Kinesin ATPase: rate-limiting ADP release.
The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from approximately equal to 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki less than 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.
Hackney
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DD
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D016547:Kinesins; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism
D009419:Nerve Tissue Proteins; D014404:Tubulin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins