Effect of the N-terminal extension in myosin essential light chain A1 on the mechanism of actomyosin ATP hydrolysis

Figures

• 

  Figure 1. Comparison of ATP hydrolysis pathways for isomyosins A1 and A2. A minimal scheme is presented in which the classical Lymn-Taylor mechanism (56) is assumed to apply to both ELCA1-containing (M1) and ELCA2-containing (M2) myosins but the fraction of hydrolysis occurring along each pathway varies depending on the ELC isoform. Under the ionic strength used in the current experiments and the exceedingly high actin concentration in muscle, the N-terminal light chain extension increases the affinity of myosin intermediates for actin and enforces comparatively more flux through an actomyosin-limited hydrolysis mechanism (indicated in bold type) that is characterized by slower cleavage (K AH, small arrow) and slower Pi dissociation (k -DAP, small arrow). M(2) will promote greater flux through a myosin-limited hydrolysis mechanism that is characterized by faster cleavage (K H) and faster Pi dissociation (k-DAP). ELC, essential light chain.

• 

  Figure 2. Diethylaminoethyl-cellulose chromatography of rabbit skeletal myosin-S1. Isoenzyme-containing fractions are pooled as indicated and processed as in Experimental procedures. Coomassie R-250-stained Laemmli gel: left-hand lane, markers (Bio-Rad, catalog no. 1610363), 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa; central lane, S1A1 and right-hand lane, S1A2. The positions of myosin-S1 heavy chain (HC), ELCs and 20 kDa marker are indicated by chevrons in right-hand margin. Differential ELC staining intensity is attributed to differences in amino acid composition, notably lysine. ELC, essential light chain

• 

  Figure 3. Chemical quench-flow analysis of ATP hydrolysis by skeletal myosin-S1 containing either ELCA1 or ELCA2. A given myosin-S1 is mixed with an equal volume of MgATP containing γ-32P-ATP in different molar ratios and allowed to incubate for 0.005 to 200 s. Samples are nonblind and nonrandomized. Fractional hydrolysis is obtained from the ratio of the charcoal-treated and total counts. Symbols, red triangles (100 μM S1 + 25 μM ATP), black circles (50 μM S1 + 12.5 μM ATP), blue squares (50 μM S1 + 200 μM ATP) and green diamonds (100 μM S1 + 400 μM ATP). Stated concentrations are final, specifically half of the starting amount. Note: the amounts of protein used did not permit technical replicates. Curves are fit by equations with two exponential terms to determine the pre-steady state and steady state rates of hydrolysis. Fitting parameters for myosin-S1A1 in plot (A): red, f(t) = 1.0-(0.62e-58.4∗t + 0.33e-0.18∗t). black, f(t) = 1.0-(0.54e-40.7∗t + 0.45e-0.16∗t). blue, f(t) = 1.0-(0.19e-43∗t + 0.78e-0.027∗t). green, f(t) = 1.0-(0.19e-43∗t + 0.71e-0.027∗t). Fitting parameters for myosin-S1A2 in plot (B): red, f(t) = 1.0-(0.69e-54.1∗t + 0.33e-0.20∗t). black, f(t) = 1.0-(0.56e-31.5∗t +…

• 

  Figure 4. F-actin and native thin filament (pCa 4) dependence of the steady state MgATPase rate of rabbit skeletal myosin-S1 containing either ELCA1 or ELCA2. Shown are averages of technical replicate titrations performed on the same day. Samples, nonblind and nonrandomized. A, unregulated actin, n = 7. B, native thin filaments (NTFs) at high [Ca(II)], n = 3. t test comparisons of rates (A1 versus A2) yield p values < 0.01 (zero–3 μM F-actin) to < 0.001 (10–40 μM F-actin). Experimental conditions, 5 mM Mops, 2 mM Mg(II), 1 mM MgATP, pH 7.00, 20 °C. Initial rates are obtained from multiple time points. In the lower F-actin concentration range, 0.12 μM myosin-S1 is used and data are collected between zero and 15 min. In the higher range, 0.061 μM myosin-S1 is used and data are collected between zero and 7.5 min. The plotted relationship for myosin-S1A1 is obtained by fitting to the appropriate steady state kinetic equation using a nonlinear least squares simplex operation (90). ELC, essential light chain.

• 

  Figure 5. Inorganic phosphate dissociation from a thin filament-myosin-S1-ADP-Pi complex at low and high [Ca(II)] and in the absence of apomyosin-S1.A, schematic of double-mixing stopped-flow instrument configuration. Syringe “I”, myosin-S1A1 or S1A2 (M); syringe “II”, ATP and syringe “III”, native cardiac thin filaments (NTFs). Delay between mixes, 1 s. Buffer, 5 mM Mops, 2 mM Mg acetate, pH 7.00, 20 °C. All solutions contained Pi binding protein, 0.01 units/ml purine nucleoside phosphorylase and 0.1 mM 7-methylguanosine. B–G, time traces. Pi release is monitored over two different time windows each of 250 data points, in accordance with the process that is being monitored. In each instance, the presented time trace is an average of recordings stemming from three or more consecutive shots (i.e. passes through the stopped-flow instrument). Concentrations are adjusted for mixing dilutions as specified in Experimental procedures. Comparison of isomyosins (nonblind) is performed on the same day. B and C, time traces for turned-off NTFs (pCa 8, zero apomyosin-S1). Final concentrations in flow cell, 0.44 μM myosin-S1, 0.44 μM ATP, 27 μM NTFs, 1.1 mM EGTA and 5 μM phosphate binding protein. The…

• 

  Figure 6. Inorganic phosphate dissociation from a thin filament-myosin-S1-ADP-Pi complex in the presence of apomyosin-S1 and at pCa 8.A, instrument configuration same as in Figure 5A with the addition of a “T”-mixer for premixing native cardiac thin filaments (NTFs) and myosin-S1A1 or A2. Stoichiometry, 1 S1 per 7 actin monomers. The myosin-S1s are treated with PMSF prior to exposure to NTFs in order to kill any residual protease and protect the thin filaments. Comparison (nonblind) is performed on consecutive days. Syringe “I”, myosin-S1A1 (A1); syringe “II”, ATP and syringe “III”, premixed NTFs (pCa 8) + myosin-S1. Delay between mixes, 1 s. Buffer, 5 mM Mops, 2 mM Mg acetate, pH 7.00, 20 °C. B and C, time traces. As per Figure 5, Pi release is monitored over two different time windows each of 250 data points. Final concentrations in flow cell, 0.44 μM myosin-S1A1, 0.44 μM ATP, 10.3 μM NTFs, 1.1 mM EGTA and 5 μM phosphate binding protein + either myosin-S1A1 (B) or myosin-S1A2 (C). The presented traces are averages of recordings stemming from three or more shots (i.e. passes through the stopped-flow instrument) and are best fits to a given double exponential equation: B, I(t) =…