Role of the regulatory light chains in skeletal muscle actomyosin ATPase and in minifilament formation.
Incubation of myosin with myopathic hamster protease results in substantial (more than 80%) removal of light chain 2 (LC2) with limited breakdown of the heavy chains. LC2-deficient myosin, purified by ion exchange chromatography, migrates as a single, monodisperse boundary in the analytical ultracentrifuge. The Ca2+- and EDTA-activated ATPases of LC2-deficient myosin are similar to those of the control and LC2-recombined myosins indicating that no denaturation occurred in its preparation. Double reciprocal plots for LC2-deficient, control, and LC2-recombined myosins reveal a biphasic behavior i.e. at actin concentrations above 11 microM, there is a sharp break in the 1/V versus 1/[actin] plots for all samples. The Vm values for LC2-deficient myosin are 50% lower (at low actin, Vm = 3.0 s-1, and at high actin, Vm = 4.2 s-1) than those for control myosin (Vm = 5.3 s-1 at low actin and 8.3 s-1 at high actin). Readdition of LC2 to LC2-deficient myosin restores the actin-activated ATPase to control levels. Electron microscopy of shadow cast preparations reveals a subtle difference between LC2-deficient myosin, and control or recombined myosin. In control and recombined myosins, S1 heads appear "pear"-shaped, whereas in LC2-deficient myosin, the S1 heads are rounder and display a "thinning" of mass in the "neck" region, suggesting that LC2 binds at the S1/S2 junction. Furthermore, removal of LC2 apparently influences the assembly of myosin into minifilaments, as revealed to a certain degree, by an increase in the width of the bare zone, accompanied by a decrease in the stability of these minifilaments.
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002458:Cell Fractionation; D006224:Cricetinae; D003599:Cytoskeleton / Q000648:ultrastructure*; D007700:Kinetics; D008854:Microscopy, Electron; D009132:Muscles / Q000201:enzymology*; D009135:Muscular Diseases / Q000378:metabolism; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium