title: n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay.
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.
authors.affiliation: Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0406, USA. rawalker@vt.edu
authors.forename: R A
authors.initials: RA
authors.lastname: Walker
authors.forename: E T
authors.initials: ET
authors.lastname: O'Brien
authors.forename: D L
authors.initials: DL
authors.lastname: Epstein
authors.forename: M P
authors.initials: MP
authors.lastname: Sheetz
mesh_terms: D000818:Animals; D001665:Binding Sites; D002465:Cell Movement; D002642:Chick Embryo; D049832:Decapodiformes; D004976:Ethacrynic Acid / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D016547:Kinesins / Q000037:antagonists & inhibitors*; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects
chemical_list: D016547:Kinesins; D004976:Ethacrynic Acid; D005033:Ethylmaleimide