n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay

R. A. Walker, E. T. O'Brien, D. L. Epstein, Michael Sheetz

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n- ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH- reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.

Original languageEnglish (US)
Pages (from-to)289-299
Number of pages11
JournalCell Motility and the Cytoskeleton
Volume37
Issue number4
DOIs
StatePublished - Aug 19 1997
Externally publishedYes

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Ethacrynic Acid
Kinesin
Ethylmaleimide
Microtubules
Sulfhydryl Compounds
Adsorption
Glass
Cysteine
Proteins
Light

Keywords

  • Ethacrynic acid
  • Kinesin
  • Microtubule
  • n-ethylmaleimide
  • Sulfhydryl

ASJC Scopus subject areas

  • Structural Biology
  • Cell Biology

Cite this

n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay. / Walker, R. A.; O'Brien, E. T.; Epstein, D. L.; Sheetz, Michael.

In: Cell Motility and the Cytoskeleton, Vol. 37, No. 4, 19.08.1997, p. 289-299.

Research output: Contribution to journalArticle

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