INTERACTION OF ETHACRYNIC-ACID WITH BOVINE BRAIN TUBULIN

Ethacrynic acid is a diuretic agent that reacts with sulfhydryl groups in proteins, and which shows promise of effectiveness in the treatment of glaucoma. Ethacrynic acid is a known inhibitor of microtubule assembly in vitro (Xu et al., Arch Biochem Biophys 296: 462-67, 1992). We have used N,N'-ethylenebis (iodoacetamide) (EBI) as a probe to examine the sulfhydryl groups of tubulin; EBI can form two intra-chain cross-links in beta-tubulin. One of these, beta*, connects Cys(239) with Cys(354); the other, beta(s), joins Cys(12) with either Cys(201) or Cys(211) (Little and Luduena, EMBO J 4: 51-56, 1985; Biochim Biophys Acta 912: 28-33, 1987). Formation of beta* inhibits microtubule assembly in vitro, consistent with the hypothesis that Cys(239) has an assembly-critical sulfhydryl (Bai et al., Biochemistry 28: 5606-5612, 1989). We have examined the interaction of ethacrynic acid with the sulfhydryl groups of bovine brain tubulin. We found that 130 mu M ethacrynic acid gave half-maximal inhibition of assembly, but had no effect on the formation of the beta* cross-link by EBI. Ethacrynic acid, however, did inhibit substantially formation of the beta(s) cross-link at this concentration and half-maximally inhibited it at approximately 185 mu M. Half-maximal inhibition of the alkylation of tubulin sulfhydryls by iodo [C-14]acetamide was obtained at an ethacrynic acid concentration in the range of 190-325 mu M. These results indicate that ethacrynic acid can inhibit microtubule assembly by reacting with sulfhydryl groups other than those of Cys(239) and Cys(354) and suggest that other sulfhydryl groups in tubulin could be assembly-critical. These results also raise the possibility that these other assembly-critical sulfhydryls may be those of Cys(12), Cys(201) or Cys(211).