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• Positive Inotropic Effects of Low dATP/ATP Ratios on Mechani...

  Positive Inotropic Effects of Low dATP/ATP Ratios on Mechanics and Kinetics of Porcine Cardiac Muscle Biophysical Journal, Volume 91, Issue 6, 15 September 2006, Pages 2216-2226 Brenda Schoffstall, Amanda Clark and P. Bryant Chase Abstract Substitution of 2′-deoxy ATP (dATP) for ATP as substrate for actomyosin results in significant enhancement of in vitro parameters of cardiac contraction. To determine the minimal ratio of dATP/ATP (constant total NTP) that significantly enhances cardiac contractility and obtain greater understanding of how dATP substitution results in contractile enhancement, we varied dATP/ATP ratio in porcine cardiac muscle preparations. At maximum Ca (pCa 4.5), isometric force increased linearly with dATP/ATP ratio, but at submaximal Ca (pCa 5.5) this relationship was nonlinear, with the nonlinearity evident at 2–20% dATP; force increased significantly with only 10% of substrate as dATP. The rate of tension redevelopment () increased with dATP at all Ca levels. increased linearly with dATP/ATP ratio at pCa 4.5 and 5.5. Unregulated actin-activated Mg-NTPase rates and actin sliding speed linearly increased with the dATP/ATP ratio (<0.01 at 10% dATP). Together these data suggest cardiac contractility is enhanced when only 10% of the contractile substrate is dATP. Our results imply that relatively small (but supraphysiological) levels of dATP increase the number of strongly attached, force-producing actomyosin cross-bridges, resulting in an increase in overall contractility through both thin filament activation and kinetic shortening of the actomyosin cross-bridge cycle. Abstract | Full Text | PDF (298 kb)

• Distance measurements near the myosin head-rod junction usin...

  Distance measurements near the myosin head-rod junction using fluorescence spectroscopy Biophysical Journal, Volume 71, Issue 1, 1 July 1996, Pages 40-47 M. Kekic, W. Huang, P.D. Moens, B.D. Hambly and C.G. dos Remedios Abstract We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region. Abstract | PDF (1043 kb)

• Elementary Steps of the Cross-Bridge Cycle in Bovine Myocard...

  Elementary Steps of the Cross-Bridge Cycle in Bovine Myocardium with and without Regulatory Proteins Biophysical Journal, Volume 82, Issue 2, 1 February 2002, Pages 915-928 Hideaki Fujita, Daisuke Sasaki, Shin’ichi Ishiwata and Masataka Kawai Abstract The role of regulatory proteins in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed by gelsolin and the actin filament was reconstituted without tropomyosin or troponin. Further reconstitution was achieved by adding tropomyosin and troponin. The effects of MgATP and phosphate (Pi) on the rate constants of exponential processes were studied in control, actin filament-reconstituted, and thin filament-reconstituted myocardium at pCa ≤4.66, pH 7.00, 25°C. In control myocardium, the MgATP association constant was 9.1±1.3mM, and the Pi association constant 0.14±0.04mM. The equilibrium constant of the cross-bridge detachment step was 2.6±0.4, and the equilibrium constant of the force generation step was 0.59±0.04. In actin filament-reconstituted myocardium without regulatory proteins, the MgATP association constant was approximately the same, and the Pi association constant increased to 2.8×. The equilibrium constant of cross-bridge detachment decreased to 0.2×, but the equilibrium constant of the force generation step increased to 4×. These kinetic constants regained control values after reconstitution of the thin filament. These results indicate that tension/cross-bridge in the presence of regulatory proteins is ∼1.5–1.7× of that in the absence of regulatory proteins. These results further indicate that regulatory proteins promote detachment of cross-bridges. Abstract | Full Text | PDF (225 kb)

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Abstract

Porcine brain myosin is a cytoplasmic protein similar to, but distinct from, its muscle counterpart. It has a high K+-ATPase activity at high ionic strength in EDTA and a low Mg+2-ATPase activity that is activated fivefold by either porcine brain or rabbit skeletal muscle actin. The molecule consists of three classes of subunits, with molecular weights of approximately 195,000 , 19,000, and 16,000. Brain myosin contains less glutamic acid, less lysine, and more threonine, serine, proline, and tyrosine than skeletal muscle myosin. The brain myosin extinction coefficient at 278 nm is 0.810 cm2/mg. Hydrodynamic studies yield an S020,w of 4.95S, a D020,w of 1.07 x 10(-7) cm2/s for brain myosin, and indicate that the molecules aggregate at high ionic strength. The molecular weight of the molecule, as calculated from extrapolation of D020,w/S20,w to zero concentration, is 444,000. The intrinsic viscosity of brain myosin is 0.191 ml/mg. These data are consistent with a highly asymmetric molecular species. Circular dichroism spectroscopy indicates that brain myosin is 58–60% alpha-helical in the presence of Ca+2 ions, and that removal of Ca+2 causes a small change in the spectrum.