Search

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition of 20 μM blebbistatin decreased tensions by ~80% in fibers, independent of phosphorylation. In blebbistatin maximal shortening velocities (V max) at 30°C, were decreased by 45% (3.2 ± 0.34 vs. 5.8 ± 0.18 lengths/s) in phosphorylated fibers but were not inhibited in dephosphorylated fibers (6.0 ± 0.30 vs. 5.4 ± 0.30). In the presence of 20 μM blebbistatin, K m for V max as a function of [ATP] was lower for phosphorylated fibers than for dephosphorylated fibers (50 ± 20 vs. 330 ± 84 μM) indicating that the apparent binding of ATP is stronger in these fibers. Phosphorylation of RLC in situ during fiber preparation or by addition of myosin light chain kinase yielded similar data. RLC phosphorylation inhibited velocity in blebbistatin at both 30 and 10°C, unlike previous reports where RLC phosphorylation only affected shortening velocities at higher temperatures.

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30°C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25°C (E. Pate, 0. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30°C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 ± 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10°C. The Km for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30°C, is 20 ± 4 µM for phosphorylated fibers and 192 ± 40 µM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30°C depends on the phosphate analog, with ~12% inhibition in fibers activated in the presence of 5 mM BeF3 and no inhibition in the presence of 0.25 mM AlF4. Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue. [ABSTRACT FROM AUTHOR]

Abstract Piperine, an alkaloid from black pepper, was found to inhibit the super-relaxed state (SRX) of myosin in fast-twitch skeletal muscle fibers. In this work we report that the piperine molecule binds heavy meromyosin (HMM), whereas it does not interact with the regulatory light chain (RLC)-free subfragment-1 (S1) or with control proteins from the same muscle molecular machinery, G-actin and tropomyosin. To further narrow down the location of piperine binding, we studied interactions between piperine and a fragment of skeletal myosin consisting of the full-length RLC and a fragment of the heavy chain (HCF). The sequence of HCF was designed to bind RLC and to dimerize via formation of a stable coiled coil, thus producing a well-folded isolated fragment of the myosin neck. Both chains were co-expressed in Escherichia coli , the RLC/HCF complex was purified and tested for stability, composition and binding to piperine. RLC and HCF chains formed a stable heterotetrameric complex (RLC/HCF) 2 which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF) 2 altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin. Highlights • The SRX is crucial for maintaining the high economy of resting muscle. • Piperine destabilizes the SRX by a previously unknown mechanism. • We found that piperine binds to the regulatory light chain (RLC) of myosin. • Piperine destabilizes and reduces the compactness of the myosin neck. • This study facilitates the search for therapies for obesity and type 2 diabetes. [ABSTRACT FROM AUTHOR]