Interaction of ethacrynic acid with bovine brain tubulin

Richard F. Ludueña, Mary Carmen Roach, David L. Epstein

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13 Scopus citations


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, βs, 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 βs 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.

Original languageEnglish (US)
Pages (from-to)1677-1681
Number of pages5
JournalBiochemical Pharmacology
Issue number9
StatePublished - Apr 29 1994


  • N,N′-ethylenebis(iodoacetamide)
  • ethacrynic acid
  • microtubule assembly
  • protein cross-linking
  • sulfhydryl groups
  • tubulin

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology


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