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. 2013 Apr 15;24(8):1103–1110. doi: 10.1091/mbc.E13-01-0030

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© 2013 Pollard and De La Cruz. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Kinetic analysis of a two-step association reaction: Mg-ATP binding to actomyosin VIIb. (A) Time courses of pyrene fluorescence after mixing 0.1 μM myosin VIIb bound to 0.1 μM pyrene-labeled actin filaments with a range of final concentrations of Mg-ATP: black, 0 μM; red, 25 μM; green, 250 μM; blue, 700 μM. The fluorescence of pyrene-labeled actin filaments is low when associated strongly with myosin without bound nucleotide (AM, black curve). The fluorescence is higher when associated with myosin in a weakly bound conformation induced by bound ATP (AM–ATP*) as observed over time for the other transients. (B) [ATP] dependence of the observed rate constant of fluorescence enhancement. The solid line through the data points is the best fit to a rectangular hyperbola, indicating a multistep mechanism, such as , where * indicates high fluorescence. The first step is rate-limiting at low concentrations of ATP. The second step is rate-limiting at high concentrations of ATP, so the observed rate constant at the plateau gives the sum of the rate constants for the second step, the reversible strong-to-weak actomyosin isomerization reaction. Figure adapted from Henn and De La Cruz (2005).

Inline graphic — Kinetic analysis of a two-step association reaction: Mg-ATP binding to actomyosin VIIb. (A) Time courses of pyrene fluorescence after mixing 0.1 μM myosin VIIb bound to 0.1 μM pyrene-labeled actin filaments with a range of final concentrations of Mg-ATP: black, 0 μM; red, 25 μM; green, 250 μM; blue, 700 μM. The fluorescence of pyrene-labeled actin filaments is low when associated strongly with myosin without bound nucleotide (AM, black curve). The fluorescence is higher when associated with myosin in a weakly bound conformation induced by bound ATP (AM–ATP*) as observed over time for the other transients. (B) [ATP] dependence of the observed rate constant of fluorescence enhancement. The solid line through the data points is the best fit to a rectangular hyperbola, indicating a multistep mechanism, such as , where * indicates high fluorescence. The first step is rate-limiting at low concentrations of ATP. The second step is rate-limiting at high concentrations of ATP, so the observed rate constant at the plateau gives the sum of the rate constants for the second step, the reversible strong-to-weak actomyosin isomerization reaction. Figure adapted from Henn and De La Cruz (2005).