Sarco/endoplasmatic reticulum calcium ATPase activity in healthy muscle and Brody disease

Figures

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  Fig. 1. Effect of incubation time on SERCA activity. Two muscle samples were measured, both showing significant slope deviation from zero with P = <0.0001 and r2 = 0.973 for sample 1 (black circles and line) and P = <0.0001 and r2 = 0.988 for sample 2 (grey circles and line). The dotted line represents the chosen incubation time for future measurements (120 min).

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  Fig. 2. Optimization of reaction conditions of the SERCA enzyme assay. Effects of homogenate (A), pH (B), KCl/Imidazole (C), ATP (D), MgCl2 (E), and CaCl2 (F) concentrations on SERCA activity. Protein normalization was not performed for these experiments, as identical volumes of the same muscle homogenate were used for each condition; results are therefore expressed in mU/mL reaction mixture rather than mU/mg protein. Black and grey circles and regression lines represent two different samples from healthy subjects. The dotted lines represent the reaction mix concentrations chosen for further measurements, which consist of 2% homogenate, 94.1 mM KCl/imidazole, 9.4 mM ATP, 0.588 mM CaCl2, and 4.7 mM MgCl2, with pH of 7.4. These concentrations were used in all sub-experiments (A-F), except for concentrations of 4.7 mM ATP and 0.518 mM CaCl2 in the pH (B), KCl/Imidazole (C), and MgCl2 (E) curves. For the regression lines we used linear regression (A + C), Bell-shaped (B + F), and Michaelis-Menten (D + E).

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  Fig. 3. SERCA activity in healthy controls and patients with Brody disease. Results are displayed for male and female subjects together (A) and separately (B + C). The grey shaded areas represent the 90% prediction bands. The horizontal dashed lines represent the lower limit of normal at 36.6 mU/mgP (mean all controls – 2xSD). For males there is a significant effect of age on SERCA activity (P = 0.007, r2 = 0.529), which is not seen in females (P = 0.097, r2 = 0.185). For both sexes together there is no age effect (P = 0.880, r2 = 0.001). Error bars represent the Standard Error of the Mean (SEM) of the duplicate measurements for each individual subject. When no error bars are shown, it indicates that there was insufficient material to perform duplicate measurements for that given subject. mgP, milligrams of protein; SERCA, sarcoplasmic/endoplasmic reticulum ATPase.