X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method

Abstract

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.

X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers.

“…Low ft relaxing solution contained 1 mM MgCl2, 2 (25 ml) was continuously recirculated with a syringe pump (13 into rigor gradually, usually within 1 hr, although this time was also affected by the gap between the Mylar windows.…”

Thermoelastic effect in chemically skinned frog skeletal muscle in rigor.

“…Sartorius muscles from Rana japonica were used. The muscle pair was isolated with pelvic bone attached and was chemically skinned by the method of MAGID and REEDY (1980). The muscle was tied to a glass rod with threads at the pelvic bone and the tibial tendon.…”

Historical perspective on heart function: the Frank–Starling Law

“…Because of its inability to diffuse into the lattice spacing, dextran could compress the sarcomere and decrease the interfilament distance (Godt and Maughan 1977). Next, it was found that variations in fiber width (as a function of lattice spacing) could be regulated by concentration of dextran (Magid and Reedy 1980). The groups of Moss et al (1983) and Stienen et al (1985) indicated that lattice spacing, rather than length, was responsible for the changes in Ca 2+ -sensitivity in membranepermeabilized skeletal muscles.…”