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Phosphorylation of myosin light chains in perfused rat heart. Effect of adrenaline and increased cytoplasmic calcium ions.

ABSTRACT: 1. A method was developed for the isolation of essentially pure myosin light chains from perfused rat heart. The phosphorylation of the P-light chains was estimated by hydrolysis and measurement of phosphate released, by electrophoresis in 8 M-urea and by 32P incorporation in perfusion with [32P]Pi. 2. In control perfusions there was 0.5-0.6 mol of phosphate/mol of P-light chain. This was not changed by perfusion with 5 microM-adrenaline for 10-40s. Perfusion for 1 min with medium containing 7.5 mM-CaCl2, or for 30s with medium containing 118 mM-KCl, also did not change the phosphorylation of P-light chains. 3. It is concluded that phosphorylation of P-light chains is not important in mediating the action of inotropic agents in the heart.

SUBMITTER: Jeacocke SA

PROVIDER: S-EPMC1161959 | biostudies-other | 1980 Jun

REPOSITORIES: biostudies-other

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Project description:We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, V(max), and K(ATPase) of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca(2+) binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218-8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.

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Phosphorylation of myosin light chains in perfused rat heart. Effect of adrenaline and increased cytoplasmic calcium ions.

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