Abstract
A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.
Highlights
A truncated motor domain of the a subunit of Dro- ionic strength [4, 5]. cDNAs for the large a subunit from five sophila kinesin was obtained by expression in Esch- species have been sequenced and they are highly homologous erichia coli and purified to homogeneity in the presence [6,7,8,9,10] with a three-domain structure [6, 11]consisting of a ofMgATP
DKH340 was eluted by a the investigation of possible inhibition by binding of tail do- gradient of 200-800 mM NaCl in MgM buffer, and the peak fractions mains, we report here the isolation and characterization of DKH340 containing the N-termina3l 40 amino residues of the were pooled and dialyzed overnight uersus KA buffer (50mM imidazole, pH 6.7,0.5mM MgCI, 0.1 mM EDTA, and 1mM 2-mercaptoethanol)
Western MTstimulated ATPase-The ATPase rate of DKH340 is blotting was performed by SDS-PAGE and electrophoretic transfer to stimulated approximately 3000-foldby MTsas indicated in Fig
Summary
The residual tubulin which remains in the supernatant following centrifugation is not capabloef stimulating the ATPase activ- leaseis therate-limitingstep ity of kinesin and is likely denatured, since no detectable rate was DKH340 in theabsence of MTs. for ATP hydrolysis by observed with the supernatants in theabsence of added MTs. Western MTstimulated ATPase-The ATPase rate of DKH340 is blotting was performed by SDS-PAGE and electrophoretic transfer to stimulated approximately 3000-foldby MTsas indicated in Fig. nitrocellulose [42] with detectioonf DKH340 by a chicken anti-kinesin 3A for results at a series ofATP concentrations.At 1 mM antibody obtained by standard methods [43] using purifiedbovine ki- MgATP, MTs stimulate the ATPase rate with a hyperbolic denesin [44] as antigen. That both the kc,, and K:‘:ATPnaevalues for stimulation by MTs are increased proportionately by increasing concentrations of
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