Effects of different enzymic treatments on the release of titin fragments from rabbit skeletal myofibrils. Purification of an 800 kDa titin polypeptide.
Ontology highlight
ABSTRACT:
SUBMITTER: Astier C
PROVIDER: S-EPMC1132341 | biostudies-other | 1993 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor. Application of a slack-test protocol revealed that the passive shortening velocity (Vp) of nonactivated cardiomyofibrils depends on: (i) initial sarcomere length, (ii) release-step amplitude, and (iii) temperature. Selective digestion of titin, with low doses of trypsin, decelerated myofibrillar passive recoil and eventually stopped it. Selective extraction of actin filaments with a Ca2+-independent gelsolin fragment greatly reduced the dependency of Vp on release-step size and temperature. These results are explained by the presence of viscous forces opposing myofibrillar passive recoil that are caused mainly by weak actin-titin interactions. Thus, Vp is determined by two distinct factors: titin elastic recoil and internal viscous drag forces. The recoil could be modeled as that of a damped entropic spring consisting of independent worm-like chains. The functional importance of myofibrillar elastic recoil was addressed by comparing instantaneous Vp to unloaded shortening velocity, which was measured in demembranated, fully Ca2+-activated, human cardiac fibers. Titin-driven passive recoil was much faster than active unloaded shortening velocity in early phases of isotonic contraction. Damped myofibrillar elastic recoil could help accelerate active contraction speed of human myocardium during early systolic shortening.
| S-EPMC240679 | biostudies-literature
Project description:The sarcomere is the smallest functional unit of myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction.
| S-EPMC5565411 | biostudies-literature
Project description:In this study, we measured the stiffness of skeletal muscle myofibrils in rigor. Using a custom-built atomic force microscope, myofibrils were first placed in a rigor state then stretched and shortened at different displacements (0.1-0.3 ?m per sarcomere) and nominal speeds (0.4 and 0.8 ?m/s). During stretching, the myofibril stiffness was independent of both displacement and speed (average of 987 nN/?m). During shortening, the myofibril stiffness was independent of displacement, but dependent on speed (1234 nN/?m at 0.4 ?m/s; 1106 nN/?m at 0.8 ?m/s). Furthermore, the myofibril stiffness during shortening was greater than that during stretching and the difference depended on speed (31% at 0.4 ?m/s; 8% at 0.8 ?m/s). The results suggest that the myofibrils exhibit nonlinear viscoelastic properties that may be derived from myofibril filaments, similar to what has been observed in muscle fibers.
| S-EPMC5770557 | biostudies-literature
Project description:A unique sequence within the giant titin molecule, the PEVK domain, has been suggested to greatly contribute to passive force development of relaxed skeletal muscle during stretch. To explore the nature of PEVK elasticity, we used titin-specific antibodies to stain both ends of the PEVK region in rat psoas myofibrils and determined the region's force-extension relation by combining immunofluorescence and immunoelectron microscopy with isolated myofibril mechanics. We then tried to fit the results with recent models of polymer elasticity. The PEVK segment elongated substantially at sarcomere lengths above 2.4 micro(m) and reached its estimated contour length at approximately 3.5 micro(m). In immunofluorescently labeled sarcomeres stretched and released repeatedly above 3 micro(m), reversible PEVK lengthening could be readily visualized. At extensions near the contour length, the average force per titin molecule was calculated to be approximately 45 pN. Attempts to fit the force-extension curve of the PEVK segment with a standard wormlike chain model of entropic elasticity were successful only for low to moderate extensions. In contrast, the experimental data also could be correctly fitted at high extensions with a modified wormlike chain model that incorporates enthalpic elasticity. Enthalpic contributions are likely to arise from electrostatic stiffening, as evidenced by the ionic-strength dependency of titin-based myofibril stiffness; at high stretch, hydrophobic effects also might become relevant. Thus, at physiological muscle lengths, the PEVK region does not function as a pure entropic spring. Rather, PEVK elasticity may have both entropic and enthalpic origins characterizable by a polymer persistence length and a stretch modulus.
| S-EPMC20927 | biostudies-literature
Project description:Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca(2+) dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca(2+) dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin C(IAEDANS), reported Ca(2+) dissociation exclusively from the structural domain of troponin C at ?0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca(2+) dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin [Formula: see text] reported Ca(2+) dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin [Formula: see text] reported a biphasic fluorescence change upon Ca(2+) dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin [Formula: see text] and troponin C(IAEDANS) at all levels of the troponin C systems. Furthermore, the rate of Ca(2+) dissociation from troponin C in the myofibrils was similar to the rate of Ca(2+) dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca(2+) dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca(2+) dissociation from troponin C may influence relaxation.
| S-EPMC3190119 | biostudies-literature
Project description:The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca(2+) and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca(2+), we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 microm in the absence of Ca(2+) at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]- and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as <or=1 nm changes in the lattice spacing.
| S-EPMC2098727 | biostudies-literature
Project description:In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before P(i) release. DMSO (5-30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto-S1 MgATPase activities in the same proportions, without altering acto-S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin-myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca(2+) and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca(2+) sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for P(i) is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP.P(i) or ATP bound to them.
| S-EPMC1222097 | biostudies-other
Project description:Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most molluscs. The basic quaternary structure of molluscan hemocyanins is a cylindrical decamer with a diameter of 35 nm which is built of 400 kDa subunits. Previously published results, however, showed that one out of two hemocyanin subunits of Rapana venosa encompasses two polypeptides, one 300 kDa and one 100 kDa polypeptide which aggregate to typical 4 MDa and 8 MDa hemocyanin (di-)decamer molecules. It was shown that the polypeptides are bound most probably by one or more cysteine disulfide bridges but it remained open if these polypeptides were coded by one or two genes. Our here presented results clearly showed that both polypeptides are coded by one gene only and that this phenomenon can also be found in the gastropod Nucella lapillus. Thus, it can be defined as clade-specific for Muricidae, a group of the very diverse Caenogastropoda. In addition, we discovered a further deviation of this hemocyanin subunit within both species, namely a region of 340 mainly hydrophilic amino acids (especially histidines and aspartic acids) which have not been identified in any other molluscan hemocyanin, yet. Our results indicate that, within the quaternary structure, these additional amino acids most probably protrude within the inner part of didecamer cylinders, forming a large extra mass of up to 800 kDa. They presumably influence the structure of the protein and may affect the functionality. Thus, these findings reveal further insights into the evolution and structures of gastropod hemocyanins.
| S-EPMC7884596 | biostudies-literature
Project description:Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca(2+)] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca(2+)-activation kinetics. Whereas the fast phase (k(obs)?1000 s(-1)) is only observed in this study, where kinetics were induced by Ca(2+), the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692-2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca(2+) ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca(2+) binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca(2+) sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.
| S-EPMC3446660 | biostudies-literature
Project description:Troponin (Tn) is the calcium-sensing protein of the thin filament. Although cardiac troponin (cTn) and skeletal troponin (sTn) accomplish the same function, their subunit interactions within Tn and with actin-tropomyosin are different. To further characterize these differences, myofibril ATPase activity as a function of pCa and labeled Tn exchange in rigor myofibrils was used to estimate Tn dissociation rates from the nonoverlap and overlap region as a function of pCa. Measurement of ATPase activity showed that skeletal myofibrils containing >96% cTn had a higher pCa 9 ATPase activity than, but similar pCa 4 activity to, sTn-containing myofibrils. Analysis of the pCa-ATPase activity relation showed that cTn myofibrils were more calcium sensitive but less cooperative (pCa50 = 6.14, nH = 1.46) than sTn myofibrils (pCa50= 5.90, nH = 3.36). The time course of labeled Tn exchange at pCa 9 and 4 were quite different between cTn and sTn. The apparent cTn dissociation rates were approximately 2-10-fold faster than sTn under all the conditions studied. The apparent dissociation rates for cTn were 5 x 10(-3) min(-1), 150 x 10(-3) min(-1), and 260 x 10(-3) min(-1), whereas for sTn they were 0.6 x 10(-3) min(-1), 88 x 10(-3) min(-1), and 68 x 10(-3) min(-1) for the nonoverlap region at pCa 9, nonoverlap region at pCa 4, and overlap region at pCa 4, respectively. Normalization of the apparent dissociation rates gives 1:30:50 for cTn compared with 1:150:110 for sTn (nonoverlap at pCa 9:nonoverlap at pCa 4:overlap at pCa 4) suggesting that calcium has a smaller influence, whereas strong cross-bridges have a larger influence on cTn dissociation compared with sTn. The higher cTn dissociation rate in the nonoverlap region and ATPase activity at pCa 9 suggest that it gives a less off or inactive thin filament. Analysis of the intensity ratio (after a short time of exchange) as a function of pCa showed that cTn had greater calcium sensitivity but lower cooperativity than sTn. In addition, the magnitude of the change in intensity ratio going from pCa 9 to 4 was less for cTn than sTn. These data suggest that the influence of calcium on cTn exchange is less than sTn even though calcium can activate ATPase activity to a similar extent in cTn compared with sTn myofibrils. This may be explained partially by cTn being less off or inactive at pCa 9. Modeling of the intensity profiles obtained after Tn exchange at pCa 5.8 suggest that the profiles are best explained by a model that includes a long-range cross-bridge effect that grades with distance from the rigor cross-bridge for both cTn and sTn.
| S-EPMC2711366 | biostudies-literature