A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs).

Abstract

The effect of the antimitotic drug taxol on the association of MAP (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of taxol at 37.degree. C, at 0.degree. and at 37.degree. C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter 2 conditions. At 37.degree. and 0.degree. C, complete assembly of both tubulin and the MAP was observed in the presence of taxol. At elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAP. The MAP could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAP of high purity from small amounts of biological material. The MAP could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAP were prepared from bovine cerebral cortex (gray matter) and from HeLa [human cervical carcinoma] cells. MAP1, MAP2, and the tau MAP, as well as species of Mr = 28,000 and 30,000 (LMW, or low MW, MAP) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 MW HeLa MAP were isolated from HeLa cells. Microtubules were also prepared for the 1st time from white matter. All of the MAP identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the 2 preparations was a 5-fold lower level of MAP 2 relative to tubulin in white matter than in gray. The high MW MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. MAP1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.