When bovine brain tubulin purified in the absence of GTP and MgCl2 is reacted with N,N'-ethylene-bis(iodacetamide) (EBI), a bifunctional analogue of iodoacetamide, three new electrophoretically distinct species of tubulin are generated, migrating ahead of β1-tubulin on gels containing Na dodecyl sulfate. All three bands appear to be derived from the β1 subunit of tubulin and not from the α or β2 subunit. Accordingly, the bands have been designated β1(s), β1( ), and β1(s ) in order of increasing electrophoretic mobility. EBI appears to introduce two intrachain cross-links into β1-tubulin; the β1(s) band contains one of these cross-links, designated β(s), and the β1( ) band contains the other cross-link, designated β* and the β1 (s*) contains both cross-links. Both cross-links appear to involve sulfhydryl groups. Colchicine, podophyllotoxin, and nocodazole completely inhibit β( ) formation while GTP, vinblastine, and maytansine enhance it. In contrast, formation of β(s) is completely blocked by guanine nucleotides and by maytansine, while vinblastine inhibits this by 70%. Colchicine, podophyllotoxin, and nocodazole enhance β(s) formation. These results show that tubulin has the unusual property of having two discrete sites which can be targeted by an alkylating agent with each site having its alkylation inhibited by a different set of ligands. The results are consistent with several models, including one where vinblastine and maytansine have overlapping binding sites on the β-subunit of tubulin relatively close to the GTP binding site.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology