1. The rate of appearance of inorganic phosphate (Pé) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P 3 -1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca¥. Pé appearance was monitored from the fluorescence signal of a Pé-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca¥ at 20°C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s¢ in the first turnover based on a myosin subfragment 1 concentration of 150 ìÒ. This was calculated from a linear regression of the fluorescence signal reporting 20-150 ìÒ of Pé release. Tension was at 67% of its isometric level by the time 150 ìÒ Pé was released. ATPase activities were 36 and 31 s¢ for Pé released in the ranges of 150-300 ìÒ and 300-450 ìÒ, respectively. The ATPase activity had a QÔÑ value of 2·9 based on measurements at 5, 12 and 20°C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12·7 s¢ at 20°C.The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s¢. 4. On photolysis of NPE-caged ATP in the absence of Ca¥ the ATPase activity was 0·11 s¢ at 20°C with no discernible rapid transient phase of Pé release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca¥ was also measured on activation from the relaxed state by photolytic release of Ca¥ from a caged Ca¥ compound, nitrophenyl-EGTA. At 5°C the ATPase rate was 5·8 and 4·0 s¢ in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pé on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0·5 mÒ) decreased the initial ATPase rate by 23%. Pé (10 mÒ) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5°C in the presence of Ca¥ the ATPase activity was biphasic being 15·0 s¢ during the first turnover (based on 180 ìÒ myosin subfragment 1). Tension was 74% of its isometric level by the time 180 ìÒ Pé was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometr...

The mechanical efficiency of muscle contraction is the ratio of work performed to the chemical energy produced by the hydrolysis of ATP. Chemical energy which is not converted into work or absorbed by the reaction is lost as heat. The efficiency of contraction is zero when no work is produced, either because the muscle does not shorten, i.e. during isometric contractions, or when the force produced by the muscle is zero, as is the case when the muscle is allowed to shorten under zero load. In the latter case, the shortening velocity is the maximum which the muscle can achieve. It is also necessary to consider the internal work which does not translate into macroscopic movement, but which may be relevant to considerations of efficiency. Here we have precisely determined the work resulting from the actomyosin ATPase activity, without interference from the effects of tendon elasticity and of ATP hydrolysis due to activation of the muscle machinery, namely calcium release and re-uptake by the sarcoplasmic reticulum. This was achieved by using segments of permeabilized muscle fibres obtained from the rabbit psoas muscle and initiating contraction by the photolytic release of ATP from the PÅ-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP; Ferenczi et al. 1984) in the presence of a saturating concentration of calcium (32 ìÒ). The ability of the muscle fibres to perform work was measured by recording the force response during a period of applied constant velocity shortening. The ends of such fibre segments may be damaged at their point of attachment to the apparatus, resulting in local stretching during force development. So we measured the sarcomere length in the segment during contraction and shortening, thus providing a direct measurement of the shortening velocity of the sarcomeres. The other aspect of efficiency calculations is the determination of chemical energy utilized. For this, we used a fluorescence assay which is sensitive to the amount of inorganic phosphate (Pé) released in the muscle fibre by the hydrolysis of ATP (He et al. 1997 1. The relationship between mechanical power output and the rate of ATP hydrolysis was investigated in segments of permeabilized fibres isolated from rabbit psoas muscle. 2. Contractions were elicited at 12°C by photolytic release of ATP from the PÅ-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP). Inorganic phosphate (Pé) release was measured by a fluorescence method using a coumarin-labelled phosphate binding protein.Force and sarcomere length were also monitored. 3. ATPase activity was determined from the rate of appearance of Pé during each phase of contraction. The ATPase rate was 10·3 s¢ immediately following release of ATP and 5·1 s¢ during the isometric phase prior to the applied shortening. It rose hyperbolically with shortening velocity, reaching 18·5 s¢ at a maximal shortening velocity > 1 ML s¢ (muscle lengths s¢). 4. Sarcomeres shortened at 0·09 ML s¢ immediately following the photolytic release of ATP and at 0·04 ML s¢ prior to the period of applied shorteni...

Energy turnover was measured during isometric contractions of intact and Triton-permeabilized white fibres from dogfish (Scyliorhinus canicula) at 12• C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (P i ) release by permeabilized fibres was recorded using the fluorescent protein MDCC-PBP, N-(2-[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3 carboxamide phosphate binding protein. The steady-state ADP release rate was measured using a linked enzyme assay. The rates decreased five-fold during contraction in both intact and permeabilized fibres. In intact fibres the rate of heat + work output by actomyosin decreased from 134 ± s.e.m. 28 µW mg −1 (n = 17) at 0.055 s to 42% of this value at 0.25 s, and to 20% at 3.5 s. The force remained constant between 0.25 and 3.5 s. Similarly in permeabilized fibres the P i release rate decreased from 5.00 ± 0.39 mmol l −1 s −1 at 0.055 s to 39% of this value at 0.25 s and to 19% at 0.5 s. The steady-state ADP release rate at 15 s was 21% of the P i rate at 0.055 s. Using a single set of rate constants, the time courses of force, heat + work and P i release were described by an actomyosin model that took account of the transition from the initial state (rest or rigor) to the contracting state, shortening and the consequent work against series elasticity, and reaction heats. The model suggests that increasing P i concentration slows the cycle in intact fibres, and that changes in ATP and ADP slow the cycle in permeabilized fibres. The rate of energy output measured as heat and work decreases substantially during an isometric tetanic contraction of skeletal muscle (Abbott, 1951;Curtin & Woledge, 1979 and references therein). This energy output comes principally from the ATPase activity of the actomyosin system and that of the sarcoplasmic reticulum Ca 2+ pump, and to a lesser extent from other energy-producing processes. Measurements of P i release by permeabilized muscle fibres have shown that the rate of the P i release step in the actomyosin cycle decreases considerably during the first few turnovers (He et al. 1998b and references therein). The ADP release has been measured much later in an isometric contraction, while force is maintained at the plateau level (Hilber et al. 2001); the rate is constant in this period, but much lower than that measured at the start of activation. What is the time course of this reduction in the rate of the actomyosin cycle during an isometric contraction? Does this reduction match the change in the rate of energy release as heat + work by intact fibres?To answer these questions we have made parallel experiments using intact and permeabilized muscle fibres. White fibres from dogfish were used because both series of experiments could be made under similar conditions, including temperature (12• C). The fish were acclimated to 12• C, which is within the normal range for the natural habitat of the dogfish. Another reason for using dogfish muscle was that f...

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The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.