Characterization of a 20S component in tubulin from mammalian brain

Abstract

Tubulin from porcine brain, purified by at least 2 cycles of assembly and disassembly, was characterized at different pH values by sedimentation velocity analysis and turbidimetric measurements. At pH 6.4 the depolymerized material was composed of 2 major species sedimenting with sO20,w values of 6 and 36 and a minor one of 20S. By raising the pH, the amount of the 20S component increased and that of the 36S decreased, but that of the 6S component was unaltered. At pH 7.6 the mixture contained 20S and 6S components but hardly any 36S. The 20S species can be separated from the 6S ones by gel filtration on agarose A-15 m at pH 7.6. On examination by EM this preparation contained far fewer double rings compared to the material at pH 6.4, but single rings could often be seen. Sodium dodecyl sulfate gel electrophoresis of the 20S and 36S components showed that they consist almost entirely of tubulin and some higher and lower molecular weight fractions. Turbidity measurements showed that the minimal protein concentration necessary for polymerization increases with increasing pH. The turbidity plateau reached at a given pH can be raised by decreasing the pH. The 20S component may be an intermediate of the 36S species. There apparently is a pH-dependent equilibrium between the 20S species and the 36S oligomers.