V P Salerno | PubMed Health

PubMed2005

Probing actomyosin interactions with 2,4-dinitrophenol.

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.

2,4-Dinitrophenol↗

PubMed1997

Specificity and kinetic effects of nitrophenol analogues that activate myosin subfragment 1.

2,4-Dinitrophenol (DNP) activates the myosin ATPase of mammalian skeletal muscle in the presence of Ca2+ or Mg2+, and inhibits it when the bivalent cations are replaced by K+ and EDTA. Activation of Mg2+ATPase is abolished by the presence of unregulated actin. 3-Nitrophenol (3-NP) is also an activator, whereas other analogues (2-nitrophenol, 2-NP, and 4-nitrophenol, 4-NP) are much less effective. Concentrations required for their half-maximal effects (K0.5) range from 2 to 15 mM for 3-NP and DNP in the presence of different cations, and the sequence for the analogues is 3-NP<=DNP<<2-NP approximately 4-NP, which is apparently unrelated to either hydrophobicity or pK. DNP and 3-NP have almost identical effects on the ATPase activity of chymotryptic subfragment 1 as they do on myosin, which is an indication that their target is the globular head region rather than the tail, or the 18 kDa (regulatory) light chain. Analysis of the ATP concentration dependence for subfragment- 1 ATPase in the presence of Ca2+ or Mg2+ shows that DNP activates only at high substrate concentrations, becoming increasingly effective with ATP concentrations in the physiological range. At low substrate concentrations, DNP inhibits hydrolysis by increasing the apparent Km for ATP at the catalytic site. In the presence of Mg2+, it mimics the effect of actin, which increases the Km and accelerates the release of products following hydrolysis. At high substrate concentrations, activation by DNP appears to involve a kinetic component with low affinity for ATP that can increase the overall reaction rate by a factor of 2- to 9-fold, depending on the bivalent cation. This low-affinity component is either induced by the drug (in the presence of Mg2+) or shifted by the drug to a lower ATP concentration range (in the presence of Ca2+).

Adenosine Triphosphate↗