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 I (SI), acto-SI and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-SI 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 (K-0.5 = 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-SI ATI'ase. Arrhenius plots for acto-SI 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. (c) 2005 Elsevier B.V. All rights reserved.