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. Author manuscript; available in PMC: 2007 May 10.
Published in final edited form as: J Biol Chem. 2006 Oct 23;281(51):39444–39454. doi: 10.1074/jbc.M608056200

FIGURE 2.

FIGURE 2

Pulse-chase kinetics of ATP binding. A preformed MT·Eg5 complex was rapidly mixed with Mg[α-32P]ATP and chased with 10 mm unlabeled MgATP. A, individual transients displaying an ATP concentration-dependent pre-steady-state burst of product formation followed by a linear phase of product formation. B, the observed exponential rates were plotted as a function of MgATP concentration and fit to a hyperbola: kmax = 52 ± 7s−1 and apparent Kd,ATP = 20.4 ± 14.5 μm. C, inset gel, not all Eg5 was bound to the MTs at the start of the experiment. Displayed are two load volumes of the supernatant and pellet fractions of centrifuged MT·Eg5 complexes. C, the burst amplitude, as micromolar ADP formed, is plotted. The hyperbola extrapolates to 4.1 μm of possible 4.5 μm maximal amplitude based on the fraction of motor that was bound to the MTs at the start of the experiment. B and C, data from individual experiments; D–F, data from multiple experiments. D, the hyperbolic fit of the observed burst rates provided k+1′= 50 ± 3s−1 and the apparent Kd,ATP, = 35 ± 7 μm. The inset displays ATP binding at low ATP concentrations where the data can be linearly fit to Equation 2. The second-order rate constant for ATP binding given by the slope is k+1 = 1.15 ± 0.1 μm−1s−1, and the off-rate provided by the y-intercept is 1.4 ± 0.6 s−1. E, the observed rates for subsequent ATP binding events obtained from the linear phase of the transients were plotted and fit to a hyperbola: kslow = 5.7 ± 0.4 μm ADP·s−1, apparent Kd,ATP = 27 ± 7 μm. F, the burst amplitudes from multiple experiments have been normalized according to the amount of Eg5 sites that partition with the MT pellet on the day of the experiment and were plotted as a function of MgATP concentration. The hyperbolic fit extrapolates to 97 ± 5% of Eg5 active sites with the apparent Kd,ATP = 57 ± 8μm. Final concentrations of the MT·Eg5 complexes were as follows: 1 μm Eg5-513/6μm MTs for 1–3 μm ATP, 2 μm Eg5-513/6 μm MTs for 2–6 μm ATP, and 5 μm Eg5-513/6 μm MTs for 5–300 μm.