title: The effects of phosphate and acidosis on regulated thin-filament velocity in an in vitro motility assay.
abstract: Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (P(i)) and hydrogen ions (H(+)) acting to directly inhibit the function of the contractile proteins; however, the molecular basis of this process remain unclear. We used an in vitro motility assay and determined the effects of elevated H(+) and P(i) on the ability of myosin to bind to and translocate regulated actin filaments (RTF) to gain novel insights into the molecular basis of fatigue. At saturating Ca(++), acidosis depressed regulated filament velocity (V(RTF)) by ≈ 90% (6.2 ± 0.3 vs. 0.5 ± 0.2 μm/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM P(i) caused V(RTF) to increase fivefold, from 0.5 ± 0.2 to 2.6 ± 0.3 μm/s at pH 6.5. Similarly, at all subsaturating Ca(++) levels, acidosis slowed V(RTF), but the addition of P(i) significantly attenuated this effect. We also manipulated the [ADP] in addition to the [P(i)] to probe which specific step(s) of cross-bridge cycle of myosin is affected by elevated H(+). The findings are consistent with acidosis slowing the isomerization step between two actomyosin ADP-bound states. Because the state before this isomerization is most vulnerable to P(i) rebinding, and the associated detachment from actin, this finding may also explain the P(i)-induced enhancement of V(RTF) at low pH. These results therefore may provide a molecular basis for a significant portion of the loss of shortening velocity and possibly muscular power during fatigue.
authors.affiliation: Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts 01003, USA. edebold@kin.umass.edu
authors.forename: Edward P
authors.initials: EP
authors.lastname: Debold
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Longyear
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Turner
mesh_terms: D000138:Acidosis / Q000503:physiopathology; D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009119:Muscle Contraction / Q000502:physiology*; D018763:Muscle Fatigue / Q000502:physiology; D009218:Myosins / Q000502:physiology*; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D014335:Tropomyosin / Q000502:physiology; D014336:Troponin / Q000502:physiology
chemical_list: D010710:Phosphates; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D002118:Calcium