Depletion of phosphate in active muscle fibers probes actomyosin states within the powerstroke.
Variation in the concentration of orthophosphate (Pi) in actively contracting, chemically skinned muscle fibers has proved to be a useful probe of actomyosin interaction. Previous studies have shown that isometric tension (Po) decreases linearly in the logarithm of [Pi] for [Pi] > or = 200 microM. This result can be explained in terms of cross-bridge models in which the release of Pi is involved in the transition from a weakly bound, low-force actin x myosin x ADP x Pi state to a strongly bound, high-force, actin x myosin x ADP state. The 200 microM minimum [Pi] examined results from an inability to buffer the intrafiber, diffusive buildup of Pi resulting from the fiber ATPase. In the present study, we overcome this limitation by employing the enzyme purine nucleoside phosphorylase with substrate 7-methylguanosine to reduce the calculated internal [Pi] in contracting rabbit psoas fibers to < 5 microM. At 10 degrees C we find that Po continues to increase as the [Pi] decreases for [Pi] > or = 100 microM. Below this [Pi], Po is approximately constant. These results indicate that the free energy drop in the cross-bridge powerstroke is approximately 9 kT. This value is shown to be consistent with observations of muscle efficiency at physiological temperatures.
Pate
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E
Department of Pure and Applied Mathematics, Washington State University, Pullman 99164, USA. epate@wsu.edu
Franks-Skiba
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K
Cooke
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R
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D010430:Pentosyltransferases; C067995:nucleoside phosphorylase; D009218:Myosins