Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

Abstract

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.

EF24, a novel curcumin analog, disrupts the microtubule cytoskeleton and inhibits HIF-1

“…The assembly of purified bovine brain tubulin, prepared as described previously, 18 was evaluated as described in detail elsewhere. 19 Briefly, 10 μM tubulin was preincubated for 15 min at 30°C in 0.8 M monosodium glutamate (2 M stock solution adjusted to pH 6.6 with HCl) and 4% (v/v) DMSO and compounds as indicated.…”

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

“…Materials-Purified bovine brain tubulin with nonradiolabeled GDP (26) or [8][9][10][11][12][13][14] C]GDP (27) Synthesis of H]Dolastatin 10 -Dolastatin 10 was first converted to 5-bromo-dolastatin 10. The dolastatin 10 (30 mg) was dissolved in 0.2 ml of acetic acid with 40 mg of silver trifluoroacetate.…”

The importance of the phenyl‐tropolone ‘aS’ configuration in colchicine's binding to tubulin

“…All other chemicals were obtained commercially. Tubuiin was purified from bovine brain using the published procedure [10] and stored in liquid nitrogen at a protein concentration of 54 mg/ml of I M sodium glutamate solution at pH 6.6 before use. Samples of tubulin for the CD experiments were prepared by rapidly thawing the stored material, followed by centrifugation, if needed to remove small amounts of denatured protein, and then diluting to a final protein concentration of approx.…”