The mechanism of breakdown of tubulin in vitro

Abstract

It is well established that in vitro tubulin is unstable in that it gradually loses its ability to bind colchicine [ 1,2] . The half time of this change has been estimated to be 11 h at 0°C [3]. During the course of experiments designed to study the assembly of microtubules, it was noted that incubation with dithiothreitol (DTT) decreased the extent of conversion of tubulin to the form that does not bind colchicine. This chance observation led to a systematic study of the effects of both reducing and oxidizing agents on the loss of colchicine binding activity by purified tubulin preparations. This loss of colchicine binding activity will be termed breakdown, although it apparently is unclear whether or not it is actually a result of the dissociation of the tubulin dimer into its two constituent monomer proteins. In contrast to the inhibitory effect of DTT, reduced glutathione (GSH) modestly increased the breakdown of tubulin occurring in 90 min. Oxidized glutathione (GSSG) had no apparent effect alone, while combinations of GSH and GSSG increased the extent of this breakdown more than did GSH alone. These results suggest (1) that the mechanism of breakdown of tubulin in vitro involves both the cleavage and the formation of disulfide bonds, and (2) that the tubulin dimer can exist in vitro in several different biochemical forms.