol, is stable and has a half-life of over 6 weeks ::