Ionic Strength and the Contraction Kinetics of Skinned Muscle Fibers

The influence of KCl concentration on the contraction kinetics of skinned frog muscle fibers at 5–7°C was studied at various calcium levels. The magnitude of the calcium-activated force decreased continuously as the KCl concentration of the bathing solution was increased from 0 to 280 mM. The shortening velocity at a given relative load was unaffected by the level of calcium activation at 140 mM KCl, as has been previously reported by Podolsky and Teichholz (1970. J. Physiol. [Lond.]. 211: 19), and was independent of ionic strength when the KCl concentration was increased from 140 to 280 mM. In contrast, the shortening velocity decreased as the KCl concentration was reduced below 140 mM; the decrease in velocity was enhanced when the fibers were only partially activated. In the low KCl range, the resting tension of the fibers increased after the first contraction cycle. The results suggest that in fibers activated at low ionic strength some of the cross bridges that are formed are abnormal in the sense that they retard shortening and persist in relaxing solution.     

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.     

Intrinsic shortening speed of temperature-jump-activated intact muscle fibers. Effects of varying osmotic pressure with sucrose and KCl

Effects of intracellular ionic strength on the isotonic contraction properties of both intact fibers and skinned fibers give insights into the cross-bridge mechanism, but presently there is fundamental disagreement in the results on the two fiber preparations. This paper, which studies the effects on contraction of varying the osmotic pressure of the bathing medium with impermeant and permeant solutes, explains the above controversy and establishes the physiological significance of the previous results on skinned fibers. Fast-twitch fibers, isolated singly from tibialis and semitendinosus muscles of frogs, were activated by a temperature-jump technique in hyperosmotic solutions with either 100 or 150 mM sucrose (impermeant), or 50 or 75 mM KCl (permeant). Intracellular ionic strength was expected to rise in these solutions from the standard value of approximately 190 to 265 mM. Cell volume and the speed of unloaded shortening both decreased with sucrose and were constant with KCl. On the other hand, isometric force decreased equally with equiosmolar addition of either solute; this is additional evidence that contractile force decreases with ionic strength and is independent of fiber volume. Therefore, for the main cross-bridges, force per bridge is constant with changes in the lateral separation between the myofilaments. The next finding, that at a fixed cell volume the contraction speed is constant with KCl, provides clear evidence in intact fibers that the intrinsic speed of shortening is insensitive to increased ionic strength. The data with KCl are in agreement with the results on skinned fibers. The results suggest that in the cross-bridge kinetics in vivo the rate-limiting step is different for force than that for shortening. On the other hand, the decrease in speed with sucrose is associated with the shrinkage in cell volume, and is explained by the possibility of an increased internal load. A major fraction of the internal load may arise from unusual interactions between the sliding filaments; these interactions are enhanced in the fibers compressed with sucrose, but this does not affect the intrinsic kinetics of the main cross-bridges.     

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 partial activation on force-velocity properties of frog skinned muscle fibers in millimolar magnesium ion

Segments of briefly glycerinated muscle fibers from Rana pipiens were activated rapidly by a brief exposure to 2.5 mM free calcium followed by a solution containing calcium buffered with EGTA to produce the desired level of force. Steps to isotonic loads were made using a servomotor, usually 3-5 s after the onset of activation. The relative isotonic forces (P/P0) and velocities from contractions obtained under similar circumstances were grouped together and fitted with hyperbolic functions. Under the condition of 6 mM MgCl2 and 5 mM ATP, there was no significant difference in the relative force-velocity relations obtained at full activation compared with those obtained at partial activation when developed force was approximately 40% of its full value. Control experiments showed that a variety of factors did not alter either the relative force-velocity relations or the finding that partial activation did not change these properties. The factors investigated included the decline in force that occurs with each successive contraction of skinned fibers, the segment length (over a range of 1-3 mm), the sarcomere length (over a range of 1.9-2.2 microns), the magnesium ion concentration (26 microM and 1.4 mM were tested), the ATP concentration, the presence of free calcium, and the age of the preparation (up to 30 h). Attempts to repeat earlier experiments by others showing a dependence of shortening velocity on activation were unsuccessful because the low ionic strength used in those experiments caused the fibers to break after a few contractions. The main conclusion, that the shortening velocity is independent of the level of activation, is consistent with the hypothesis that the cross-bridges act independently and that activating calcium acts only as an all-or-none switch for individual cross-bridge attachment sites, and does not otherwise influence the kinetics of cross-bridge movement.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Isometric force development, isotonic shortening, and elasticity measurements from Ca(2+)-activated ventricular muscle of the guinea pig

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.     

Calcium sensitivity of vertebrate skeletal muscle myosin

The calcium sensitivity of vertebrate skeletal muscle myosin has been investigated. Adenosinetriphosphatase (ATPase) activity was assayed in a reconstituted system composed of either purified rabbit myosin plus actin or myosin plus actin, tropomyosin, and troponin. The calcium sensitivity of actomyosin Mg-ATPase activity was found to be directly affected by the ionic strength of the assay medium. Actomyosin assayed at approximately physiological ionic strength (120 mM KCl) demonstrated calcium sensitivity which varied between 6 and 52%, depending on the myosin preparation and the age of the myosin. Mg-ATPase activity was increased when calcium was present in the assay medium at physiological ionic strength. Conversely, actomyosin Mg-ATPase activity assayed at a lower ionic strength (15 mM KCl) was inhibited by addition of calcium. Addition of tropomyosin and troponin to the assay increased the calcium sensitivity of the system at the physiological ionic strength still further (up to 99% calcium sensitivity) and conferred calcium sensitivity on the system at the lower ionic strength (greater than 90% calcium sensitivity). A correlation also existed between myosin's calcium sensitivity and the phosphorylated state of light chain 2.     

Swelling of Skinned Muscle Fibers of the Frog: Experimental Observations

Frog skeletal muscle fibers, mechanically skinned under water-saturated silicone oil, swell upon transfer to aqueous relaxing medium (60 mM KCl; 3 mM MgCl₂; 3 mM ATP; 4 mM EGTA; 20 mM Tris maleate; pH = 7.0; ionic strength 0.15 M). Their cross-sectional areas, estimated with an elliptical approximation, increase 2.32-fold (±0.54 SD). Sarcomere spacing is unaffected by this swelling. Addition of 200 mM sucrose to relaxing medium had no effect on fiber dimensions, whereas decreasing pH to 5.0 caused fibers to shrink nearly to their original (oil) size. Decreasing MgCl₂ to 0.3 mM caused fibers to swell 10%, and increasing MgCl₂ to 9 mM led to an 8% shrinkage. Increasing ionic strength to 0.29 M with KCl caused a 26% increase in cross-sectional area; decreasing ionic strength to 0.09 M had no effect. Swelling pressure was estimated with long-chain polymers, which are probably excluded from the myofilament lattice. Shrinkage in dextran T10 (number average mol wt 6,200) was transient, indicating that this polymer may penetrate into the fibers. Shrinkage in dextran T40 (number average mol wt 28,000), polyvinylpyrrolidone (PVP) K30 (number average mol wt 40,000) and dextran T70 (number average mol wt 40,300) was not transient, indicating exclusion. Maximal calcium-activated tension is decreased by 21% in PVP solutions and by 31% in dextran T40 solutions. Fibers were shrunk to their original size with 8 × 10^-2 g/cm³ PVP K30, a concentration which, from osmometric data, corresponds to an osmotic pressure (II/RT) of 10.5 mM. As discussed in the text, we consider this our best estimate of the swelling pressure. We find that increasing ionic strength to 0.39 M with KCl decreases swelling pressure slightly, whereas decreasing ionic strength to 0.09 M has no effect. We feel these data are consistent with the idea that swelling arises from the negatively charged nature of the myofilaments, from either mutual filamentary repulsion or a Donnan-osmotic mechanism.     

Cross-linkings between spectrin and band 3 in human erythrocyte membranes

A specific structural association between spectrin component 1 and band 3 in human erythrocyte membrane has been demonstrated by covalent cross-linkings, specific labeling, and the technique of two-dimensional gel electrophoresis. A complex of 330,000 daltons, representing 1 + 3, was produced in mildly oxidized membranes at physiologic pH and isotonic conditions but not at hypotonic conditions (< 10 mM KCl or NaCl). The yield of this complex decreased dramatically as the monovalent cation concentration decreased from 90 mM to 30 mM. The presence of Mg⁺⁺ or Ca⁺⁺ (2 mM) at low ionic strength promoted 1 + 3 cross-linking in an amount similar to that produced at isotonic conditions. The specific segment of band 3 involved in the cross-linking was also investigated by means of chymotrypsin digestion of band 3 in the intact red cells. The results showed the cross-links between spectrin component 1 and the 55,000-dalton fragment of band 3 at physiologic pH and isotonic conditions. This is consistent with the idea that band 3 is anchored on or contacted with the submembrane meshwork at the cytoplasmic membrane surface.     

Overrelaxation of muscle models in media with high concentrations of calcium ions and exposed to MgATP in "contraction-inducing" concentrations]

Cross-striation pattern and sarcomere length in isolated myofibrils (both glycerinated and fresh) as well as isometric tension of glycerinated fibers of rabbit m. psoas are unaffected by an evaluation in ionic strength of CaCl2 up to 0.2 in the absence of ATP. An addition of MgATP (1 to 3mM) to the Ca2+ media induces the changes which have been shown to be characteristic of overrelaxation [1, 2]: A band shortening occurs followed by a complete plastification of the fibres. A tentative mechanism of the process is discussed in terms of spontaneous rearrangement of calcium myosinate packing in thick filaments that follows disrupting of rigor crossbridges with thin filaments under the action of ATP. Released calcium myosinate heads fail to form "active" bridges with actin; thick filaments undergo a conformational change resulted in their shattening due to increase in the equilibrium region of LMM tail overlap. The effects do not depend on ionic strength only: on replacing CaCl2 by KCl at equal ionic strength 0.2, an addition of ATP induces normal contraction instead of overrelaxation. A possibility is discussed that in a living muscle overrelaxation could provide a siding to prevent damage in case of emergency.     

Effects of ionic strength on calcium binding to rabbit skeletal myofibrils,thin filaments and myosin

Calcium binding by rabbit skeletal myosin, thin filaments and myofibrils was measured in solutions with and without 2 mM MgATP and with ionic strengths adjusted with KCl to 0.05, 0.10 and 0.14 M. Free Mg²⁺ was held constant at 1 mM, pH at 7.0 and temperature at 25 °C. In the presence of MgATP, the relation between free Ca²⁺ and myofibrillar bound calcium shifted to the left as ionic strength was decreased from 0.14 to 0.05 M. In the absence of MgATP, myofibrillar calcium binding was enhanced over a wide range of free Ca²⁺ concentration, but calcium binding was no longer a function of ionic strength. Similarly, calcium binding by thin filaments and myosin was unaffected by changes in ionic strength from 0.05 to 0.14 M. In view of evidence that cross-bridge connections between thick and thin filaments increase as ionic strength decreases, our results suggest that these connections enhance myofibrillar calcium binding. These results thus confirm previous data of Bremel and Weber (Bremel, R. D. and Weber, A. (1972) Nature New Biol. 238, 97–101) who first showed that nucleotide-free cross-bridge connections enhance thin filament calcium binding.     

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.     

Tension in Skinned Frog Muscle Fibers in Solutions of Varying Ionic Strength and Neutral Salt Composition

The maximal calcium-activated isometric tension produced by a skinned frog single muscle fiber falls off as the ionic strength of the solution bathing this fiber is elevated declining to zero near 0.5 M as the ionic strength is varied using KCl. When other neutral salts are used, the tension always declines at high ionic strength, but there is some difference between the various neutral salts used. The anions and cations can be ordered in terms of their ability to inhibit the maximal calcium-activated tension. The order of increasing inhibition of tension (decreasing tension) at high ionic strength for anions is propionate^- SO₄^-- < Cl^- < Br^-. The order of increasing inhibition of calcium-activated tension for cations is K⁺ Na⁺ TMA⁺ < TEA⁺ < TPrA⁺ < TBuA⁺. The decline of maximal calcium-activated isometric tension with elevated salt concentration (ionic strength) can quantitatively explain the decline of isometric tetanic tension of a frog muscle fiber bathed in a hypertonic solution if one assumes that the internal ionic strength of a muscle fiber in normal Ringer's solution is 0.14–0.17 M. There is an increase in the base-line tension of a skinned muscle fiber bathed in a relaxing solution (no added calcium and 3 mM EGTA) of low ionic strength. This tension, which has no correlate in the intact fiber in hypotonic solutions, appears to be a noncalcium-activated tension and correlates more with a declining ionic strength than with small changes in [MgATP], [Mg], pH buffer, or [EGTA]. It is dependent upon the specific neutral salts used with cations being ordered in increasing inhibition of this noncalcium-activated tension (decreasing tension) as TPrA⁺ < TMA⁺ < K⁺ Na⁺. Measurements of potentials inside these skinned muscle fibers bathed in relaxing solutions produced occasional small positive values (<6 mV) which were not significantly different from zero.     

The effects of ADP and phosphate on the contraction of muscle fibers.

The products of MgATP hydrolysis bind to the nucleotide site of myosin and thus may be expected to inhibit the contraction of muscle fibers. We measured the effects of phosphate and MgADP on the isometric tensions and isotonic contraction velocities of glycerinated rabbit psoas muscle at 10 degrees C. Addition of phosphate decreased isometric force but did not affect the maximum velocity of shortening. To characterize the effects of ADP on fiber contractions, force-velocity curves were measured for fibers bathed in media containing various concentrations of MgATP (1.5-4 mM) and various concentrations of MgADP (1-4 mM). As the [MgADP]/[MgATP] ratio in the fiber increases, the maximum velocity achieved by the fiber decreases while the isometric tension increases. The inhibition of fiber velocities and the potentiation of fiber tension by MgADP is not altered by the presence of 12 mM phosphate. The concentration of both MgADP and MgATP within the fiber was calculated from the diffusion coefficient for nucleotides within the fiber, and the rate of MgADP production within the fiber. Using the calculated values for the nucleotide concentration inside the fiber, observed values of the maximum contraction velocity could be described, within experimental accuracy, by a model in which MgADP competed with MgATP and inhibited fiber velocity with an effective Ki of 0.2-0.3 mM. The average MgADP level generated by the fiber ATPase activity within the fiber was approximately 0.9 mM. In fatigued fibers MgADP and phosphate levels are known to be elevated, and tension and the maximum velocity of contraction are depressed. The results obtained here suggest that levels of MgADP in fatigued fibers play no role in these decreases in function, but the elevation of both phosphate and H+ is sufficient to account for much of the decrease in tension.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

The effect of mepyramine on the development of morphine tolerance and physical dependence in mice.

Latency relaxation (LR) as well as resting tension and twitch tension of frog toe muscles are studied in an isotonic solution (= 1 T) and in solutions made hypotonic by leaving out the appropriate amounts of NaCl and KCl (0.54 T and 0.76 T). In hypotonic solutions there is an increase in peak twitch tension as well as a decrease in the depth of the LR: the resting tension is increased at sarcomere lengths which are greater than 2.8 μm and is decreased at sarcomere lengths which are less than this value. The behaviour of twitch tension is discussed with respect to the influence of the sarcoplasmic ionic strength on the interaction between the contractile filaments. Concerning the decrease in both the LR and the resting tension, it is assumed that these effects are induced osmotically, the tension of the membranes of the longitudinal sarcoplasmic reticulum being the particular parameter which is influenced.     

The ADP release step of the smooth muscle cross-bridge cycle is not directly associated with force generation.

When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a approximately 3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748-751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15 mM EDTA. The addition of 2 mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200 mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.     

High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state

The effects of varying pH and ionic strength on the force-velocity relations and tension transients of skinned rabbit skeletal muscle were studied at 1-2 degrees C. Both decreasing pH from 7.35 to 6.35 and raising ionic strength from 125 to 360 mM reduced isometric force by about half and decreased sarcomere stiffness by about one-fourth, so that the stiffness/force ratio was increased by half. Lowering pH also decreased maximum shortening velocity by approximately 29%, while increasing ionic strength had little effect on velocity. These effects on velocity were correlated with asymmetrical effects on stiffness. The increase in the stiffness/force ratio with both interventions was manifest as a greater relative force change associated with a sarcomere length step. This force difference persisted for a variable time after the step. At the high ionic strength the force difference was long- lasting after stretches but relaxed quickly after releases, suggesting that the structures responsible would not impose much resistance to steady-state shortening. The opposite was found in the low pH experiments. The force difference relaxed quickly after stretches but persisted for a long time after releases. Furthermore, this force difference reached a constant value of approximately 8% of isometric force with intermediate sizes of release, and was not increased with larger releases. This value was almost identical to the value of an internal load that would be sufficient to account for the reduction in maximum velocity seen at the low pH. The results are interpreted as showing that both low pH and high ionic strength inhibit the movement of crossbridges into the force-generating parts of their cycle after they have attached to the actin filaments, with very few other effects on the cycle. The two interventions are different, however, in that detained bridges can be detached readily by shortening when the detention is caused by high ionic strength but not when it is caused by low pH.     

Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.