title: Isolation of a 45-kDa fragment from the kinesin heavy chain with enhanced ATPase and microtubule-binding activities.
abstract: Kinesin is a microtubule-activated, mechanochemical ATPase capable of moving particles along microtubules and making microtubules glide along a solid substrate. In this study we used limited proteolysis to study the structure of bovine brain kinesin, a heterotetramer composed of two heavy (120-kDa) and two light (62-kDa) chains. alpha-chymotrypsin, trypsin, and subtilisin all produced a protease-resistant 45-kDa fragment from the kinesin heavy chain. As isolated by gel-filtration chromatography, this fragment contains both the microtubule-binding site and the ATP catalytic site of the molecule. Proteolytic cleavage stimulated microtubule-dependent Mg2+-ATPase activity 4- to 5-fold up to 75-120 mumol ATP/min/mg. Cleavage also increased the affinity of the fragment for microtubules at least 10-fold. Since the purified fragment does not support the gliding of flagellar axonemes, we propose that cleavage of the heavy chain uncouples ATPase activity from its translocator activity, which may require other parts of the molecule.
authors.affiliation: Department of Molecular Biology, Biology Faculty, Moscow State University, Union of Soviet Socialist Republics.
authors.forename: S A
authors.initials: SA
authors.lastname: Kuznetsov
authors.forename: Y A
authors.initials: YA
authors.lastname: Vaisberg
authors.forename: S W
authors.initials: SW
authors.lastname: Rothwell
authors.forename: D B
authors.initials: DB
authors.lastname: Murphy
authors.forename: V I
authors.initials: VI
authors.lastname: Gelfand
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002918:Chymotrypsin; D016547:Kinesins; D007700:Kinetics; D008868:Microtubule Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism
chemical_list: D008868:Microtubule Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D014404:Tubulin; D002918:Chymotrypsin; D000251:Adenosine Triphosphatases; D016547:Kinesins