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 β-tubulin. One of these, β^*, connects Cys²³⁹ with Cys³⁵⁴; the other, β^s, joins Cys¹² with either Cys²⁰¹ or Cys²¹¹ (Little and Ludueña, EMBO J 4: 51-56, 1985; Biochim Biophys Acta 912: 28-33, 1987). Formation of β^* inhibits microtubule assembly in vitro, consistent with the hypothesis that Cys²³⁹ 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 μM ethacrynic acid gave half-maximal inhibition of assembly, but had no effect on the formation of the β^* cross-link by EBI. Ethacrynic acid, however, did inhibit substantially formation of the β^s cross-link at this concentration and half-maximally inhibited it at approximately 185 μM. Half-maximal inhibition of the alkylation of tubulin sulfhydryls by iodo [¹⁴C]acetamide was obtained at an ethacrynic acid concentration in the range of 190-325 μM. These results indicate that ethacrynic acid can inhibit microtubule assembly by reacting with sulfhydryl groups other than those of Cys²³⁹ and Cys³⁵⁴ 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 Cys¹², Cys²⁰¹ or Cys²¹¹.