Properties of disulfide-linked tubulin purified on hydroxyapatite and its comparison with intact and dissociated microtubules using limited tryptic digestion

Abstract

This investigation pursued aspects of tubulin structure using limited tryptic digestion as a sensitive monitor of conformational state. A novel form of tubulin was analyzed that had been purified on hydroxyapatite in sodium phosphate buffer and exhibited interchain disulfide crosslinking. Circular dichroism spectra of this protein were highly reproducible and indicate it assumes a stable native state. Limited proteolysis of bovine brain microtubule protein in its assembled or disassembled states yielded a prominent 41,000-molecular-weight product as long as it was stabilized by GTP-containing buffers. Hydroxyapatite-purified tubulin yielded entirely different limited digestion products, with species having molecular weights of 48,000 and several between 35,000 and 19,000. Limited proteolysis was found to offer a useful probe of tubulin structure and to produce fragments which might prove valuable in analyzing its protein substructure. Discussion is given on the relevance of these results to the structural state assumed by tubulin and to the conformation of disulfide-linked tubulin reported to reside in the postsynaptic density.