Proximity of regulatory light chains in scallop myosin

The distance between the regulatory light chains of the two heads of the scallop myosin molecule was estimated with the aid of two photolabile cross-linkers, benzophenone maleimide and p-azidophenacylbromide. These cross-linkers selectively alkylate thiol groups and have a maximum length of about 9 A. One of the two regulatory light chains of scallop myosin was removed by treatment of myofibrils at 10 degrees C with EDTA and replaced with a foreign regulatory light chain carrying a cross-linker. Cross-linking between the scallop and foreign regulatory light chains was effected by photolysis. This was demonstrated by incubating nitrocellulose transfers of sodium dodecyl sulfate/polyacrylamide gels of the photolyzed hybrid myofibrils with specific antibodies against the different light chains, followed by fluorescein isothiocyanate-125I-labeled secondary antibody. Scallop regulatory light chains cross-linked extensively (20 to 50%) with Mercenaria regulatory light chains (cysteine in position approximately 50) in solutions that induce rigor in skinned fibers (no ATP) and in relaxing solutions (ATP but no Ca2+). Neither the regulatory light chains of chicken skeletal myosin (cysteines 129 and 157) nor those of gizzard myosin (cysteine 108) were cross-linked to scallop regulatory light chains in either medium. These results indicate that the N-terminal portions of the myosin regulatory light chains can approach each other within 9 A or less, while the distance between the C-terminal halves exceeds 9 A, and support the view that the N termini of the regulatory light chains point toward the myosin rod. Since the relative distance between the regulatory light chains of the two myosin heads is not altered between rigor and rest, we suggest that motion of the essential light chains is mainly responsible for the observed difference in the relative positions of the regulatory and essential light chains between conditions of rigor and rest.     

Regulation of scallop myosin by mutant regulatory light chains

Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression. Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity. The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity. E. coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus. The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase. A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues. This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. Several other point mutations do not alter light chain function. The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.     

The light chains of scallop myosin as regulatory subunits

In molluscan muscles contraction is regulated by the interaction of calcium with myosin. The calcium dependence of the aotin-activated ATPase activity of scallop myosin requires the presence of a specific light chain. This light chain is released from myosin by EDTA treatment (EDTA-light chains) and its removal desensitizes the myosin, i.e. abolishes the calcium requirement for the actin-activated ATPase activity, and reduces the amount of calcium the myosin binds; the isolated light chain, however, does not bind calcium and has no ATPase activity. Calcium regulation and calcium binding is restored when the EDTA-light chain is recombined with desensitized myosin preparations. Dissociation of the EDTA-light chain from myosin depends on the concentration of divalent cations; half dissociation is reached at about 10^?5 M-magnesium or 10^?7 M-calcium concentrations. The EDTA-light chain and the residual myosin are fairly stable and the components may be kept separated for a day or so before recombination.Additional light chains containing half cystine residues (SH-light chains) are detached from desensitized myosin by sodium dodecyl sulfate. The EDTA-light chains and the SH-light chains have a similar chain weight of about 18,000 daltons; however, they differ in several amino acid residues and the EDTA-light chains contain no half cystine. The SH-light chains and EDTA-light chains have different tryptic fingerprints. Both light chains can be prepared from washed myofibrils.Densitometry of dodecyl sulfate gel electrophoresis bands and Sephadex chromatography in sodium dodecyl sulfate indicate that there are three moles of light chains in a mole of purified myosin, but only two in myosin treated with EDTA. The ratio of the SH-light chains to EDTA-light chains was found to be two to one in experiments where the total light-chain complements of myosin or myofibril preparations were carboxymethylated. A similar ratio was obtained from the densitometry of urea-acrylamide gel electrophoresis bands. We conclude that a myosin molecule contains two moles of SH-light chain and one mole of EDTA-light chain, and that the removal of a single EDTA-light chain completely desensitizes scallop myosin.Heavy meromyosin and S-1 subfragment can be prepared from scallop myosin. Both of these preparations bind calcium and contain light chains in significant amounts. The heavy meromyosin of scallop is extensively degraded; the S-1 preparation, however, is remarkably intact. Significantly, heavy meromyosin has a calcium-dependent actin-activated ATPase while the S-1 does not require calcium and shows high ATPase activity in its absence. These results suggest that regulation involves a co-operativity between the two globular ends of the myosin.Desensitized scallop myosin and scallop S-1 preparations can be made calcium sensitive when mixed with rabbit actin containing the rabbit regulatory proteins. This result makes it unlikely that specific light chains of myosin are involved in the regulation of the vertebrate system.The fundamental similarity in the contractile regulation of molluscs and vertebrates is that interaction between actin and myosin in both systems requires a critical level of calcium. We propose that the difference in regulation of these systems is that the interaction between myosin and actin is prevented by blocking sites on actin in the case of vertebrate muscles, whereas in the case of molluscan muscles it is the sites on myosin which are blocked in the absence of calcium.     

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.     

Characterization of regulatory light chain-a myosin kinase from smooth muscle of scallop, Patinopecten yessoensis

The protein kinase that phosphorylates the regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was isolated from scallop smooth muscle (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163). The enzymatic properties of this kinase (aMK) were investigated using RLC-a as the substrate. The Km value for ATP was 6.5 microM in the presence of 27 microM RLC-a at pH 7.0, and that for RLC-a was 133 microM in the presence of 1 mM ATP. The Vm value at saturation of both RLC-a and ATP was 0.25 s-1 at pH 7.0. The pH activity curve for aMK was bell-shaped with a maximum at around pH 7.8. The aMK activity was inhibited strongly by an increase in the KCl concentration. aMK required Mg2+, but was inhibited by high concentrations of Mg2+. The optimum activity was seen at 3 mM MgCl2. The mode of inhibition of the aMK activity by Ca2+ was studied. Assuming that the binding of Ca2+ to aMK induces the inhibition, the dissociation constant of Ca2+ was estimated to be 64 microM. aMK also phosphorylated LC20 of chicken gizzard myosin at a similar rate to that for RLC-a and the DTNB light chain of rabbit skeletal muscle myosin at a more lower rate. The helix and beta-sheet contents of aMK were estimated to be 19 and 30%, respectively, from the CD spectrum.     

Akazara scallop myosins hybridized with DTNB-light chains of skeletal myosin and with regulatory light chains of gizzard myosin

Two different hybrid myosins were obtained by combining "desensitized" myosin (DM) of Akazara scallop striated adductor with rabbit skeletal DTNB-light chains (DTNB-LC) and with chicken gizzard regulatory light chains (GR-LC). Using the two hybrid myosins, the following were found: (a) DTNB-LC has an inhibitory effect on the Mg-ATPase activities of Akazara DM and acto-DM both in the absence of calcium and in its presence. (b) DTNB-LC also has an enhancing effect on the superprecipitation activity of acto-DM. (c) The Mg-ATPase activities of DM and acto-DM are made sensitive to calcium by GR-LC, regardless of whether GR-LC is phosphorylated or unphosphorylated. (d) However, the Mg-ATPase activity of acto-myosin hybridized with phosphorylated GR-LC is definitely higher than that of acto-myosin hybridized with unphosphorylated GR-LC.     

A cAMP-dependent regulatory protein for RLC-a myosin kinase catalyzing the phosphorylation of scallop smooth muscle myosin light chain

A cAMP-dependent regulatory protein which modulates the phosphorylation of scallop myosin regulatory light chain-a (RLC-a) by RLC-a myosin kinase (aMK) (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163) was purified from the scallop smooth muscle. RLC-a is abundant in the opaque portion of scallop smooth muscle, one of the catch muscles. The regulatory protein for aMK was purified by employing successively DEAE Toyopearl ion exchange chromatography, Sepharose 4B-8(6-aminohexylamino)cAMP affinity chromatography, and Sephadex G 100 gel filtration. The molecular mass of the regulatory protein was 41 kDa, based on the mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. With increasing amounts of the regulatory protein, the aMK activity decreased, and complete inhibition was observed at the concentration of twice that of aMK. The aMK activity inhibited by the regulatory protein was restored by the addition of cAMP. These results suggest that aMK is similar to a catalytic subunit of cAMP-dependent protein kinase, and the protein reported here is similar to its regulatory subunit. aMK may exist as an inactive form, as a combination with this regulatory protein, in vivo and be deinhibited by an increase in the intracellular concentration of cAMP. We discuss a possible correlation between the phosphorylation of RLC-a in myosin catalyzed by aMK and the catch state of the opaque portion of scallop smooth muscle.     

Ca2(+)-dependent protein phosphatase which dephosphorylates regulatory light chain-a in scallop smooth muscle myosin

Ca2(+)-dependent protein phosphatase was purified from scallop adductor smooth muscle by a combination of DEAE-Toyoperal 650S ion exchange chromatographies and gel filtration on Sephacryl S-300. The phosphatase consisted of two subunits having molecular weights of 60 and 19 kDa. Phosphorylated regulatory light chain-a (RLC-a) was dephosphorylated by this phosphatase both in free and bound states in myosin prepared from the opaque portion of scallop smooth muscle (opaque myosin). The dephosphorylation was activated by Ca2+. The half maximal activation was a 1 microM free Ca2+ in the presence of calmodulin and 7 microM free Ca2+ in the absence of calmodulin. Opaque myosin phosphorylated at the heavy chain was not dephosphorylated with this phosphatase. p-Nitrophenyl phosphate was dephosphorylated. In addition to Ca2+, the phosphatase activity for RLC-a was activated by Mn2+, while p-nitrophenylphosphatase activity was activated by Mg2+ more strongly than by Mn2+. The pH-activity curves showed a maximum at pH 7 in the presence of Mn2+, but at around pH 8 in the presence of Mg2+. This phosphatase is similar to phosphatase 2B or calcineurin. The possible regulatory function of this phosphatase in scallop catch muscle is discussed.     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

Modification of interface between regulatory and essential light chains hampers phosphorylation-dependent activation of smooth muscle myosin

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.     

The N-terminal lobes of both regulatory light chains interact with the tail domain in the 10 S-inhibited conformation of smooth muscle myosin

In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P(i) is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554-1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains ( Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384 ). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428-1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.     

Regulatory light chain-a myosin kinase (aMK) catalyzes phosphorylation of smooth muscle myosin heavy chains of scallop, Patinopecten yessoensis

Regulatory light chain-a myosin kinase (aMK), which phosphorylates one of the myosin regulatory light chains, RLC-a, contained in the catch muscle of scallop, was also found to phosphorylate heavy chains of scallop myosin. After incubation of myosin isolated from the opaque portion of scallop smooth muscle (opaque myosin) with aMK in the presence of [gamma-32P]ATP, about 2 mol of 32P was incorporated per mol of the myosin. The radioactivity was mostly found in the heavy chain at 0.26 M KCl. The pH-activity curve and MgCl2 requirement for the heavy chain phosphorylation were similar to those for RLC-a phosphorylation. In contrast, the dependency of activity on KCl concentration was different from that for RLC-a. The heavy chain phosphorylation activity decreased with increase in KCl concentration up to 0.06 M, and then increased at concentrations over 0.06 M to a maximum at around 0.26 M KCl. This complicated profile probably reflects the solubility of myosin, and the phosphorylation site may be located in the rod portion insoluble at low KCl concentrations. Phosphorylation of heavy chain did not change the solubility of the opaque myosin molecule at all. The acto-opaque myosin ATPase activity in the presence of Ca2+ was found to be decreased to less than one-fourth by the heavy chain phosphorylation.     

Interaction between the heavy and the regulatory light chains in smooth muscle myosin subfragment 1.

The interaction between the heavy and the regulatory light chains within chicken gizzard myosin heads was investigated by using a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC). The chicken gizzard subfragment 1 (S-1) used was treated with papain so that the heavy chain was partly cleaved into the NH2-terminal 72K and the COOH-terminal 24K fragments and the regulatory light chain into the 16K fragment. S-1 was reacted with EDC either alone or in the presence of ATP or F-actin. In all cases, the 16K fragment of the regulatory light chain formed a covalent cross-link with the 24K heavy chain fragment but not with the 72K fragment. The 38K cross-linked peptide, which was the product of cross-linking between the 16K light chain and the 24K heavy chain fragments, was isolated and further cleaved with cyanogen bromide and arginylendopeptidase. Smaller cross-linked peptides were purified by reverse-phase HPLC and then characterized by amino acid analysis and sequencing. The results indicated that cross-linking occurred between Lys-845 in the heavy chain and Asp-168, Asp-170, or Asp-171 in the regulatory light chain. The position of the cross-linked lysine was only three amino acid residues away from the invariant proline residue mapped as the S-1-rod hinge by McLachlan and Karn [McLachlan, A. D., & Karn, J. (1982) Nature (London) 299, 226-231]. We propose that the COOH-terminal region of the regulatory light chain is located in the neck region of myosin and that this region and the phosphorylation site of the regulatory light chain together may play a role in the phosphorylation-induced conformational change of gizzard myosin.     

Phosphorylation of regulatory light chain a (RLC-a) in smooth muscle myosin of scallop, Patinopecten yessoensis

One of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.     

Hybrids of Physarum myosin light chains and desensitized scallop myofibrils

The two light chains of Physarum myosin have been purified in a 1:1 ratio with a yield of 0.5-1 mg/100 g of plasmodium and a purity of 40- 70%; the major contaminant is a 42,000-dalton protein. The 17,700 Mr Physarum myosin light chain (PhLC1) binds to scallop myofibrils, providing the regulatory light chains (ScRLC) have been removed. The 16,500 Mr light (PhLC2) does not bind to scallop myofibrils. The calcium control of scallop myosin ATPase is lost by the removal of one of the two ScRLC's and restored equally well by the binding of either PhLC1 or rabbit skeletal myosin light chains. When both ScRLC's are removed, replacement by two plasmodial light chains does not restore calcium control as platelet or scallop light chains do. Purified plasmodial actomyosin does not bind calcium in 10(-6) M free calcium, 1 mM MgCl2. No tropomyosin was isolated from Physarum by standard methods. Because the Physarum myosin light chains can substitute only partially for light chains from myosin linked systems, because calcium does not bind to the actomyosin, and because tropomyosin is apparently absent, the regulation of plasmodial actomyosin by micromolar Ca++ may involve other mechanisms, possibly phosphorylation.     

Two isoforms of regulatory light chain of abalone smooth muscle myosin

Abalone myosin contains two kinds of light chain, regulatory light chain (LC2) and essential light chain (LC1) according to SDS-PAGE. Three distinct light chain bands were observed on polyacrylamide gel electrophoresis of purified abalone myosin in the presence of urea (urea-PAGE). The slower two components showed had mobility on SDS-PAGE and they also showed regulatory activity as the regulatory light chain. They were termed LC2-a and LC2-b in order of increasing mobility on urea-PAGE and isolated by DE-32 ion exchange column chromatography in the presence 8 M urea. The ratio of LC2-a and LC2-b in the central portion of adductor muscle of abalone (LC2-a: LC2-b = 7:3) was different from that (1:1) in the peripheral portion. These results suggest that the two light chains are isoforms of the regulatory light chain. The amino acid compositions of LC2-a and LC2-b were very similar to each other except for the Cys content. The UV absorption spectra were also quite similar, as were the UV difference absorption spectra induced by Ca2+. Phosphorylation was not detectable with the myosin light chain kinase of chicken gizzard. The Ca2+ concentration dependencies of Mg-ATPase activity of LC2-a or LC2-b hybridized abalone myosin (a-myosin, b-myosin) were similar to each other in the absence of rabbit F-actin, but differed in the presence of actin. The b-myosin had a higher maximum value of actomyosin ATPase activity and a lower apparent binding constant of actin and myosin than a-myosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective purification of the 20,000-Da light chains of smooth muscle myosin

The 20,000-Da light chains of gizzard smooth muscle myosin have been purified to homogeneity. Actomyosin, prepared by MgATP extraction of myofibrils, was denatured in 8 M urea, 1 M guanidine HCl, and 0.05% sodium dodecyl sulfate. Myosin heavy chains were precipitated with ethanol and the light chain enriched fraction was dialyzed and subjected to chromatography on DEAE-Sephacel. Fractions containing the 20,000-Da light chains were further purified by hydrophobic chromatography on phenyl-Sepharose. The 20,000-Da light chains eluted at low ionic strength from the phenyl-Sepharose column were judged to be greater than 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained only 0.04 mol of phosphate/mol of light chain. The yield of light chains was calculated to be 219 +/- 17 mg/kg of starting gizzard smooth muscle. This method may be useful for preparation of homogeneous 20,000-Da smooth muscle myosin light chains in the quantities necessary for study of contractile systems.