Essential light chain exchange in scallop myosin

Ashiba, Gaku; Szent‐Györgyi, Albert

doi:10.1021/bi00344a048

Cited by 25 publications

(12 citation statements)

References 37 publications

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“…When scallop R-LC was removed totally by EDTA treatment prior to TFP addition, E-LC removal remained refractory to TFP treatment, although some small losses (Ͻ5%) did occur (data not shown). Such losses are consistent with data suggesting that prior R-LC removal destabilizes E-LC attachment to the myosin heavy chain (21,29). For smooth muscle myosin, a combined treatment of 5-10 mM TFP and 4.5 M ammonium chloride facilitates E-LC exchange and even complete removal of both R-LCs and E-LCs (21,30).…”

Section: Discussionsupporting

confidence: 88%

Locking Regulatory Myosin in the Off-state with Trifluoperazine

Patel

Margossian²,

Chantler

2000

Journal of Biological Chemistry

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Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 M trifluoperazine, a concentration 10-fold less than that required for lightchain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased ␣-helicity could be induced in the isolated essential light chain through addition of 100 -200 M trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the Cterminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the offstate. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.Trifluoperazine (TFP), 1 a member of the phenothiazine class of drugs, is one of the strongest antagonists of calmodulin action known, capable of binding to calmodulin in the presence of calcium and preventing its stimulatory effects (1-3). The structure of the TFP⅐calmodulin complex in the presence of calcium has been determined at 2.45-Å resolution (4,5), where it was shown that TFP induces a profound conformational change in calmodulin, converting the elongated dumbbell to a compact globular structure, analogous to the form obtained through the binding of calmodulin to a target peptide (6, 7). This effect was accomplished when a single TFP molecule bound to the predominant binding site, a hydrophobic pocket within the C-terminal domain (4). Additional TFP-binding sites are apparent when the TFP concentration is raised (5). Recently, TFP-binding sites on the related calcium-binding protein troponin C have also been identified (8).Myosin light chains belong to the same large family of calcium-binding proteins as calmodulin, to which they display a similar overall structure (9, 10). There are two types of light chain, termed regulatory (R-LC) and essential (E-LC); one member of each type binds to each of the two heads of conventional myosin II. In those regulatory myosins that are activated directly by calcium binding (for review, see Ref. 11), the exact relationship of the heavy chain to the light chains, in the presence of calcium, is now known in detail, the structure of the regulatory domain of scallop myosin having first been established at 2.8-Å resolution (12) and then refined to 2.0-Å resolution (13). Solut...

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Section: Discussionsupporting

confidence: 88%

Locking Regulatory Myosin in the Off-state with Trifluoperazine

Patel

Margossian²,

Chantler

2000

Journal of Biological Chemistry

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“…However, Okamoto et al (1986) have shown that MLC1 binds near the site of ATP binding on smooth muscle myosin. In the scallop it has been shown that exchange of MLC1 with exogenously added MLC1 can occur only in the absence of MLC2 (Ashiba and Szent-Gyorgyi, 1985). This finding, coupled with the observation that the 1.…”

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confidence: 95%

Identification of sequences necessary for the association of cardiac myosin subunits.

McNally

Bravo-Zehnder

Leinwand

1991

The Journal of Cell Biology

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Abstract. To begin to understand the nature of myosin subunit assembly, we determined the region of a vertebrate sarcomeric myosin heavy chain required for binding of light chain 1. We coexpressed in Escherichia coli segments of the rat alpha cardiac myosin heavy chain which spanned the carboxyl terminus of subfragment 1 and the amino terminus of subfragment 2 with a full-length rat cardiac myosin light chain 1. A 16 amino acid region of the myosin heavy chain (residues 792-808) was shown to be required for myosin light chain 1 binding in an immunoprecipitation assay.M YosiN is an asymmetric hexamer comprised of two heavy chains (210 kD) and two pairs of light chains (18-25 kD). In smooth muscle, invertebrate muscle and vertebrate nonmuscle myosin, myosin light chain 2 (MLC2) ~ regulates the actin-activated ATPase of myosin in response to the calcium concentration of the cell (for reviews see Kamm and Stull, 1985;Citi and Kendrick-Jones, 1988). The role of MLC1 in myosin function is less clear (see Wagner and Giniger, 1981;Sivaramakrishnan and Burke, 1982). However, isoforms of MLC1 have been shown to be associated with different shortening velocities of skeletal muscle (Sweeney et al., 1988).The interactions of the subunits of myosin are not precisely defined, but both biochemical and electron microscopic studies suggest that MLCs bind near the junction of the subfragments 1 and 2 ($1 and $2). Scallop myosin reacted with anti-light chain antibodies demonstrates MLC2 binding near the head-rod junction (Flicker et al., 1983). Similar positions were noted for vertebrate MLC2, with MLC1 binding distally to MLC2, in the S1 head (Tokunaga et al., 1987;Katoh and Lowey, 1989). Proteolytic peptides of myosin heavy chain (MHC) from the carboxyl terminus of the S1 head bind MLC (Szentkiralyi, 1984;Mitchell et al., 1986). Burke et al. (1983) also implicated the carboxyl terminus of the S1 head by abolishing MLC1 binding with proteolytic digestion of a 3-kD segment from the S1-$2 junction. Sellers and Harvey (1984) used gel overlay to assign the light chain binding sites to 26 kD at the carboxyl terminus of smooth muscle myosin $1. However, Okamoto et al. (1986) have shown that MLC1 binds near the site of ATP binding on smooth muscle myosin. In the scallop it has been shown that exchange of MLC1 with exogenously added MLC1 can occur only in the absence of MLC2 (Ashiba and Szent-Gyorgyi, 1985). This finding, coupled with the observation that the 1. Abbreviations used in this Paper: MHC, myosin heavy chain; MLC, myosin light chain; S1 and $2, subfragment 1 and 2, respectively. sulfhydryl groups of scallop MLC1 are only available for labeling in the absence of MLC2, suggests that MLC1 lies between MLC2 and MHC (Walliman and Szent-Gyorgyi, 1981;Hardwicke et al., 1982).The use of recombinant DNA methods to produce myosin segments that have precisely defined sequences would be of great use in mapping the interactions of myosin subunits. A portion of the Caenorhabditis elegans unc54 MHC gene corresponding to S1 was expressed in Es...

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“…2B), indicating that oxygen-containing side A 1 (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) I (57-68) Hybrid RDs containing the cardiac ELC, the CI-SCII,SCIII,SCIV chimera, and the C1,CII-SCIII,SCIv chimera were formed and were isolated as stable complexes by ion-exchange chromatography ( Fig. 2A).…”

Section: Methodsmentioning

confidence: 99%

Role of essential light chain EF hand domains in calcium binding and regulation of scallop myosin.

Fromherz

Szent‐Györgyi

1995

Proc. Natl. Acad. Sci. U.S.A.

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The specific Ca2+ binding site that triggers contraction of molluscan muscle requires the presence of an essential light chain (ELC) from a Ca2+ binding myosin. Muscle myosins are highly conserved in overall morphology and subunit composition, consisting of two heavy chains, two essential light chains (ELCs), and two regulatory light chains (RLCs). The C-terminal portions of the heavy chains are wrapped around each other in an a-helical coiled-coil "tail," and the N termini form two pear-shaped globular "heads." Each head contains the actin binding sites, ATPase activity, and binding sites for one of each type of light chain. Despite these similarities, myosins from different muscle types are functionally distinct. A distinguishing feature of molluscan myosins is that each head possesses one specific, high-affinity

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Essential light chain exchange in scallop myosin

Cited by 25 publications

References 37 publications

Locking Regulatory Myosin in the Off-state with Trifluoperazine

Locking Regulatory Myosin in the Off-state with Trifluoperazine

Identification of sequences necessary for the association of cardiac myosin subunits.

Role of essential light chain EF hand domains in calcium binding and regulation of scallop myosin.

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