Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport.
N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.
Pfister
K K
KK
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235.
Wagner
M C
MC
Bloom
G S
GS
Brady
S T
ST
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001370:Axonal Transport; D049832:Decapodiformes; D005033:Ethylmaleimide / Q000494:pharmacology; D016547:Kinesins; D008868:Microtubule Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000493:pharmacokinetics
D008868:Microtubule Proteins; D013438:Sulfhydryl Compounds; D000251:Adenosine Triphosphatases; D016547:Kinesins; D005033:Ethylmaleimide