Key Points
- Determine whether calcium alone fully activates the troponin-tropomyosin-actin complex or whether rigor cross-bridge binding is required to turn on acto-S-1 ATPase activity.
- Measured acto-S-1 ATPase activity in reconstituted troponin-tropomyosin-actin complexes (regulated actin) under varying calcium concentrations and rigor cross-bridge binding states.
- Evaluated the binding affinity of S-1.ATP to regulated actin and quantified the rate of inorganic phosphate (Pi) release during the catalytic cycle.
- Calcium alone failed to fully activate acto-S-1 ATPase; binding of rigor cross-bridges in the presence of calcium produced an almost 10-fold increase in ATPase activity at low actin concentrations.
- S-1.ATP binding affinity was nearly identical between maximally turned-off and maximally turned-on regulated actin, indicating activation does not depend on substrate binding.
- Activation markedly accelerated the rate of inorganic phosphate (Pi) release, shifting the rate-limiting kinetic step upstream of Pi release.
Structured PICO
PPopulationIn vitro model using myosin subfragment 1 (S-1) and troponin-tropomyosin-actin complex (regulated actin)
IInterventionBinding of rigor cross-bridges in the presence of Ca2+
OOutcomeacto.S-1 ATPase activitysurrogate
The binding of rigor cross-bridges synergizes with Ca2+ to fully turn on regulated actin ATPase activity, suggesting a cooperative mechanism in muscle contraction.