Abstract
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 language | English (US) |
|---|---|
| Pages (from-to) | 1677-1681 |
| Number of pages | 5 |
| Journal | Biochemical Pharmacology |
| Volume | 47 |
| Issue number | 9 |
| DOIs | |
| State | Published - Apr 29 1994 |
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Keywords
- ethacrynic acid
- microtubule assembly
- N,N′-ethylenebis(iodoacetamide)
- protein cross-linking
- sulfhydryl groups
- tubulin
ASJC Scopus subject areas
- Pharmacology
Cite this
Interaction of ethacrynic acid with bovine brain tubulin. / Ludueña, Richard F.; Roach, Mary Carmen; Epstein, David L.
In: Biochemical Pharmacology, Vol. 47, No. 9, 29.04.1994, p. 1677-1681.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Interaction of ethacrynic acid with bovine brain tubulin
AU - Ludueña, Richard F.
AU - Roach, Mary Carmen
AU - Epstein, David L.
PY - 1994/4/29
Y1 - 1994/4/29
N2 - 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.
AB - 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.
KW - ethacrynic acid
KW - microtubule assembly
KW - N,N′-ethylenebis(iodoacetamide)
KW - protein cross-linking
KW - sulfhydryl groups
KW - tubulin
UR - http://www.scopus.com/inward/record.url?scp=0028360795&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028360795&partnerID=8YFLogxK
U2 - 10.1016/0006-2952(94)90547-9
DO - 10.1016/0006-2952(94)90547-9
M3 - Article
C2 - 8185683
AN - SCOPUS:0028360795
VL - 47
SP - 1677
EP - 1681
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
SN - 0006-2952
IS - 9
ER -