The myosin neck, which is supported by the interactions between light chains and the underlying alpha-helical heavy chain, is thought to act as a lever arm to amplify movements originating in the globular motor domain. Here, we studied the role of the cardiac myosin regulatory light chains (RLCs) in the capacity of myosin to produce force using a novel optical-trap-based isometric force in vitro motility assay. We measured the isometric force and actin filament velocity for native porcine cardiac (PC) myosin, RLC-depleted PC (PCdepl) myosin, and PC myosin reconstituted with recombinant bacterially expressed human cardiac RLC (PCrecon). RLC depletion reduced unloaded actin filament velocity by 58% and enhanced the myosin-based isometric force similar to 2-fold. No significant change between PC and PCdepl preparations was observed in the maximal rate of actin-activated myosin ATPase activity. Reconstitution of PCdepl myosin with human RLC partially restored the velocity and force levels to near untreated values. The reduction in unloaded velocity after RLC extraction is consistent with the myosin neck acting as a lever, while the enhancement in isometric force can be directly related to enhancement of unitary force. The force data are consistent with a model in which the neck region behaves as a cantilevered beam.
Tuesday, October 13, 2009
Removal of the cardiac myosin regulatory light chain increases isometric force production
Kiran Pant, James Watt, Michael Greenberg, Michelle Jones, Danuta Szczesna-Cordary, and Jeffrey R. Moore
The myosin neck, which is supported by the interactions between light chains and the underlying alpha-helical heavy chain, is thought to act as a lever arm to amplify movements originating in the globular motor domain. Here, we studied the role of the cardiac myosin regulatory light chains (RLCs) in the capacity of myosin to produce force using a novel optical-trap-based isometric force in vitro motility assay. We measured the isometric force and actin filament velocity for native porcine cardiac (PC) myosin, RLC-depleted PC (PCdepl) myosin, and PC myosin reconstituted with recombinant bacterially expressed human cardiac RLC (PCrecon). RLC depletion reduced unloaded actin filament velocity by 58% and enhanced the myosin-based isometric force similar to 2-fold. No significant change between PC and PCdepl preparations was observed in the maximal rate of actin-activated myosin ATPase activity. Reconstitution of PCdepl myosin with human RLC partially restored the velocity and force levels to near untreated values. The reduction in unloaded velocity after RLC extraction is consistent with the myosin neck acting as a lever, while the enhancement in isometric force can be directly related to enhancement of unitary force. The force data are consistent with a model in which the neck region behaves as a cantilevered beam.
The myosin neck, which is supported by the interactions between light chains and the underlying alpha-helical heavy chain, is thought to act as a lever arm to amplify movements originating in the globular motor domain. Here, we studied the role of the cardiac myosin regulatory light chains (RLCs) in the capacity of myosin to produce force using a novel optical-trap-based isometric force in vitro motility assay. We measured the isometric force and actin filament velocity for native porcine cardiac (PC) myosin, RLC-depleted PC (PCdepl) myosin, and PC myosin reconstituted with recombinant bacterially expressed human cardiac RLC (PCrecon). RLC depletion reduced unloaded actin filament velocity by 58% and enhanced the myosin-based isometric force similar to 2-fold. No significant change between PC and PCdepl preparations was observed in the maximal rate of actin-activated myosin ATPase activity. Reconstitution of PCdepl myosin with human RLC partially restored the velocity and force levels to near untreated values. The reduction in unloaded velocity after RLC extraction is consistent with the myosin neck acting as a lever, while the enhancement in isometric force can be directly related to enhancement of unitary force. The force data are consistent with a model in which the neck region behaves as a cantilevered beam.
Subscribe to:
Post Comments (Atom)
No comments:
Post a Comment