scholarly journals Evaluation of the symmetric model for myosin-linked regulation: effect of site-directed mutations in the regulatory light chain on scallop myosin

2003 ◽  
Vol 374 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Melanie COLEGRAVE ◽  
Hitesh PATEL ◽  
Gerald OFFER ◽  
Peter D. CHANTLER
Keyword(s):  
Light Chain ◽  
Structural Difference ◽  
Regulatory Light Chain ◽  
Site Directed Mutagenesis ◽  
Light Chains ◽  
Asymmetric Structure ◽  
Symmetric Model ◽  
Regulatory Light Chains ◽  
Hybrid Molecules ◽  
Muscle Myosin

Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium. It has been proposed that the two heads of scallop myosin are symmetrically arranged and interact through their regulatory light chains [Offer and Knight (1996) J. Mol. Biol. 256, 407–416], the interface being strengthened in the inhibited state. By contrast, vertebrate smooth-muscle myosin is activated by phosphorylation. Its structure in the inhibited state has been determined from two-dimensional crystalline arrays [Wendt, Taylor, Trybus and Taylor (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361–4366] and is asymmetric, requiring no interaction between regulatory light chains. Using site-directed mutagenesis of the scallop regulatory light chain, we have tested the symmetric model for scallop adductor muscle myosin. Specifically, we have made myosin hybrid molecules from scallop (P. maximus) myosin, in which the normal regulatory light chains have been replaced by expressed light chains containing mutations in three residues proposed to participate in the interaction between regulatory light chains. The mutations were R126A (Arg126→Ala), K130A and E131A; made singly, in pairs or all three together, these mutations were designed to eliminate hydrogen bonding or salt linkages between heads, which are key features of this model. Functional assays to address the competence of these hybrid myosins to bind calcium specifically, to exhibit a calcium-regulated myofibrillar Mg-ATPase and to display calcium-dependent actin sliding were performed. We conclude that the symmetrical model does not describe the inhibited state of scallop regulatory myosin and that an asymmetric structure is a plausible alternative.

scholarly journals The mechanism of regulatory light chain dissociation from scallop myosin

1986 ◽  
Vol 233 (1) ◽  
pp. 179-186 ◽  
Author(s):  
A J Bennett ◽  
C R Bagshaw
Keyword(s):  
Light Chain ◽  
Structural Information ◽  
Regulatory Light Chain ◽  
Light Chains ◽  
Heavy Meromyosin ◽  
Regulatory Light Chains ◽  
First Order ◽  
High Affinity Site ◽  
Refractory State ◽  
Kinetics Of

The dissociation of the regulatory light chains from scallop myosin subfragments, on addition of EDTA, was investigated by using the fluorophore 8-anilinonaphthalene-1-sulphonate as a probe. The rate of this process (0.014 s-1) was partially limited by the rate of Mg2+ dissociation (0.058 s-1) from the non-specific high-affinity site. The dissociation of the regulatory light chain subfragment 1 was less extensive than from heavy meromyosin. Reassociation of the scallop regulatory light chain was induced on addition of Mg2+, but it appeared to be limited by a first-order step. The nature of this step was revealed by the kinetics of Mercenaria regulatory light chain association. Scallop heavy meromyosin, denuded of its regulatory light chains, exists in a refractory state, whose reversal to the nascent state limits the rate of light chain association (0.006 s-1). The formation of the refractory state is the driving force for the net dissociation of regulatory light chains from scallop heavy meromyosin. This mechanism is discussed with reference to existing structural information on light-chain-denuded myosin.

scholarly journals Function of the NH2-terminal domain of the regulatory light chain on the regulation of smooth muscle myosin.

1994 ◽  
Vol 269 (45) ◽  
pp. 28173-28180
Author(s):  
M Ikebe ◽  
R Ikebe ◽  
H Kamisoyama ◽  
S Reardon ◽  
J P Schwonek ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Regulatory Light Chain ◽  
Smooth Muscle Myosin ◽  
Terminal Domain ◽  
Muscle Myosin

scholarly journals Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

1985 ◽  
Vol 100 (6) ◽  
pp. 2025-2030 ◽  
Author(s):  
H Takano-Ohmuro ◽  
T Obinata ◽  
M Kawashima ◽  
T Masaki ◽  
T Tanaka
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Striated Muscles ◽  
Two Dimensional Gel Electrophoresis ◽  
Embryonic Chicken ◽  
Muscle Myosin

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

scholarly journals Properties of the non-specific calcium-binding sites of rabbit skeletal-muscle myosin

1980 ◽  
Vol 185 (1) ◽  
pp. 265-268 ◽  
Author(s):  
J Wikman-Coffelt
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Binding Sites ◽  
Calcium Binding ◽  
Light Chains ◽  
Binding Properties ◽  
Skeletal Muscle Myosin ◽  
Heavy Chains ◽  
Calcium Binding Sites ◽  
Muscle Myosin

The non-specific Ca2+-binding sites of skeletal-muscle myosin are located on the light chains; with the dissociation of light chains there is a corresponding decrease in the number of Ca2+-binding sites on light-chain-deficient myosin. The released light chains have a decreased binding affinity. Myosin heavy chains indirectly influence the Ca2+-binding properties of light chains by increasing the affinity of light chains for bivalent cations; this influence varies with pH. Because of light-chain dissociation at low Ca2+ and/or Mg2+ concentrations, anomalies may exist when analyses of non-specific Ca2+-binding properties of myosin are assessed by dialysis equilibrium.

Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins

Nature ◽  
1986 ◽  
Vol 322 (6074) ◽  
pp. 80-83 ◽  
Author(s):  
Fernando C. Reinach ◽  
Kiyoshi Nagai ◽  
John Kendrick-Jones
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Regulatory Light Chain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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