Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions.

Abstract

Key words: fast and slow skeletal muscle myosin, myosin filaments, Ca 2+ -induced structural transitions, method of slow skeletal muscle myosin preparation. In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca 2+ -induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca 2+ /+ Ca 2+ ). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca 2+ ) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosus proprius muscle). In the presence of 0.1 mM free Ca 2+ , 3 mM Mg 2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca 2+ . In the absence of Ca 2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an ax-Vol. 47 No. 4/2000 1007-1017 QUARTERLY .

Cardiac myosin filaments are directly regulated by calcium

“…Given our observation of Ca 2+ -induced off -to- on transitions of the thick filament in the absence of active force, and inspired by previous work suggesting that there might be direct effects of Ca 2+ on thick filaments ( Morimoto and Harrington, 1974 ; Metzger and Moss, 1992 ; Podlubnaya et al, 2000 ), we explored whether these structural transitions can be translated into functional alterations. The thin filament inhibitor (MYK-7660) we used in this study has fluorescence in the 400–500 nm range, making performing conventional SRX/DRX assays on muscle tissue impractical.…”

Calcium activation through thick and thin?

“…In the current manuscript, the muscle and myosin filament preparations were incubated in calcium prior to making the experimental measurements. These steady-state conditions are very different than those observed in vivo, where calcium is quickly released into the sarcomere and rapidly removed, as discussed ( Podlubnaya et al, 2000 ). However, studies performed by Dr. Matsubara and colleagues during the 1970s using intact muscle preparations may provide insight into the positioning of the myosin heads within the sarcomere during a contraction, and the impact of calcium on these structural interactions in vivo.…”

“…These observations that calcium can impact the structure of the thick filament are consistent with previous structural data from vertebrate myosin thick filaments. Dr. Craig and colleagues demonstrated that myosin molecules in relaxed native cardiac thick filament preparations adopt the IHM in the absence of calcium ( Zoghbi et al, 2008 ), while Dr. Podlubnaya et al (2000) demonstrated that addition of 0.1 mM calcium to synthetic thick filaments preparations resulted in the protrusion of the myosin heads from the thick filament backbone.…”

The effects of the interaction of myosin essential light chain isoforms with actin in skeletal muscles.

“…Dephosphorylated fast skeletal myosin was purified from rabbit back muscles, as described previously (Stêpkowski et al, 1985). Slow skeletal myosin was isolated from myofibrils by the method described by Tartakowski (1978) from rabbit semimembranosus proprius muscle with the modifications described by Podlubnaya et al (2000). F-actin was prepared according to the method of Strzelecka-Go³aszewska et al (1975).…”