Abstract
DKH392 is a construct which contains the first 392 amino acids of the alpha-subunit of Drosophila kinesin and is dimeric in solution (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). The ATPase rate of DKH392 was 0.005 s-1 in the absence of MTs. One ADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis. Microtubules accelerated the rate of ADP release and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (kcat = approximately 45 s-1). The KMT0.5,ATPase value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lower concentrations of DKH392. Physical binding of DKH392 to microtubules in the presence of 1 mM MgATP paralleled saturation of the stimulation of the ATPase activity by microtubules indicating that the rate-limiting step in microtubule-stimulated ATP hydrolysis occurs while DKH392 is bound to the microtubule. These results suggest that microtubule-stimulated ATP hydrolysis by DKH392 may be processive with the hydrolysis of multiple ATP molecules during each diffusional encounter of DKH392 with a microtubule.
Highlights
ATPase rate of D m 3 9 2 was 0.005 s-l in the absence of MTs.OneADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis.Microtubulesaccelerated the rate of ADP release and increased the rate of steady state ATP hydrolysis byalmost10,000-fold
K, values of both DKH340 and DKH392 are independent of concentration
The discoverythat DKH392 is a dimer in solution introduced DKH392 is at least in part due to mutual depletion, since the possibility that theremay be differencesin theproperties of DKH392 is bound to the MTs during steadystate ATP hydrolythe two active sites in the dimer caused by asymmetric inter- sis and the KFZAwasevalue is not in largeexcess of the level of actions
Summary
ATP hydrolysis by DKH392 may be processive with the Protein preparations and generalmethods were as described previhydrolysis of multipleATP molecules during each diffu- ously (4, 7). All reactions and incubations were performed at 25 “C in sional encounter of D M 3 9 2 with a microtubule. 25A25 buffer (4) supplemented with 0.1 mg/ml bovine serum albumin unless otherwise indicated. Kinesin is a motor protein which produces movement along MTs’ (see Ref. 1).-terminal region of thea-subunits constitutes the heaodr motor domain which has MT-stimulated dehydrogenase at 2 pg/mleach, 2 m~ phosphoenolpyruvate, 0.3 mM NADH, and MgATP as indicated. The concentration of DKH392 is expressed as the molar concentration of 44-kDa head domains. The minimal motor unit consist of the first 340 amino acids, and this domain has been expressed in Escherichia coli as DKH340 (4) and other constructs containing the head domain have been produced and characterized(3,5). SUK4was isolated from ascites fluid by three cycles of precipitation with 50% saturated ammonium sulfate followed by chromatography on DEAE-“Gel blue (10).After SDS-polyacrylamide gel electrophoresis,proteinbands were electrophoretically transferred to nitrocellulose (11)using a Mini Trans-blot apparatus (Bio-Rad)
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