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• Articles by Y. Umazume
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  Compliance of thin filaments in skinned fibers of rabbit skeletal muscle Biophysical Journal, Volume 69, Issue 3, 1 September 1995, Pages 1000-1010 H. Higuchi, T. Yanagida and Y.E. Goldman Abstract The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction. Abstract | PDF (1154 kb)

• Ca- and Cross-Bridge-Dependent Changes in N- and C-Terminal ...

  Ca- and Cross-Bridge-Dependent Changes in N- and C-Terminal Structure of Troponin C in Rat Cardiac Muscle Biophysical Journal, Volume 80, Issue 1, 1 January 2001, Pages 360-370 Donald A. Martyn, Michael Regnier, Daguang Xu and Albert M. Gordon Abstract Linear dichroism of 5′-tetramethylrhodamine (5′ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the -terminus with a cysteine inserted at site 98 (cTnC-C35S,C84S,S98C, cTnC(C98)). With 5′ATR-labeled cTnC(C84) and cTnC(C98) dichroism increased with increasing [Ca], while rigor cross-bridges caused dichroism to increase more with 5′ATR-labeled cTnC(C84) than cTnC(C98). The pCa values and from Hill analysis of the Ca-dependence of force and dichroism were 6.4 (±0.02) and 1.08 (±0.04) for force and 6.3 (±0.04) and 1.02 (±0.09) (=) for dichroism in cTnC(C84) reconstituted trabeculae. Corresponding data from cTnC(C98) reconstituted trabeculae were 5.53 (±0.03) and 3.1 (±0.17) for force, and 5.39 (±0.03) and 1.87 (±0.17) (=5) for dichroism. The contribution of active cycling cross-bridges to changes in cTnC structure was determined by inhibition of force to 6% of pCa 4.0 controls with 1.0mM sodium vanadate (Vi). With 5′ATR-labeled cTnC(C84) Vi caused both the pCa of dichroism and the maximum value at pCa 4.0 to decrease, while with 5′ATR-labeled cTnC(C98) the pCa of dichroism decreased with no change of dichroism at pCa 4.0. The dichroism of 5′ATR-labeled cTnC(C35) was insensitive to either Ca or strong cross-bridges. These data suggest that both Ca and cycling cross-bridges perturb the -terminal structure of cTnC at Cys-84, while -terminal structure is altered by site II Ca-binding, but not cross-bridges. Abstract | Full Text | PDF (211 kb)

• Influence of Length on Force and Activation-Dependent Change...

  Influence of Length on Force and Activation-Dependent Changes in Troponin C Structure in Skinned Cardiac and Fast Skeletal Muscle Biophysical Journal, Volume 80, Issue 6, 1 June 2001, Pages 2798-2808 Donald A. Martyn and A.M. Gordon Abstract Linear dichroism of 5′ tetramethyl-rhodamine (5′ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5′ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5′ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5′ATR probe attached to Cys 84 increased during Ca activation of force. Force and dichroism-[Ca] relations were fit with the Hill equation to determine the pCa and slope (). Increasing SL increased the Ca sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca sensitivity of dichroism. In contrast, increasing SL increased the Ca sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca sensitivity of dichroism, decreased dichroism at saturating [Ca], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca sensitivity of force are not caused by corresponding changes in Ca binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges. Abstract | Full Text | PDF (217 kb)

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Abstract

Skinned fibers of frog semitendinosus muscle could easily be stretched up to 8 mum or more in sarcomere length. Such extremely stretched fibers gave quite sharp optical diffraction patterns. The intensities of all observable diffraction lines were found to increase on application of electric field (10 similar to 100 V/cm) parallel to the fiber axis, provided that there was no overlap between thin and thick filaments. By use of a polarizing microscope, it was concluded that I-bands were mainly responsible for this intensity increase. By application of square pulses, the time course of the intensity increase and decay was followed. The analysis based on a simple model suggests: (a) Each thin filament has a permanent dipole movement and the movement directs from Z-bands to the free end of the thin filament. (b) The flexural rigidity of thin filaments is estimated to be similar to 3 with 10–17 dyn with cm-2. The present fibers will provide various applications in physiochemical studies of in vivo thin and thick filaments.