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Probing actomyosin interactions with 2,4-dinitrophenol
A S Ribeiro1, V P Salerno, M Sorenson
1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 RJ, Brazil.
Abstract:
Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.
Insights
2,4-Dinitrophenol (DNP) impacts muscle contraction by increasing the affinity of actin to myosin while slowing turnover. This leads to stabilized weakly bound cross-bridges, reducing muscle force.
Area of Science:
- Muscle physiology
- Biochemistry
- Molecular motors
Background:
- Understanding chemomechanical coupling in muscle contraction requires studying intermediates post-ATP cleavage.
- 2,4-Dinitrophenol (DNP) accelerates isolated myosin ATPase activity but inhibits muscle contraction.
Purpose of the Study:
- Investigate the action of DNP on myosin subfragment 1 (S1), acto-S1, and skinned fibers in the presence of actin.
- Elucidate the mechanism by which DNP affects muscle contraction and cross-bridge dynamics.
Main Methods:
- Utilized myosin subfragment 1 (S1), acto-S1, and mammalian skinned fibers.
- Measured acto-S1 affinity and turnover rates in the presence of DNP.
- Analyzed isometric force reduction and Pi release kinetics.
- Performed Arrhenius plot analysis and studied the effect of Pi concentration on force.
Main Results:
- DNP increased acto-S1 affinity and reduced maximum turnover rate.
- Isometric force in skinned fibers was reduced by DNP.
- DNP reduced the ratio of actin-activated Pi release to acto-S1 turnover by 30-fold.
- DNP and meta-nitrophenol (MNP) stabilize weakly bound cross-bridges (AM.ADP.Pi).
- MNP increased apparent affinity for Pi.
Conclusions:
- DNP's inhibition of muscle contraction stems from a combination of enhanced S1 activity and reduced acto-S1 activity.
- DNP and MNP likely stabilize the weakly bound AM.ADP.Pi state, impacting the muscle contraction cycle.
- The findings provide insight into chemomechanical coupling mechanisms in skeletal muscle.
