Separation of tubulin from microtubule-associated proteins on phosphocellulose. Accompanying alterations in concentrations of buffer components.
Tubulin can be conveniently separated from the microtubule-associated proteins by chromatography on phosphocellulose [Weingarten, M. D., Lockwood, A. H., Hwo, S.-Y., & Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1858]. Concentrations of Mg2+, GTP, GDP, and glycerol were measured in the various fractions collected during several such separations. The phosphocellulose was found to sequester Mg2+ under the conditions employed for the separation and to retard GTP, GDP, and glycerol relative to tubulin. Polymerization of the resulting phosphocellulose-tubulin was found to be inhibited by this Mg2+ depletion. The composition of the buffer in which tubulin emerges from the chromatographic column was found to depend in a sensitive way upon the retardation of the other components of the buffer and to be a sensitive function of the width of the pooled tubulin peak and of the ratio of the volume of the chromatographic column to that of the column load. The bearing of these findings on interpretation of existing literature is briefly discussed. Efficient separation of tubulin from microtubule-associated proteins can also be obtained on phosphocellulose columns that have been saturated with Mg2+. Such saturation of the column, or addition of Mg2+ to the collected fractions, followed by equilibration of the tubulin with known buffer, is suggested as an improvement to the preparative scheme.
D002852:Chromatography, Ion Exchange; D005990:Glycerol; D006023:Glycoproteins / Q000302:isolation & purification*; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium; D008870:Microtubules / Q000032:analysis*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D014404:Tubulin / Q000302:isolation & purification*
D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium; D005990:Glycerol