ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay

Abstract

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...

Muscle Contraction

“…The Ql0 of 3·9 for the ATPase rate is higher than previously reported for fibres (e.g. 2'9, He et al 1997) but lower than that of the actin-activated ATPase of myosin subfragment-l in solution (5'9 from 5-15 °C and 4·3 from 15-25 °C, Brenner & Eisenberg, 1986). The QlO of the fibre ATPase rate was approximately constant in the range 5-25 °C, in contrast with the QlO for force, which was higher at lower temperature.…”

Trading force for speed: Why superfast crossbridge kinetics leads to superlow forces

“…In this case the cycling rate (i.e., ATPase rate) for the active head, and thus both f and g, would double. Third, crossbridge kinetics may be substantially faster during the first few crossbridge cycles after activation (i.e., during the k develop measurement) than during steady contraction (42). Lastly, crossbridge attachment may be reversible with a rate constant of f Ϫ (24,43).…”

High mechanical efficiency of the cross-bridge powerstroke in skeletal muscle

“…Due to the limited amount of ATP in the fibre and the low temperature at which the present experiments were done, the maximum power output of the fibres in the present study (0.6 W l -1 ) was more than one order of magnitude smaller than the value obtained from Ps Pi rabbit psoas fibres (28 W l -1 at 12°C) (He et al, 1997) even when the high Q10 value (>5) (He et al, 2000) is taken into consideration. The maximum mean rate of ATP utilization for the first 1 s after activation (0.80 s -1 per cross-bridge, Fig.…”

“…The maximum mean rate of ATP utilization for the first 1 s after activation (0.80 s -1 per cross-bridge, Fig. 4) was also markedly smaller than the value of 18.5 s -1 per crossbridge (at 12°C) (He et al, 1997). Meanwhile the amount of ATP utilized for the first 1 s after activation (Pu) increased with decreasing load P, reaching a maximum at P=0 without any sign of leveling off (Fig.…”