Twitch fibres were isolated from the semitendinosus muscles of frogs. The sarcolemma was made more permeable by a 30 min soak in a solution containing 47.3% glycerine (v/v), 2 mM-EGTA and 10 mM phosphate buffer, pH 7. This was followed by a 30-60 min soak in a solution containing the non-ionic detergent Lubrol-WX. The fibres were then placed in a relaxing medium containing (in mM): KCl, 100; MgCl(2), 1; ATP, 4; EGTA, 2; imidazole buffer, 10; pH 7.0.2. A piece of fibre about 1-2 mm long treated as described in (1) was attached to a servo apparatus. This apparatus made it possible either to hold fibre length constant giving isometric conditions, or alternatively to hold the force constant while measuring isotonic length changes. A special network made it possible to switch control from isometric to isotonic conditions so that afterloaded contractions with a shortening stop could be carried out.3. Contractions were induced at about 4 degrees C by lowering the pCa in the relaxing solution to various levels determined by the ratio of calcium and EGTA added. Contractions were never observed above pCa 7. The steady force generated reached a maximum over the range of pCa 6.09 to 5.49. The relationship between steady force generated and pCa is S-shaped and very steep, implying that multiple interacting binding sites for calcium are involved in the force generating process.4. The relative force-velocity relation is the same at pCa 6.09 and 5.49 where the steady force is at a maximum. The data points can be well fitted by a hyperbola in which the extrapolated value for V(max) is 2.39 muscle lengths/sec. The values obtained for the Hill parameters a/P(0) and b are within the range of those reported for living electrically excited frog muscle.5. The relative force-velocity points obtained at higher pCa values at which the steady force was on average 37% of that developed at pCa 5.49 can also be fitted by a hyperbola. However, the extrapolated value for V(max) is only 1.12 muscle lengths/sec. The value for a/P(0) is increased slightly and the value for b is markedly decreased.6. Evidence is presented against the possibility that an unrecognized fixed internal load is responsible for the change in the relative force-velocity relation obtained at high pCa.7. The relative force-velocity relation does not change appreciably over at least part of the range of sarcomere lengths in which the force generated varies linearly with overlap provided the pCa is held constant.8. The results support the view that lowering the pCa produces a mechanical state equivalent to that produced by tetanic electrical stimulation.9. Some models for calcium activation are discussed. It is concluded that a model based on calcium binding to troponin on the thin filaments is difficult to reconcile with all of the experimental evidence. There is additional evidence for believing that activating calcium may directly influence the cross-bridges.
F J Julian (Fri,) studied this question.