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 [calf brain] microtubule-associated proteins (MAP) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAP by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAP-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAP) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtuble 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 MAP were further purified by DEAE-cellulose chromatogrpahy into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAP) contained proteins with the electrophoretic mobility of MAP-1, MAP-2 and .tau. factor. The polymerization reaction induced by the unbound component (flow-through MAP) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAP contained significantly more ATPase, tubulin-independent GTPase and, especially, nucleosidediphosphate kinase activity than the DEAE-MAP, preparation of a MAP fraction without these enzymes required heat treatment.