Effect of Microtubule-associatedProteinson the Interactionof

Bovine brain tubulin was purified by the polymerization method of Shelanski et a!. (19) with some modification as described previously (14). The homogenization buffer was 20 mM 2-(N-morpholino)ethanesulfonate, 70 m@i NaCI, 1 mM EGTA, and 0.5 mM MgCI2, pH 6.4. GTP (0.5 mM) and glycerol (4 M) were added for polymerization. The protein was purified through 2 polymerization cycles and was stored at —80° in the buffer containing 2 M glycerol. Tubulin lacking associated proteins (65 tubulin) was prepared by polymerizing the twice cycledproteinin0.4 MPIPES:10% DMSO, pH 6.9, and passing the resuspended pellet through phosphocellulose (8). MAP's were prepared from purified tubulin by passing the protein through a phosphocellulose column at room tempera ture. Twice-cycled tubulin (30 to 50 mg) was first dialyzed against 20 mM PIPES, pH 6.9, for 2 hr in the cold to remove glycerol. The dialyzed protein was loaded onto a 4x 1.2-cm phosphocellulose column at room temperature, allowed to stand 30 mm, and then eluted with 20 m@iPIPES, pH 6.9, followed by 20 mM PIPES:0.8 M NaCI, pH 6.9. MAP's are eluted with the high-salt buffer. The fractions containing MAP's were pooled and concentrated about 2-fold by centrifugation at low speed through an Amicon membrane cone to a final concentration of 3 to 4 mg/mI. The concentrated protein was then dialyzed against 20 mt@i PIPES, pH 6.9, in the cold for 1.5 to 2 hr with fresh changes of buffer every 15 to 20 mm. The dialyzed protein could be kept for 2 or 3 days at 4°but gave maximal activity in the polymerization reaction if used fresh. Assembly reaction buffer contained 0.1 M PIPES (pH 6.9), 0.5 mM MgCI2, 1.0 mM EGTA, and 0.5 mt,i GTP. Tubulin and MAP concentrations are given in the figure and chart legends. The polymerization reaction was started by addition of GTP and was measured by following the increase in absorbance at 350 nm using a Gilford 2000 spectrophotometer. Samples for electron microscopy were negatively stained with 2% uranyl acetate. Ten-id samples were applied to Formvarand carbon coated grids and after 10 to 15 sec were washed with the uranyl acetate solution. One mm later, the grids were dried with filter paper. VCR sulfate (Oncovin) was obtained from Lilly, Indianapolis, Ind.; PIPES, 2-(N-morpholino)ethanesulfonate, GTP, and EGTA were purchased from Sigma Chemical Co., St. Louis, Mo.