The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

Pavlov, Dmitry; Gerson, Jack H.; Yu, Tianwei; Tobacman, Larry S.; Homsher, Earl; Reisler, Emil

doi:10.1074/jbc.m210889200

Cited by 5 publications

(2 citation statements)

References 43 publications

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“…This rate is much less than that of MgADP (Weiss et al, 2001;Siemankovski et al, 1985). Further in work in our group (Pavlov et al, 2003) we find that V o in 1-2 mM e-ATP at 258C is only ;30% (1.3 mm s ÿ1 ) of that seen in ATP and V o is the same in the presence and absence of regulatory proteins. Therefore, the features of acto-myosin interaction that produce the effects seen in MgATP are ineffective in Mg-e-ATP.…”

Section: Discussionmentioning

confidence: 60%

Regulatory Proteins Alter Nucleotide Binding to Acto-Myosin of Sliding Filaments in Motility Assays

Homsher

Nili

Chen

et al. 2003

Biophysical Journal

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The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin.

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Section: Discussionmentioning

confidence: 60%

Regulatory Proteins Alter Nucleotide Binding to Acto-Myosin of Sliding Filaments in Motility Assays

Homsher

Nili

Chen

et al. 2003

Biophysical Journal

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“…The presence of caldesmon did not affect the ADP release rate even though a quaternary complex was observed in co-sedimentation experiments. This contrasts skeletal muscle where the rate of ADP release was found to be increased by Ca 2ϩ binding to the troponin-tropomyosin complex (20,57).…”

mentioning

confidence: 99%

The Mechanism of Smooth Muscle Caldesmon-Tropomyosin Inhibition of the Elementary Steps of the Actomyosin ATPase

Alahyan

Webb

Marston

et al. 2006

Journal of Biological Chemistry

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Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1⅐ADP; however the rate of phosphate release from the actin-S1⅐ADP⅐P i complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.Smooth muscle contractility and actomyosin ATP hydrolysis are regulated by Ca 2ϩ at two levels. The first involves phosphorylation of the myosin light chain by myosin light chain kinase upon stimulation by Ca 2ϩ -calmodulin. The second occurs via the thin filament-associated protein caldesmon. Caldesmon (CaD) 2 binds to actin, calmodulin, and tropomyosin (1) and inhibits the actin activation of both smooth and skeletal muscle myosin ATPase in vitro (2) and force production when added to smooth and skeletal muscle fibers (3-5). Caldesmon also regulates the actin activation of myosin I ATPase in smooth muscle (6). The smooth muscle thin filament is Ca 2ϩ -regulated and is made up of actin, tropomyosin, caldesmon, and a Ca 2ϩ -binding protein in molar ratios ϳ14:2:1:1 (7). Previous studies of the molecular mechanism of caldesmon inhibition of the actomyosin ATPase have focused on its effect on the ATPase steady state parameters K m and V max (8) and on the equilibrium binding of myosin to actin filaments (9 -12). A detailed mechanism by which caldesmon regulates the actin activation of myosin ATPase and force production has not been demonstrated by such experiments but two potentially conflicting models have been developed.A competition model proposes that ATPase inhibition by caldesmon is caused by a reduction in the formation of the weakly bound actomyosin complex as a...

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Skeletal regulatory proteins enhance thin filament sliding speed and force by skeletal HMM

Clemmens

Regnier

2004

J Muscle Res Cell Motil

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At saturating calcium and nucleotide concentrations, troponin (Tn) and tropomyosin (Tm) enhance the in vitro motility speed of individual actin filaments, suggesting the roles of these thin filament proteins in regulating contraction may include a modulation of crossbridge kinetics. Using a homogeneous complement of fast rabbit skeletal proteins, we examined if Tn and Tm modify specific transitions in the crossbridge cycle by varying skeletal muscle crossbridge kinetics and measuring actin filament sliding speed and steady-state force using the in vitro motility and microneedle assays, respectively. Skeletal regulatory proteins increased the force and sliding speed of actin filaments sliding on skeletal HMM. Faster crossbridge cycling with increased temperature or with substitution of dATP as the contractile substrate resulted in both increased sliding speed and force of unregulated filaments, while the addition of regulatory proteins diminished or eliminated this increase. In contrast, regulatory proteins did not influence filament mechanics when crossbridge cycling was slowed with lowered ATP concentration. The results are most simply explained if addition of the Tn and Tm complex to actin enhances both the transition rate of the force-generating actomyosin isomerization (or the preceding transition) and the apparent crossbridge detachment rate, but that the relative influence of Tn and Tm is dependent on the external load.

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The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

Cited by 5 publications

References 43 publications

Regulatory Proteins Alter Nucleotide Binding to Acto-Myosin of Sliding Filaments in Motility Assays

Regulatory Proteins Alter Nucleotide Binding to Acto-Myosin of Sliding Filaments in Motility Assays

The Mechanism of Smooth Muscle Caldesmon-Tropomyosin Inhibition of the Elementary Steps of the Actomyosin ATPase

Skeletal regulatory proteins enhance thin filament sliding speed and force by skeletal HMM

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