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• Articles by M. Gagelmann
• Articles by K. Güth

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• Characterization of the myosin adenosine triphosphate (M.ATP...

  Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers Biophysical Journal, Volume 54, Issue 1, 1 July 1988, Pages 135-148 M. Schoenberg Abstract In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30–40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2–3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states. Abstract | PDF (1735 kb)

• A Quantitative Analysis of Cardiac Myocyte Relaxation: A Sim...

  A Quantitative Analysis of Cardiac Myocyte Relaxation: A Simulation Study Biophysical Journal, Volume 90, Issue 5, 1 March 2006, Pages 1697-1722 S.A. Niederer, P.J. Hunter and N.P. Smith Abstract The determinants of relaxation in cardiac muscle are poorly understood, yet compromised relaxation accompanies various pathologies and impaired pump function. In this study, we develop a model of active contraction to elucidate the relative importance of the [Ca] transient magnitude, the unbinding of Ca from troponin C (TnC), and the length-dependence of tension and Ca sensitivity on relaxation. Using the framework proposed by one of our researchers, we extensively reviewed experimental literature, to quantitatively characterize the binding of Ca to TnC, the kinetics of tropomyosin, the availability of binding sites, and the kinetics of crossbridge binding after perturbations in sarcomere length. Model parameters were determined from multiple experimental results and modalities (skinned and intact preparations) and model results were validated against data from length step, caged Ca, isometric twitches, and the half-time to relaxation with increasing sarcomere length experiments. A factorial analysis found that the [Ca] transient and the unbinding of Ca from TnC were the primary determinants of relaxation, with a fivefold greater effect than that of length-dependent maximum tension and twice the effect of tension-dependent binding of Ca to TnC and length-dependent Ca sensitivity. The affects of the [Ca] transient and the unbinding rate of Ca from TnC were tightly coupled with the effect of increasing either factor, depending on the reference [Ca] transient and unbinding rate. Abstract | Full Text | PDF (437 kb)

• Formation of ATP-insensitive weakly-binding crossbridges in ...

  Formation of ATP-insensitive weakly-binding crossbridges in single rabbit psoas fibers by treatment with phenylmaleimide or para-phenylenedimaleimide Biophysical Journal, Volume 61, Issue 2, 1 February 1992, Pages 358-367 V.A. Barnett, A. Ehrlich and M. Schoenberg Abstract Chaen et al. (1986. J. Biol. Chem. 261:13632–13636) showed that treatment of relaxed single muscle fibers with para-phenylenedimaleimide (pPDM) results in inhibition of a fiber's ability to generate active force and a diminished ATPase activity. They postulated that the inhibition of force production was due to pPDM's ability to prevent crossbridges from participating in the normal ATP hydrolysis cycle. We find that the crossbridges produced by pPDM treatment of relaxed muscle cannot bind strongly to the actin filaments in rigor, but do bind weakly to the actin filaments in the presence and also absence of ATP. After pPDM treatment, fiber stiffness, as measured using ramp stretches of varying duration, is ATP-insensitive and identical to that of untreated relaxed fibers (both at high [165 mM] and low [40 mM] ionic strength). These results suggest that the pPDM-treated crossbridges, in both the presence and absence of ATP, are locked in a state that resembles the weakly-binding myosin ATP state of normal crossbridges. Their resemblance to the ATP-crossbridges of relaxed untreated fibers is quite strong; both bind to actin about equally tightly and have similar attachment and detachment rate constants. We also found that crossbridges are locked in a weakly-binding state after treatment with N-phenylmaleimide (NPM). In muscle fibers, this method of producing weakly-binding crossbridges appears preferable to pPDM treatment because, unlike treatment with pPDM, it does not increase the fiber's resting tension and stiffness and it does not disrupt the titin band seen on SDS-PAGE. Abstract | PDF (1203 kb)

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Abstract

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775–776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.