Skinned Frog Muscle Fibers Research Articles

Role of calcium and cross bridges in determining rate of force development in frog muscle fibers.

The influence of Ca2+ and force-generating cross bridges on the kinetics of force development was examined in skinned frog muscle fibers activated by photolytic release of Ca2+ from caged Ca2+ at 10 degrees C. Isometric force development was fit by a double exponential equation with rates of 44.4 s-1 (kc1) and 6.1 s-1 (kc2); kc1 was not significantly different from the rate of force development observed in intact fibers. Maximum activation by caged Ca2+ from preexisting submaximal force produced rates of contraction similar to those observed with maximum activation from zero force. Decreasing the Ca2+ level to an extent that resulted in 50% of maximum force development produced an approximately sevenfold decrease in kc1 and no change in kc2. Partial extraction of troponin C reduced kc1 only slightly (by 16%), whereas decreasing the number of force-generating cross bridges by vanadate did not decrease kc1. Neither treatment altered kc2. Thus the rate of force development increases dramatically with increases in Ca2+ level.

Approximating the isometric force-calcium relation of intact frog muscle using skinned fibers

In previous papers we used estimates of the composition of frog muscle and calculations involving the likely fixed charge density in myofibrils to propose bathing solutions for skinned fibers, which best mimic the normal intracellular milieu of intact muscle fibers. We tested predictions of this calculation using measurements of the potential across the boundary of skinned frog muscle fibers bathed in this solution. The average potential was -3.1 mV, close to that predicted from a simple Donnan equilibrium. The contribution of ATP hydrolysis to a diffusion potential was probably small because addition of 1 mM vanadate to the solution decreased the fiber actomyosin ATPase rate (measured by high-performance liquid chromatography) by at least 73% but had little effect on the measured potential. Using these solutions, we obtained force-pCa curves from mechanically skinned fibers at three different temperatures, allowing the solution pH to change with temperature in the same fashion as the intracellular pH of intact fibers varies with temperature. The bath concentration of Ca2+ required for half-maximal activation of isometric force was 1.45 microM (22 degrees C, pH 7.18), 2.58 microM (16 degrees C, pH 7.25), and 3.36 microM (5 degrees C, pH 7.59). The [Ca2+] at the threshold of activation at 16 degrees C was approximately 1 microM, in good agreement with estimates of threshold [Ca2+] in intact frog muscle fibers.

Freeze-Fracture Studies on the Cross-Bridge Angle Distribution at Various States and the Thin Filament Stiffness in Single Skinned Frog Muscle Fibers

To give information about the changes of cross-bridge (myosin head) orientation during muscle contraction, mechanically skinned frog muscle fibers were rapidly frozen at various states, and the cross-bridges were observed on freeze-etch replicas. The number of cross-bridges per unit length of thick filament in relaxed state was less than one-third of that in contracting and rigor states. The interval between adjacent cross-bridges was maximum around 35 nm, a value close to the crossover repeat of actin helix. The cross-bridge angle distribution, as measured with a digital image processor, showed a peak around 90 degrees in all the states examined. The proportion of cross-bridges with angles around 90 degrees decreased either after stretch or after release of fibers in rigor state. The axial spacing of actin monomers in the thin filament was found to increase with increasing rigor force, to give the thin filament stiffness (times unit length) of about 1.8 x 10(4) pN. These results are discussed in connection with the mechanical properties of cross-bridges.

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments.

The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 microns remained at about 0.8 microns from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 microns. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 microns of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Sarcoballs: direct access to sarcoplasmic reticulum Ca2+-channels in skinned frog muscle fibers

In skeletal muscle, twitch contraction is caused by the rapid release of Ca2+ from the sarcoplasmic reticulum (SR) (Endo, M. 1977. Physiol. Rev. 57:71-108) via Ca2+ conducting channels in the SR membrane (Smith, J. S., R. Coronado, and G. Meissner, 1985. Nature (Lond.). 316:446-449; Suarez-Isla, B. A., G. Orozco, P. F. Heller, and J. P. Froehlich. 1986. Proc. Natl. Acad. Sci. USA. 83:7741-7745). To facilitate study of these and other intracellular channels, we have developed a method which allows direct patch-clamp recording of currents through SR channels in native membrane. The Ca2+-release channel studied using this method exhibits two predominant conductance levels (80-100 pS and 120-160 pS), conducts Ca2+ preferentially over K+ (PCa/Pk = 6.5), is highly voltage sensitive, blocked on one side by ruthenium red (1 microM), and displays enhanced activity in the presence of caffeine (5 mM). Studied in skinned fibers, this channel appears fundamentally similar to homologous channels from isolated rabbit SR incorporated into bilayers, with some distinct differences.

Mechanism of contracture on cooling of caffeine-treated frog skeletal muscle fibres.

1. In order to clarify the mechanism of contracture on cooling of caffeine-treated intact muscle fibres, the temperature dependence of a calcium (Ca2+) release mechanism, 'Ca2+-induced Ca2+ release', of the sarcoplasmic reticulum (SR) was examined in skinned frog muscle fibres. 2. Skinned fibres in a solution containing 1.2 mM-caffeine and 0.7 mM-EGTA (Mg2+, 1.5 mM, Mg-ATP, 3.5 mM, pH 7), contracted on cooling (from 22 to 2 degrees C) due to Ca2+ release from the SR. 3. The rate of Ca2+ release from skinned fibre SR in a medium which contained Ca2+ ions (with 10 mM-EGTA) and no ATP salts, was determined under various conditions using the 'caffeine method.' 4. In the absence of Mg2+ ions, adenine nucleotides and caffeine, the rates at room temperature (21-22 degrees C) were 3-4 times greater than those at a lower temperature (1.5-3 degrees C), at any concentrations of Ca2+ ions external to the SR. 5. In the presence of Mg2+ ions (1.5 mM) and beta,gamma-methylene ATP (1 mM), the effect of temperature on the rates disappeared in Ca2+-containing media, although the effect remained in Ca2+-free medium. 6. When caffeine (1.2 mM), which is a potentiator of the Ca2+-induced Ca2+ release, was added to the test medium with Mg2+ and beta,gamma-methylene ATP, the resulting potentiating effect was several times greater than that at lower temperature. 7. In order to examine the temperature dependence of the Ca2+ pump activity of the SR, the initial rate of Ca2+ uptake by the empty SR was determined under various conditions in the presence of Mg2+ ions (1.5 mM) and Mg-ATP (3.5 mM). The Q10 of the pump activity was around 2.0 at the Ca2+ ion concentrations examined (less than 10(-6) M). 8. A numerical model based on the results obtained, together with some reasonable assumptions, suggested that both suppression of the Ca2+ pump and enhancement of the Ca2+ release contribute to the cooling contracture of caffeinized fibres.

The influence of free calcium on the maximum speed of shortening in skinned frog muscle fibres.

The influence of [Ca2+] on the maximum velocity of shortening (Vmax) was examined in mechanically skinned Rana pipiens and Rana temporaria fibres using improved force clamps and the slack test techniques. All measurements were made at 7.5 degrees C. At low relative loads (P/P0 less than 0.1), maximally activated R. pipiens fibres shortened more rapidly than did submaximally activated fibres. At higher relative loads, however, little difference in the speed of shortening was observed. Vmax (determined by the slack test) of R. pipiens fibres increased as the level of activation increased. Over sarcomere lengths 1.8-2.1 microns it was 2.28 muscle lengths/s (m.l./s) (S.E. of mean +/- 0.25, n = 5) at 20-35% activation, 2.89 m.l./s (+/- 0.22, n = 7) at 40-60% activation, and 4.18 m.l./s (+/- 0.25, n = 6) at 100% activation. At longer sarcomere lengths (2.2-2.6 microns), higher Vmax values were observed at all levels of activation, but the influence of Ca2+ on Vmax persisted. Vmax was 3.54 m.l./s (+/- 0.41, n = 4) at 20-30% activation and 5.15 m.l./s (+/- 0.22, n = 5) at 100% activation. In R. temporaria fibres, Vmax (determined by force clamps over sarcomere lengths 1.8-2.1 micron) also increased as the level of activation increased, from 3.47 m.l./s (+/- 0.06, n = 6) at 13-29% activation to 5.62 m.l./s (+/- 0.17, n = 6) at 100% activation. Vmax was also determined (using the slack test) in mechanically and chemically skinned rabbit soleus fibres. Vmax at 15 degrees C (1.05 m.l./s, +/- 0.11, n = 5) at full activation decreased by more than 3-fold as the level of activation was reduced to 10%. We conclude that the level of activation influences the Vmax of skinned skeletal muscle fibres. This has now been demonstrated in three different preparations and by a variety of techniques. This effect is most pronounced at low relative loads, and might not be observed if there are experimental limitations which prevent making velocity measurements at low relative loads.

The maximum speed of shortening in living and skinned frog muscle fibres.

This study was performed to determine whether Viso (the maximum speed of shortening extrapolated from force-velocity curves) equalled Vu (the unloaded speed of shortening determined by the slack test) in both living fibres from Rana temporaria and mechanically skinned fibres from Rana pipiens. In living fibres (R. temporaria) we obtained improved estimates of Viso by performing force clamps (isotonic) and length ramps (isovelocity) down to very low loads (0.005 isometric tension, P0). Force-velocity characteristics determined by force clamps and length ramps were the same. The hyperbolic Hill curves deviated from the force-velocity data at both high and low loads and underestimated Viso by varying degrees. A better estimate of Viso was obtained by linear extrapolation of data at loads from 0.005-0.02 P0 and the mean Viso at 7.5 degrees C was 4.08 muscle lengths/s +/- 0.11 (mean +/- S.E., n = 14). Improved estimates of Vu in living fibres were obtained by photographically calibrating the slack test. The mean Vu was 4.05 muscle lengths/s +/- 0.13 (mean +/- S.E., n = 14) and the intercept was 0.0156 fibre lengths (L0) +/- 0.0013 (mean +/- S.E., n = 14). The step-ramp photographic method, in which the motor speed is matched to Vu, was developed as an independent way to measure Vu in living fibres. Vu measured in this way agreed well with Vu measured by the slack test. In all living fibres, the improved estimates of Vu agreed well with the improved estimates of Viso. Vu/Viso = 0.99 +/- 0.01 (mean +/- S.E., n = 14). In mechanically skinned R. pipiens fibres, force clamps were performed down to loads of 0.01 mN. The force-velocity curve of the skinned fibres differed in shape from that of the living fibres. Although there was significant deviation from the Hill equation at low loads, the data at high loads were well fitted by the Hill curve. Viso determined by extrapolating the Hill equation to zero load was 5.87 muscle lengths/s +/- 0.38 (mean +/- S.E., n = 9) at 7.5 degrees C. In five fibres, the linear extrapolation of low loads (0.01-0.05 P0) showed that the Hill equation underestimated the true Viso by 6%. The slack test with mechanically skinned fibres was calibrated by taking a series of photographic exposures of the fibre at various times following each length step. Vu = 6.12 muscle lengths/s +/- 0.44 (mean +/- S.E., n = 10) and the intercept was 0.0585 L0 +/- 0.0069 (mean +/- S.E., n = 10).(ABSTRACT TRUNCATED AT 400 WORDS)

Contraction kinetics of intact and skinned frog muscle fibers and degree of activation. Effects of intracellular Ca2+ on unloaded shortening.

This study addresses a long-standing controversy on the effects of the degree of activation on cross-bridge kinetics in vivo, by utilizing isolated intact and skinned fiber preparations. Steady force levels ranging from 0.1 to 0.76 P0 were achieved at 0 degrees C with temperature-step stimulation of intact fibers by varying the amount of caffeine in the bathing medium. The speed of unloaded shortening (by slack test) was found to be practically constant, which suggests that intracellular Ca2+ in the intact preparation has relatively little effect on isotonic shortening. Along with the results on tetanically stimulated fibers (force, P0), we observed a minor but significant trend for the speed to decline with lowered force levels. This trend is explained by the presence of a constant internal load equaling approximately 1% P0. The effect of Ca2+ on the shortening behavior of skinned fibers was examined at 0 and 10 degrees C. At 0 degrees C, there was practically no effect of Ca2+ on the shortening response in slack tests. At 10 degrees C, there was also no Ca2+ effect during the first activation cycle, but in subsequent cycles the speed of shortening was reduced during partial activation, which indicates that there were permanent changes in the fiber properties under these experimental conditions. The latter result could be explained if the internal load had increased to approximately 5% P0 in the modified skinned fiber (compared with 1% P0 in intact fiber). These findings show that isotonic contraction of frog fibers is intrinsically unaffected by the variations in intracellular Ca2+ that modulated the force over a nearly complete range. The results provide support for the idea that Ca2+ influences the force development in vivo by on-off switching mechanisms.

Magnesium ion-dependent contraction of skinned frog muscle fibers in calcium-free solution

Skinned frog fibers were reversibly activated in Ca-free solutions containing 0 mM KCl, 23 microM free Mg, and having an ionic strength of approximately 50 mM. Contractile force was nearly maximal at 22 degrees - 25 degrees C and decreased at lower temperatures. Maximal force in Ca-free solution at 50 mM ionic strength was close to twice the calcium-activated force with pCa 5 and 190 mM ionic strength. The force in Ca-free solution could be reduced to zero by raising the concentration of free Mg from 23 microM to 1.0 mM at the same ionic strength (50 mM). On stretching the fiber from 2.0 to 3.2 micron the force decreased; this effect was similar to that seen with Ca-activated fiber and the data support the idea that Ca-free tension is made at the cross-bridge level. Isotonic contraction during Ca-free activation showed a velocity transient as in Ca-activated fiber at 190 mM ionic strength, but the transient in the present case was very much prolonged. This finding suggests that contraction mechanisms for force generation and for shortening are essentially the same in the two conditions, but that certain rate constants of cross-bridge turnover are slower for the Ca-free contraction. Also, the results indicate that, in low ionic strength, Ca binding to thin filaments is not essential for unmasking the cross-bridge attachment sites, which suggests that the steric blocking mechanism is modified under these conditions.

Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog.

Increasing temperature (4-22 degrees C) increases the Ca2+ concentration required for activation of mechanically skinned frog muscle fibers. The pCa required for 50% maximal force (pCa50) was inversely proportional to absolute temperature. Assuming that relative force is directly related to fractional occupancy of the Ca2+-binding sites on troponin that regulate force, the shift was consistent with a Gibbs free energy change of binding (delta G) of about -7.8 kcal/mol. This is close to the delta G for Ca2+ binding to the calcium-specific sites on troponin C reported by others. Decreasing Mg2+ from 1 mM to 60 microM shifts the force-pCa curves at either 4 or 22 degrees C to higher pCa, but the shift of pCa50 with temperature over this range (0.4 log units) was the same at low and high Mg2+. Maximal force increased with temperature for the entire range 4-22 degrees C with a Q10 of 1.41, and over the restricted range 4-15 degrees C with a Q10 of 1.20. From the dual effects of temperature on Ca2+ activation and maximal force, one would expect that force would respond differently to temperature change at high or low Ca2+. At high Ca2+, a temperature increase will lead to an increased force. However, at low to intermediate Ca2+ levels (below the intersection of the force-pCa curves for the initial and final temperatures), steady state force should decrease with increasing temperature. The inverse responses should occur with a decrease in temperature. These responses are observed when temperature is changed by rapid solution exchange.

Inhibition of force production in compressed skinned muscle fibers of the frog.

Calcium-activated force development in skinned frog muscle fibers is inhibited by osmotically compressing the fiber, probably owing to a decrease in spacing between the myofilaments. This inhibition depends upon sarcomere length in that fibers at long lengths must be compressed further than those at short lengths to achieve the same degree of inhibition. As a result, this length dependency of inhibition tends to compensate for the reduction of force due solely to the decrease in interfilament spacing which occurs with stretch in intact fibers.

Radial forces within muscle fibers in rigor.

Considering the widely accepted cross-bridge model of muscle contraction (Huxley. 1969. Science [Wash. D. C.]. 164:1356-1366), one would expect that attachment of angled cross-bridges would give rise to radial as well as longitudinal forces in the muscle fiber. These forces would tend, in most instances, to draw the myofilaments together and to cause the fiber to decrease in width. Using optical techniques, we have observed significant changes in the width of mechanically skinned frog muscle fibers when the fibers are put into rigor by deleting ATP from the bathing medium. Using a high molecular weight polymer polyvinylpyrrolidone (PVP-40; number average mol. wt. (Mn) = 40,000) in the bathing solution, we were able to estimate the magnitude of the radial forces by shrinking the relaxed fiber to the width observed with rigor induction. With rigor, fiber widths decreased up to approximately 10%, with shrinking being greater at shorter sarcomere spacing and at lower PVP concentrations. At higher PVP concentrations, some fibers actually swelled slightly. Radial pressures seen with rigor in 2 and 4% PVP ranged up to 8.9 x 10(3) N/m2. Upon rigor induction, fibers exerted a longitudinal force of approximately 1 x 10(5) N/m2 that was inhibited by high PVP concentrations (greater than or equal to 13%). In very high PVP concentrations (greater than or equal to 20%), fibers exerted an anomalous force, independent of ATP, which ranged up to 6 x 10(4) N/m2 at 60% PVP. Assuming that all the radial force is the result of cross-bridge attachment, we calculated that rigor cross-bridges exert a radial force of 0.2 x 1.2 x 10(-9) N per thick filament in sarcomeres near rest length. This force is of roughly the same order of magnitude as the longitudinal force per thick filament in rigor contraction or in maximal (calcium-activated) contraction of skinned fibers in ATP-containing solutions. Inasmuch as widths of fibers stretched well beyond overlap of thick and thin filaments decreased with rigor, other radially directed forces may be operating in parallel with cross-bridge forces.

Cross bridge slippage in skinned frog muscle fibres.

Mechanically skinned single fibres of the semitendinosus muscles of Rana esculenta were investigated at ca. 4 degrees C. The fibres were activated by a Ca2+ jump technique, which allowed the development of a steady isometric tension within several seconds of entering a calcium rich solution at 4 degrees C. Sequences of length changes of different duration and amplitude were applied to the fibre. It could be demonstrated that the fibre behaved as a Hookean spring in the case of small amplitude length changes (up to 0.5% L0, ramp duration 0.5 ms) and that a sequence of length changes induced reversible changes in fibre state. In contrast, large stretches (greater than 1% L0) induced a muscle "give" if the stretch were not immediately preceded by a release. The data was interpreted on the basis of a strain induced detachment of cross bridges in combination with a rapid reattachment of presumably the same cross bridges in a discharged position. The rates of strain induced detachment and reattachment depended on the stretch amplitude. At amplitudes exceeding 2% L0 the rates were estimated to be at least several thousands per second.

Chloride and osmotic contractures in skinned frog muscle fibers

Single contractures were elicited in segments of skinned frog muscle fibers when the segments were moved from relaxing-loading solutions to various test solutions. The effective test solutions produced an increase in the concentration of chloride ions in the myofilament space, [Cl] ms , and/or presumably caused the sarcoplasmic reticulum to undergo a change in volume. The contractures were quantified in terms of their maximum tension and time-integral. Two outer segments from each fiber underwent a contracture in a control solution (chloride ions were substituted for all of the methanesulfonate ions in the relaxing solution). The mean values of tension and area in the control contractures of each fiber were divided into the corresponding values from a test contracture obtained in the central segment of the same fiber. Test contractures obtained upon increasing [Cl] ms and increasing the product, [K] ms ×[Cl] ms , were compared to contractures that were obtained by increasing [Cl] ms while keeping [K] ms ×[Cl] ms constant. The former contractures were greater in magnitude for a given [Cl] ms . Whereas the former solutions may have caused an increase in the volume of the sarcoplasmic reticulum and altered the electrical potential across the membranes of the sarcoplasmic reticulum as well, only a change in potential was presumed to have occurred in the latter solutions. Other types of contractures were investigated to show that both swelling of the sarcoplasmic reticulum and changes in the electrical potential of its membranes can cause release of calcium ions and elicit contractures in skinned fibers.

Sarcomere length effects on the Sr 2+- and Ca 2+-activation curves in skinned frog muscle fibres

1. 1. A procedure recently described to produce rapid changes in [Ca 2+ and [Sr 2+ within the whole cross-section of skinned muscle preparations (Moisescu, D.G. (1976) Nature 262, 610–613, and Moisescu, D.G. and Thieleczek, R. (1978) J. Physiol. 275, 241–262) has enabled us to obtain whole Ca 2+- or Sr 2+-activation curves at different sacromere lengths with the same preparation. 2. 2. The maximal isometric force response was found to be very similar in Ca 2+- and Sr 2+-buffered solutions for otherwise identical conditions. 3. 3. The change in sarcomere length between approx. 2.2 and 2.6 μm reversibly shifted both the Ca 2+- and the Sr 2+-activation curves by approx. 0.1 log units towards lower concentrations of the activator, without affecting their shape. 4. However, the change in sarcomere length in the range above 2.6 μm did not have an effect upon the relative isometric force response-pCa (and -pSr) relationship. 5. 4. All the Ca 2+- and Sr 2+-activation curves present a similar steepness and indicate that the relative isometric force increases from approx. 10 to 90% if the concentration of the activator is increased 3-fold. 6. 5. The half time for force development in these experiments did not appear to be influenced by the length of the sarcomeres. 7. 6. A potentiometric method for determining the apparent affinity constants of Ca 2+, Mg 2+ and Sr 2+ to EGTA and ATP under various conditions is described.

Contraction transients of skinned muscle fibers: effects of calcium and ionic strength.

Calcium and ionic strength are both known to modify the force developed by skinned frog muscle fibers. To determine how these parameters affect the cross-bridge contraction mechanism, the isotonic velocity transients following step changes in load were studied in solutions in which calcium concentration and ionic strength were varied. Analysis of the motion showed that calcium has no effect on either the null time or the amplitude of the transients. In contrast, the transient amplitude was increased in high ionic strength and was suppressed in low ionic strength. These results are consistent with the idea that calcium affects force in skeletal muscle by modulating the number of force generators in a simple switchlike "on-off" manner and that the steady force at a given calcium level is proportional to cross-bridge number. On the other hand, the effect of ionic strength on force is associated with changes in the kinetic properties of the cross-bridge mechanism.

Calcium ionophores and tension production in skinned frog muscle fibers

Antibiotics, X-537A and A23187, were added in micromolar concentrations to selected bathing solutions of skinned frog muscle fibers, and they were shown to affect the production of tension in the skinned fibers. Segments of skinned fibers were bathed in a buffered calcium solution with a pCa near the threshold for contraction. When a segment was moved to a lightly buffered calcium solution with a pCa higher than the threshold for contraction but one containing either antibiotic, a transient contracture of the segment resulted. if the procedure was repeated in the same segment, no contracture was produced. The results are consistent with the idea that the antibiotics function as calcium ionophores in the membrane of the sarcoplasmic reticulum of skinned fibers.

Effect of calcium ions on rigor contractions in skinned frog muscle fibers

Rigor contractions were examined in skinned frog muscle fibers. The concentrations of calcium ions, pCa = 9.0−5.0, in the solutions which caused rigor were shown to affect the magnitude and time course of the contractions.

Calcium-activated tension of skinned muscle fibers of the frog. Dependence on magnesium adenosine triphosphate concentration.

The influence of MgATP on the Ga(++)-activated isometric tension of skinned frog muscle fibers was examined in solutions containing: Mg(++) = 5 mM, creatine phosphate (CP) = 14.5 mM, creatinephosphokinase (CPK) = 1 mg/ml, total EGTA = 7 mM, CaCl(2), KCl, imidazole >/= 20 mM so that ionic strength = 0.15, pH = 7.00, and MgATP = 2 mM, 0.1 mM, or 20 microM. CP and CPK were necessary for these experiments as determined experimentally by their effect on the tension-Ca(++) relation, which was saturated for CP >/= 14.5 mM. This was interpreted to mean that sufficient CP was present to effectively buffer MgATP intracellularly. Decreasing MgATP shifts the tension-pCa curve to higher pCa (-log Ca(++)) so that, for half-maximal tension: pCa(1/2) = 4.5 for MgATP = 2 mM, pCa(1/2) = 5.1 for MgATP = 0.1 mM, and pCa(1/2) = 5.8 for MgATP = 20 microM; maximum isometric tension is the same in all cases, however. If MgATP was decreased to 1 microM, tension at Ga(++) > 10(-8) M was 84% of the maximum Ca(-+)-activated tension in 2 mM MgATP and increased only slightly to 90% for pCa = 4.5. Weber (1970, In The Physiology and Biochemistry of Muscle as Food, Volume 2, E. J. Briskey, R. G. Cassens, and B. B. Marsh, University of Wisconsin Press, Madison, Wis.), using similar solutions, observed similar shifts in half-maximal calcium activation of rabbit myofibril ATPase rates. In explanation, Weber and Bremel (1971, In Contractility of Muscle Cells and Related Processes, R. J. Podolsky, editor, Prentice-Hall, Inc., Englewood Cliffs, N.J.; Bremel and Weber, 1972, Nat. New Biol., 238:97) have described a mechanism whereby, at low ATP, "rigor complexes" are formed between myosin and thin filament actin and, in turn, alter the calcium affinity of one class of the two Ca(++)-binding sites on troponin, so that the thin filament is "turned on" for contraction at lower Ca(++) levels. Tension data from skinned fibers substantially supports this hypothesis. A stability constant for CaEGTA of 2.62 x 10(10) M(-1) was determined, with the help of F. N. Briggs, in solutions similar to those used for skinned fibers and was the same for 100 and 300 mM KCl.