Effect of strain on actomyosin kinetics in isometric muscle fibers

Investigations were conducted into the biochemical and mechanical states of cross-bridges during isometric muscle contraction. Rapid length steps (3 or 6 nm hs(-1)) were applied to rabbit psoas fibers, permeabilized and isometric, at either 12 degrees C or 20 degrees C. Fibers were activated by photolysis of P(3)-1-(2-nitrophenyl)-ethyl ester of ATP infused into rigor fibers at saturating Ca(2+). Sarcomere length, tension, and phosphate release were recorded-the latter using the MDCC-PBP fluorescent probe. A reduction in strain, induced by a rapid release step, produced a short-lived acceleration of phosphate release. Rates of the phosphate transient and that of phases 3 and 4 of tension recovery were unaffected by step size but were elevated at higher temperatures. In contrast the amplitude of the phosphate transient was smaller at 20 degrees C than 12 degrees C. The presence of 0.5 or 1.0 mM added ADP during a release step reduced both the rate of tension recovery and the poststep isometric tension. A kinetic scheme is presented to simulate the observed data and to precisely determine the rate constants for the elementary steps of the ATPase cycle.     

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.     

The Apparent Rates of Crossbridge Attachment and Detachment Estimated from Atpase Activity in Insect Flight Muscle

The ATPase activity of single fibers of small fiber bundles (one to three fibers) of insect flight muscle was measured when fibers were repetitively released and restretched by 1.5% of their initial length. The ATPase activity increased with increasing duration of release-restretch pulses applied at a constant repetition frequency, reaching a maximum at a duration of ~20 ms. For a given duration, the average ATPase activity also increased with increasing frequency of applied length changes and reached a maximum (200% of the isometric ATPase) at a frequency of ~50 Hz. The data could be fitted to a two-state model in which the apparent rate of crossbridge detachment is enhanced when the crossbridges are mechanically released. Estimates of the apparent rates of attachment and detachment in the isometrically contracting state and of the enhanced detachment rate of unloaded crossbridges were derived from fits to the two-state model. After short pulses of releasing and restretching the fiber the force was low and increased after the restretch in a roughly exponential manner to the initial level. The rate at which force increased after a release-restretch pulse was similar to the sum of the apparent attachment and detachment rates for the isometrically contracting muscle derived from the ATPase activity measurements.     

Adenosine triphosphate hydrolysis by purified rubisco activase

Activation of ribulose bisphosphate carboxylase/oxygenase (rubisco) in vivo is mediated by a specific protein, rubisco activase. In vitro, activation of rubisco by rubisco activase is dependent on ATP and is inhibited by ADP. Purified rubisco activase hydrolyzed ATP with a specific activity of 1.5 mumol min-1 mg-1 protein, releasing approximately stoichiometric amounts of ADP and Pi. Hydrolysis was highly specific for ATP-Mg and had a broad pH optimum, with maximum activity at pH 8.0-8.5. ATPase activity was inhibited by ADP but not by molybdate, vanadate, azide, nitrate, or fluoride. Addition of rubisco in either the inactive or activated form had no significant effect on ATPase activity. Incubation of rubisco activase in the absence of ATP resulted in loss of both ATPase and rubisco activation activities. Both activities were also heat labile, with 50% loss in activity after 5 min at 38 degrees C and complete inhibition following treatment at 43 degrees C. Both activities showed a sigmoidal response to ATP concentration, with half-maximal activity at 0.053 mM ATP. Rubisco activation activity was dependent on the concentrations of both ATP and ADP. The results suggest that ATPase activity is an intrinsic property of rubisco activase.     

Probing actomyosin interactions with 2,4-dinitrophenol

Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.     

A non-cross-bridge stiffness in activated frog muscle fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14 degrees C at approximately 2.1 microm sarcomere length on fibers bathed in Ringer's solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed "static tension," and the ratio between the force and the accompanying sarcomere length change was termed "static stiffness." The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1-2.8 microm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.     

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.     

Temperature dependence of mammalian muscle contractions and ATPase activities

Isolated rat and mouse extensor digitorum longus (EDL) and soleus muscles were studied under isometric and isotonic conditions at temperatures from approximately 8 degrees -38 degrees C. The rate constant for the exponential rise of tension during an isometric tetanus had a Q10 of approximately 2.5 for all muscles (corresponding to an enthalpy of activation, delta H = 66 kJ/mol, if the rate was determined by a single chemical reaction). The half-contraction time, contraction time, and maximum rate of rise for tension in an isometric twitch and the maximum shortening velocity in an isotonic contraction all had a similar temperature dependence (i.e., delta H approximately 66 kJ/mol). The Mg++ ATPase rates of myofibrils prepared from rat EDL and soleus muscles had a steeper temperature dependence (delta H = 130 kJ/mol), but absolute rates at 20 degrees C were lower than the rate of rise of tension. This suggests that the Mg++ ATPase cycle rate is not limiting for force generation. A substantial fraction of cross-bridges may exist in a resting state that converts to the force-producing state at a rate faster than required to complete the cycle and repopulate the resting state. The temperature dependence for the rate constant of the exponential decay of tension during an isometric twitch or short tetanus (and the half-fall time of a twitch) had a break point at approximately 20 degrees C, with apparent enthalpy values of delta H = 117 kJ/mol below 20 degrees C and delta H = 70 kJ/mol above 20 degrees C. The break point and the values of delta H at high and low temperatures agree closely with published values for the delta H of the sarcoplasmic reticulum (SR) Ca++ ATPase. Thus, the temperature dependence for the relaxation rate of a twitch or a short tetanus is consistent with that for the reabsorption rate of Ca++ into the SR.     

Effects of pH on myofibrillar ATPase activity in fast and slow skeletal muscle fibers of the rabbit.

In permeabilized single fibers of fast (psoas) and slow (soleus) muscle from the rabbit, the effect of pH on isometric myofibrillar ATPase activity and force was studied at 15 degrees C, in the pH range 6.4-7.9. ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH, present in the bathing solution. NADH absorbance at 340 nm was determined inside a measuring chamber. To measure ATP turnover in single soleus fibers accurately, a new measuring chamber (volume 4 microliters) was developed that produced a sensitivity approximately 8 times higher than the system previously used. Under control conditions (pH 7.3), the isometric force was 136 and 115 kN/m2 and the ATP turnover was 0.43 and 0.056 mmol per liter fiber volume per second in psoas and soleus fibers, respectively. Over the pH range studied, isometric force increased monotonically by a factor 1.7 for psoas and 1.2 for soleus fibers. In psoas the isometric ATPase activity remained constant, whereas in soleus it slightly decreased with increasing pH. The pH dependency of relative tension cost (isometric ATPase activity divided by force) was practically identical for psoas and soleus fibers. In both cases it decreased by about a factor 0.57 as pH increased from 6.4 to 7.9. The implications of these findings are discussed in terms of cross-bridge kinetics. For both fiber types, estimates of the reaction rates and the distribution of cross-bridges and of their pH dependencies were obtained. A remarkable similarity was found between fast- and slow-twitch fibers in the effects of pH on the reaction rate constants.     

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.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers.

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca2+ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca2+ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechano-chemical cycle are Ca2+ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.     

Troponin I serines 43/45 and regulation of cardiac myofilament function

We studied Ca(2+) dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.     

Temperature dependence of nucleotide association and kinetic characterization of myo1b

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 degrees C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide-inaccessible state to a nucleotide-accessible state (k(alpha)). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2-3-fold greater than other characterized myosins (e.g., myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (Vmax and K(ATPase)). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to k(alpha) suggesting that a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.     

Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1

We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V(max) and K(actin) of the actin-activated Mg(2+) ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 microM, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 microm s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 microm s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V(max) value of the actin-activated Mg(2+) ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca²⁺ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca²⁺ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechanochemical cycle are Ca²⁺ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Strain sensitivity and turnover rate of low force cross-bridges in contracting skeletal muscle fibers in the presence of phosphate.

Inorganic phosphate (Pi) decreases the isometric tension of skinned skeletal muscle fibers, presumably by increasing the relative fraction of a low force quaternary complex of actin, myosin, ADP, and Pi (A.M.ADP.Pi). At the same time, Pi gives rise to a fast relaxing mechanical component as detected by oscillations at 500 Hz. To characterize the dynamic properties of this A.M.ADP.Pi complex, the effect of Pi on the tension response to stretch was investigated with rabbit psoas fibers. A ramp stretch applied in the presence of 20 mM Pi increased tension more than in the control solution (0 mM Pi) but reduced the fast relaxing component to the control level. Thus, a stretch seems to convert the low force, fast relaxing A.M.ADP.Pi complex to a high force, slow relaxing form. However, the Pi-induced enhancement of the tension response was not observed until the fibers were stretched more than 0.4% of their length, suggesting that a critical cross-bridge extension of approximately 4 nm is required for this conversion. The rate constant of the attachment/detachment of this low force complex was estimated from the velocity dependence of the enhancement. It was approximately 10 s-1, in marked contrast to the A.M.ADP.Pi complex under low salt, relaxed conditions (approximately 10,000 s-1). The enhancement of the tension response was not observed when isometric tension was reduced by lowering free calcium, implying that calcium and Pi affect different steps in the actomyosin ATPase cycle during contraction.     

Reptilian skeletal muscle: contractile properties of identified, single fast-twitch and slow fibers from the lizard Dipsosaurus dorsalis

Contractile properties and innervation patterns were determined in identified single fibers from the iliofibularis muscle of the desert iguana, Dipsosaurus dorsalis. Single fibers from both the red and white regions of the iliofibularis muscle were dissected along their length under oil and a portion was mounted on transducers for determination of maximum isometric tension (Po) and unloaded shortening velocity (Vmax) using the slack test method. Fibers were chemically skinned and activated by high Ca++. The remaining portion of the muscle fiber was mounted on a glass slide and histochemically treated to demonstrate myosin ATPase activity. Fibers studied functionally could therefore be classified as fast or slow according to their myosin ATPase activity, and they could also be classified metabolically according to the region of the muscle from which they were dissected. Fast-twitch glycolytic (FG) fibers from the white region and fast-twitch oxidative, glycolytic (FOG) and slow fibers from the red region had shortening velocities at 25 degrees C of 7.5, 4.4, and 1.5 l X s-1, respectively. Po did not differ in the three fiber types, averaging 279 kN X m-2. In a second experiment, 10 microns sections were examined every 30 microns through the proximal-most 7.5 mm of the iliofibularis muscle for motor endplates. Sections were stained to demonstrate regions of acetylcholinesterase activity. Fibers with visible endplates were classified in serial sections by histochemical treatment for myosin ATPase and succinic dehydrogenase. All slow fibers examined (n = 22) exhibited multiple endplates, averaging one every 725 microns.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of sarcomere length and stretching on the rate of ATP splitting in glycerinated rabbit psoas muscle fibers.

The effect of sarcomere length and stretching on the tension and the rate of ATP splitting was studied using small fiber bundles from glycerinated rabbit psoas muscle. The rate of ATP slitting was determined by measuring ADP production, while the tension development in response to a contracting solution (at pCa 5.3) was recorded in the same preparation. The isometric tension developed by the preparation decreased when the sarcomere length was increased. The decrease of tension development was accompanied by a decrease in the rate of ATP splitting. If a preparation exerting steady isometric tension was stretched by 5--10% at a velocity of 0.1 mm/s, the rate of ATP splitting was increased after stretching, while the steady isometric tension attained after stretching was also higher than the initial value. The extent of the excess ATP splitting caused by stretching decreased with increasing sarcomere length. These results suggest that the rate of the interaction cycle between actin and myosin molecules may increase as a result of stretching.