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.
Ribeiro
A S
AS
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 RJ, Brazil.
Salerno
V P
VP
Sorenson
M
M
D019297:2,4-Dinitrophenol / Q000737:chemistry*; D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009218:Myosins / Q000737:chemistry; D009596:Nitrophenols / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D013696:Temperature
D000199:Actins; D009596:Nitrophenols; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins; D019297:2,4-Dinitrophenol