Increase in ATP consumption during shortening in skinned fibres from rabbit psoas muscle: effects of inorganic phosphate.

Abstract

1. The influence of inorganic phosphate (P(i)) on the relationship between ATP consumption and mechanical performance under isometric and dynamic conditions was investigated in chemically skinned single fibres or thin bundles from rabbit psoas muscle. Myofibrillar ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH. NADH absorbance at 340 nm was determined inside a miniature (4 microliters) measuring chamber. 2. ATP consumption due to isovelocity shortenings was measured in the range between 0.0625 and 1 L0 s‐1(L0: fibre length previous to shortening, corresponding to a sarcomere length of 2.64 microns), in solutions without added P(i) and with 30 mM P(i). To get an estimate of the amount of ATP utilized during the shortening phase, quick releases of various amplitudes were also performed. 3. After quick releases, sufficiently large that force dropped to zero, extra ATP was hydrolysed which was largely independent of the amplitude of the release and of the period of unloaded shortening. This extra amount, above the isometric ATP turnover, corresponded to about 0.7 and 0.5 ATP molecules per myosin head at 0 and 30 mM P(i), respectively. 4. ATP turnover during the isovelocity shortenings was higher than isometric turnover and increased with increasing shortening velocity up to about 2.7 times the isometric value. At low and moderate velocities of shortening (< 0.5 L0 s‐1), P(i) reduced ATP turnover during isovelocity shortening and isometric ATP turnover to a similar extent, i.e. a decrease to about 77% between 0 and 30 mM added P(i). 5. The extra ATP turnover above the isometric value, resulting from isovelocity shortenings studied at different speeds, was proportional to the power output of the preparation, both in the absence and presence of added [P(i)]. 6. The effect of shortening velocity and [P(i)] on energy turnover can be understood in a cross‐bridge model that consists of a detached, a non‐ or low‐force‐producing, and a force‐producing state. In this model, mass action of P(i) influences the equilibrium between the force‐producing and the non‐or‐low‐force‐producing cross‐bridges, and shortening enhances cross‐bridge detachment from both attached states.

Chemo‐mechanical energy transduction in relation to myosin isoform composition in skeletal muscle fibres of the rat

“…The apparatus used for the mechanical and energetic measurements was similar to that used in previous studies (Potma & Stienen, 1996) with slight modifications. The apparatus was composed of: (1) a large chamber filled with relaxing solution where the fibre segment could be mounted horizontally between the hook of the force transducer (AE801, Sensonor, Horten, Norway) and the hook of the displacement generator (Model 101, Ling Dynamic Systems, Royston, UK); (2) a second chamber with transparent walls where the fibre length and thickness could be measured and the sarcomere length could be determined by laser diffraction; (3) a third chamber (80 ìl) containing relaxing solution plus enzymes; (4) a fourth chamber (10 ìl) containing activating solution; and (5) a small chamber (volume 4 ìl) also containing activating solution where ATP consumption could be measured.…”

“…The fibre segment was mounted between the force transducer and the displacement generator as previously described (Potma & Stienen, 1996). While sarcomere length was monitored with laser diffraction, the segment length was adjusted to a sarcomere length of 2·7 ìm.…”

“…In skinned fibres myofibrillar ATP consumption can be directly determined by means of an enzymatic coupling of ATP resynthesis with oxidation of NADH which is present in the bathing solution (Glyn & Sleep, 1985;Stienen, Roosemalen, Wilson & Elzinga, 1990). A recent improvement of the technique (Potma & Stienen, 1996) has increased the resolution to enable the determination of ATP consumption during single isovelocity shortenings. This study aimed to assess whether MHC isoforms or MLC isoforms or both influence the ATP consumption during shortening and the efficiency of the conversion of chemical into mechanical energy.…”

Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

“…While there is little structural evidence for a lowaffinity M.ADP state (13), indirect evidence exists suggesting that the release of P i and ADP can indeed occur quite rapidly off actin under certain conditions. For example, elevated levels of P i have a much greater effect on force than the ATPase rate in muscle fibers (30,31), suggesting product release continues to be quite rapid off actin. In fact, efforts to model these data appear to require the relatively rapid release of P i and ADP from myosin while off actin (19) to accurately fit the ATPase results.…”

“…One prediction of the proposed mechanism that P i can rebind to the AM.ADP state and cause dissociation from strongly bound state, while still being able to complete the ATPase cycle, is that the process would decrease myosin's duty ratio (the proportion of the ATPase cycle spent strongly bound to actin). This assumes, of course, that the total cycle time is either unchanged or increased, which seems robust based on the effects of P i of myosin's ATPase rate in fibers (30,31). Therefore, to test this prediction of our model, we measured the effect of P i on myosin's duty ratio at 2 mM ATP.…”