Creatine Kinase Equilibration and ΔGATP over an Extended Range of Physiological Conditions: Implications for Cellular Energetics, Signaling, and Muscle Performance

Figures

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  Figure A1. Nonlinear least squares regression estimation of Keq″ [pMg, T] for all twelve sets of conditions employed in this study. Total concentrations of ADP, ATP, Cr, and PCr were measured by HPLC (as illustrated in Figure 3), although only ADP and Cr are plotted here because they vary most widely due to the amount of Cr added. Keq″ [pMg, T] was obtained by fitting each dataset to Equation (12) using values of β, as illustrated in Figure 4 (data for the pMg 3.0 and 30 °C condition are plotted in Figure 5). Within each panel, the colors correspond to pH (blue, pH 8; cyan, pH 7.5; green, pH 7; orange, pH 6.5; red, pH 6). The panel columns correspond to free Mg2+ (left-to-right: pMg 2.0, pMg 3.0, pMg 4.0) and the panel rows correspond to the temperature (top-to-bottom: 10 °C, 20 °C, 30 °C, 40 °C). Axis scales were kept the same in all panels for ease of comparison among conditions. All data (all Cr and pH values) at each combination of constant pMg and temperature were used to simultaneously obtain one estimate of Keq″ [pMg, T]. A summary of the results of these analyses (Keq″ [pMg, T]) is shown in Figure 6.

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  Figure A2. Isometric force–pCa relationships for single permeabilized fibers from (A) rabbit psoas and (B) rabbit soleus muscles (Section 4.4.1) at 0.1 mM Pi (open squares and solid line in panel (A); red squares and dashed line in panel (B)) or 20 mM Pi (gray squares and dotted line in panel (A); dark red squares and dotted line in panel (B)). Measurements are from N = 4 psoas fibers from two rabbits in panel (A) and N = 3 soleus fibers from two rabbits in panel (A). Not shown are data points at pCa ≥ 8 that corresponded to normalized force = 0, as the absolute value (passive force) for each fiber was subtracted from all force values before plotting. Parameter estimates for nonlinear regressions on the Hill Equation (Equation (A2)) are provided in Table A1.

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  Figure A3. Dependence of isometric force on (A,B) [Pi] and (C,D) [sulfate] for single permeabilized fibers from (A,C) rabbit psoas and (B,D) rabbit soleus muscles (Section 4.4.1). Each point represents the normalized maximum force (normalized as described for the data in Figure A2) for one muscle fiber at one concentration of Pi (A,B) or sulfate (C,D). Measurements are from N = 28 psoas fibers from ten rabbits in panel (A), N = 19 soleus fibers from six rabbits in panel (B), N = 7 psoas fibers from one rabbit in panel (C), and N = 7 soleus fibers from one rabbit in panel (D). The lines in panels (A,B) were fitted to Equation (A3) using nonlinear least squares regression, while the lines in panels (C,D) were fitted using linear least squares regression constrained to pass through normalized force = 1.0 at [sulfate] = 0.

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  Figure A4. Dependence of the velocity of unloaded shortening (VUS) on [Pi] for single permeabilized fibers from (A) rabbit psoas (open squares) and (B) rabbit soleus muscles (red squares) (Section 4.4.1). Each point represents the normalized value of VUS (normalized to VUS obtained at 0.1 mM Pi in the same fiber, comparable to normalization of force data as described in Figure A2 and Figure A3) at maximum Ca2+-activation. Measurements are from N = 18 psoas fibers from eight rabbits in panel (A) and N = 9 soleus fibers from six rabbits in panel (B). The control values of VUS were 3.3 ± 0.8 FL s−1 (mean ± SD) for psoas fibers and 0.7 ± 0.2 FL s−1 (mean ± SD) for soleus fibers. The lines are linear least squares regression fits constrained to pass through VUS = 1 at 0.1 mM Pi.

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  Figure 1. 31P-NMR spectroscopy of model solutions containing inorganic phosphate (Pi), phosphocreatine (PCr), ATP (left-to-right, the peaks correspond to the γ, α, and β phosphate resonances), ADP (only visible at pH 7.5 and 8.0; left-to-right, the peaks correspond to the β and α phosphate resonances) in the presence of CK and with 50 mM added Cr at pMg 3.0, 30 °C, and pH 6.0, 6.5, 7.0, 7.5, or 8.0 (bottom-to-top stacked plot). Details of solution composition were as described in Section 4.1. An external standard (H3PO4, chemical shift δ = 0) was placed in a small capillary and centered in the coil’s sensitive volume. Spectra were acquired on a Varian 600 MHz spectrometer at the phosphorus frequency (242 MHz) using an 8000 Hz sweep width. Data are the sum of 1024 transients collected with a 1.0 s recycle delay and a π/2 pulse width and 1024 complex points and zero-filled once to a total of 2048 data points and exponentially filtered prior to the Fourier transform. Note that Pi chemical shift (δPi) moves from right to left along the δ-axis with increasing pH (bottom-to-top), corresponding to deprotonation of H2PO4− with pKa around neutral pH. Additionally, note that the γATP and βATP peaks…

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  Figure 2. (A–C) Experimentally determined 31P-NMR chemical shift of Pi (δPi) titrated between pH 6–8 and (D–F) calculated pH-dependence of [H2PO4−]/Σ[Pi]. (A,D) pMg 2; (B,E) pMg 3; (C,F) pMg 4. In all panels, blue is 10 °C, green is 20 °C, yellow is 30 °C, and red is 40 °C. In panels (A–C), all data in each panel (single pMg) were simultaneously fitted to Equation (8) by nonlinear least squares regression, while in panels (D–F) all values in each panel (single pMg) were simultaneously fitted to Equation (9) by nonlinear least squares regression; regression parameter estimates are provided in Table 1. Note the qualitative correspondence between the left and right panels.

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  Figure 3. HPLC quantitation of (A,B) PCr, ADP, and ATP by anion exchange chromatography and (C,D) Cr by cation exchange chromatography (Section 4.3) in solutions designed to mimic the cytosol under different metabolic conditions (Section 4.1). Representative sample #226 (panels (A,C); low ADP; no added Cr) was held at 10 °C, pMg 4 and pH 8. Representative sample #80 (panels (B,D); high ADP; 50 mM added Cr) was held at 40 °C, pMg 2 and pH 8. Both samples initially contained 1 mg/mL rabbit CK. At equilibrium as determined by monitoring the reactions using 31P-NMR spectroscopy (Figure 1), each reaction was stopped with addition of SDS at 2% per mg CK (Section 4.2). For both HPLC methods, detection was by optical absorbance at 210 nm and peak areas were quantified against calibration curves determined using known standards. Numerical scales on vertical (optical absorbance) axes correspond to detector output in millivolts. Note that scales for the vertical (optical absorbance) axes are the same for panels (A,B) and for panels (C,D).

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  Figure 4. Calculated pH dependence and range of the stoichiometric coefficient of proton consumption (β) by ADP rephosphorylation via transfer of Pi from PCr (Equation (4)). pH dependence of β was predicted according to the ion binding equilibria (Section 4.1) for all conditions of this study. The smallest range of predicted values as a function of pH was obtained at pMg 4.0 and 10 °C (dashed line), while the largest range of predicted values as a function of pH was obtained at pMg 3.0 and 30 °C (solid line); all other predicted values of β fell within the range between the two lines shown.

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  Figure 5. Nonlinear least squares regression estimation of Keq″ for model solutions at pMg 3.0 and 30 °C. Total concentrations of ADP, ATP, Cr, and PCr were measured by HPLC (Figure 3), although only ADP and Cr are plotted here because they vary most widely by the amount of Cr added. All data (all Cr and pH values) were used to simultaneously obtain a single estimate of Keq″ [pMg 3.0, 30 °C] by nonlinear least squares regression fitting of the data to Equation (12) using values of β, as illustrated in Figure 4. Colors correspond to pH (blue, pH 8; cyan, pH 7.5; green, pH 7; orange, pH 6.5; red, pH 6). Comparable analyses were performed for each combination of temperature and pMg, resulting in a total of 12 plots (Figure A1) and regression estimates of Keq″ [pMg, T] (Figure 6).

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  Figure 6. Creatine kinase Keq″ as a function of free Mg2+ (pMg) and temperature at Γ/2 = 0.25 M. Points are regression estimates of Keq″ (Equations (10) and (11)) obtained as illustrated (Figure 5 and Figure A1). Error bars are the SE regression for each estimate of Keq″. The results of a multiple linear least squares regression on all of the Keq″ data (Equation (13)) are shown by thick blue lines. This simple relationship allows for prediction of [ADP] (which is not always directly measurable, i.e., as illustrated in Figure 1 and Figure 3) over a wide range of conditions, ranging from those relevant to intact tissue in vivo to experiments with permeabilized muscle fibers.

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  Figure 7. Variation of maximum Ca2+-activated force with ΔGATP when ΔGATP was altered by varying [Pi] in (A) intact mouse soleus muscles (red filled triangles with blue borders and with dashed red line) or (B) single skinned fibers from rabbit psoas (open squares with solid gray line) and soleus (red filled squares with dashed red line) muscles. [Pi] was varied in intact muscles (panel (A)) by altering the substrate supplied extracellularly [49], and the force and biochemical data are from that study; [Pi] in the presence of pyruvate was set to 1 mM in the plot, with the upper limit determined [49]. [Pi] for skinned fiber experiments (panel (B)) was 0.1–36 mM. The psoas force at the first Pi concentration in panel (B) was previously published in Chase and Kushmerick [35], and the psoas force at the second Pi concentration in panel (B) was previously published in Chase and Kushmerick [36]; all other data in panel (B) are previously unpublished. Measurements on skinned fibers in panel (B) were from N = 28 psoas fibers from ten rabbits and N = 19 soleus fibers from six rabbits. As described in the text, the force for the psoas fibers in panel (B) was normalized to 1 mM Pi, while the soleus…

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  Figure 8. Maximum Ca2+-activated isometric force variation with (A) ΔGATP and (B) [ADP] when pH was altered. We reanalyzed previously published data from (panel (A)) isolated perfused biceps (open circles with blue error bars connected by solid gray line) and soleus (red filled circles with blue error bars connected by red dashed line) muscles from cat [67] and (panels (A,B)) single skinned fibers from rabbit psoas (open squares fit with solid gray lines) and soleus (red filled squares fit with dashed red lines) muscles [35]. Hypercapnic (acidic cytoplasmic pH 6.48–6.6) force data from cat muscles were normalized to the normocapnic condition (higher force; cytoplasmic pH 7.09–7.11) for each muscle type. Skinned fiber force data were normalized to that at pH 7.1 for the same fiber. Lower forces were associated with acidic (lower) pH and higher forces were associated with basic (higher) pH [35,67]. The points represent the average ± SD. ΔGATP was estimated according to Equation (16) and [ADP] was estimated according to Equation (12) using values of Keq″ from Equation (13). In panel (A) smooth curves were drawn through the points, while in panel (B) the lines represent nonlinear regression…