Table 1.
Composition of the solutions
| Solution name | MgCl2(mM) | Na2ATP(mM) | EGTA(mM) | HDTA(mM) | CaEGTA(mM) | KProp(mM) |
|---|---|---|---|---|---|---|
| P1 experiments (pH 7.1) | ||||||
| Control (0 P1) | ||||||
| Relaxing | 6.80 | 6.09 | 5 | 15 | — | 27.9 |
| Pre-activating | 6.75 | 6.14 | 0.5 | 19.5 | — | 28.0 |
| Activating (pCa 4.8) | 6.71 | 6.15 | — | 15 | 5 | 27.8 |
| 30 mM P1 | ||||||
| Relaxing | 7.77 | 6.09 | 5 | — | — | 13.3 |
| Pre-activating | 7.72 | 6.09 | 0.5 | 4.5 | — | 13.4 |
| Activating (pCa 4.8) | 7.68 | 6.15 | — | — | 5 | 13.3 |
| Fatigue (30 mM P1, pH 6.2) | ||||||
| Relaxing | 6.80 | 8.14 | 5 | 7 | — | 31.1 |
| Pre-activating | 6.79 | 8.14 | 0.5 | 11.5 | — | 31.1 |
| Activating (pCa 3.9) | 6.79 | 8.57 | — | 7 | 5 | 29.9 |
| pH series | ||||||
| Control (pH 7.1) | ||||||
| Relaxing | 7.32 | 6.14 | 5 | — | — | 91.0 |
| Pre-activating | 7.32 | 6.14 | 0.5 | 4.5 | — | 91.0 |
| Activating (pCa 4.8) | 7.29 | 6.20 | — | — | 5 | 90.8 |
| pH 6.2 | ||||||
| Relaxing | 6.79 | 8.15 | 5 | — | — | 116.4 |
| Pre-activating | 6.78 | 8.15 | 0.5 | 4.5 | — | 116.4 |
| Activating (pCa 3.9) | 6.78 | 8.60 | — | — | 5 | 115.0 |
| pH 7.4 | ||||||
| Relaxing | 7.59 | 5.97 | 5 | — | — | 74.4 |
| Pre-activating | 7.48 | 5.97 | 0.5 | 4.5 | — | 74.6 |
| Activating (pCa 5.0) | 7.40 | 6.00 | — | — | 5 | 74.6 |
All solutions contained in addition 4 mg ml−1 pyruvate kinase (500 U ml−1, Sigma) and 0.24 mg ml−1 LDH (870 U ml−1, Sigma), with 10 mM phosphoenol pyruvate, 5 mM sodium azide, 10 μM oligomycin B, 0.8 mM NADH, 0.2 mM p1,p5-di(adenosin-5’)pentaphosphate, 5 mM caffeine and 100 mM Bes. The pH was adjusted with KOH. Potassium propionate (KProp) and HDTA were added to adjust ionic strength. CaEGTA was made by dissolving equimolar amounts of CaCO3 and EGTA. The pH 6.2 and fatigue solutions contained elevated enzyme concentrations (see Methods). The solutions used at pH 6.8 and pH 6.5 were obtained by appropriate mixing of the control (pH 7.1) and the pH 6.2 solutions.