The mechanism of regulatory light chain dissociation from scallop myosin.

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.     

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.     

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.     

Crystal structure of a phosphorylated light chain domain of scallop smooth-muscle myosin

We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg¹⁶, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.     

Reversible changes in the ATPase activity and in the regulatory light chain content upon heat (30 degrees C)-treatment of "Akazara" striated adductor myosin

Myosin from striated adductor muscle of "Akazara" scallop was incubated at 30 degrees C for 5 min in a medium containing 2 mM MgCl2 and various concentrations of Ca2+ ions. It was observed that the 30 degrees C-treatment resulted in a decrease in the Ca2+-sensitivity of myosin-ATPase as well as in the release of the regulatory light chain (EDTA-LC) of myosin. The 30 degrees C-treated myosin was then subjected to a cooling treatment, being kept for 18 h at 0 degrees C. It was found that EDTA-LC recombined with myosin and that Ca2+-sensitivity of myosin-ATPase was restored. It was also found that Ca2+ alone was about 70 times more effective than Mg2+ alone in preventing the heat-induced release of EDTA-LC from occurring and also in recombination of EDTA-LC with the heat-treated myosin.     

Hybrid myosin light chains containing a calcium-specific site from troponin C.

Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule. For example, we have demonstrated that the presence of high-affinity Ca2+/Mg(2+)-binding site in the N-terminal domain of the RLC is essential for the regulation of myosin-actin interaction [Reinach, F. C., Nagai, K. & Kendrick-Jones, J. (1986) Nature 322, 80-83]. To explore further the role of this metal-binding site in the RLC and generate an RLC with a Ca(2+)-specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid 'genes'. In these, the first domain containing the high-affinity Ca2+/Mg(2+)-binding site in the RLC was replaced with that containing the lower-affinity, Ca(2+)-specific, regulatory site from troponin C (TnC). In two of these hybrids, we replaced only the Ca(2+)-binding EF hand, while in the other two the EF hand and the N-terminal helix of TnC were transplanted. These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium-binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour. The results obtained show that the calcium-binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained. As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto-myosin, they were unable to regulate it. These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation. Our results suggest that the specificity and affinity of the calcium-binding site in troponin C is dependent on both intra- and inter-domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.     

Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand.

The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.     

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.     

Essential light chain exchange in scallop myosin

The exchange of essential light chains (SH-LCs) of scallop myosin was followed with the aid of scallop SH-LC alkylated with 14C-labeled iodoacetate. More than 70% of the SH-LCs were exchanged in myosin preparations that were desensitized by removal of both regulatory light chains (R-LCs) with ethylenediaminetetraacetic acid (EDTA) treatment. Although desensitized myosin solubilized with 0.6 M NaCl or with 10 mM adenosine 5'-triphosphate (ATP) in the absence of salt equilibrated rapidly with SH-LCs even in the cold, exchange in myosin filaments required elevated temperatures. Equilibration of the SH-LCs in desensitized preparations did not depend on ATP or magnesium ions but was significantly accelerated by actin. The desensitized myosin preparations containing alkylated SH-LCs (approximately 1 mol of thiol alkylated/mol of SH-LC) readily recombined with R-LCs. The preparations regained fully the calcium dependence of the actin-activated magnesium adenosinetriphosphatase (Mg-ATPase), contained R-LCs and SH-LCs in equimolar amounts, and had an ATPase activity similar to that of untreated myosin preparations. R-LCs interfered with the equilibration of the SH-LCs. In intact myosin preparations, the exchange of SH-LCs was slow and was frequently associated with the dissociation of the R-LCs. The blocking action of the R-LC on SH-LC exchange agrees with evidence showing that the two light chain types interact and suggests that parts of the SH-LC may lie between the R-LC and the heavy chain of myosin.     

Effects of removal and reconstitution of myosin regulatory light chain and troponin C on the Ca(2+)-sensitive ATPase activity of myofibrils from scallop striated muscle

In order to examine the involvement of troponin-linked Ca(2+)-regulation, in addition to well-known myosin-linked Ca(2+)-regulation, in the contraction of molluscan striated muscle, myofibrils from Ezo-giant scallop striated muscle were desensitized to Ca(2+) by removing both myosin regulatory light chain and troponin C by treatment with a strong divalent cation chelator, CDTA. The ATPase level in the desensitized myofibrils was about half the maximum level in intact myofibrils regardless of the Ca(2+)-concentration at 25 and 15 degrees C. In the absence of Ca(2+), the ATPase of the desensitized myofibrils was suppressed by myosin regulatory light chain but not affected by troponin C at either temperature. The ATPase was activated at higher Ca(2+)-concentrations by both myosin regulatory light chain and troponin C, but the activating effects of these two proteins were affected differently by temperature. The activation of ATPase by myosin regulatory light chain was much greater than that by troponin C at 25 degrees C, whereas the activation by troponin C was much greater than that by myosin regulatory light chain at 15 degrees C. The maximum activation was only obtained in the presence of both myosin regulatory light chain and troponin C at these temperatures. These findings strongly suggest that the contraction of scallop striated muscle is regulated through both myosin-linked and troponin-linked Ca(2+)-regulation, and that the troponin-linked Ca(2+)-regulation is more significant at lower temperature.     

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.     

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.     

Changes in reactivities of scallop adductor myosin with 5,5'-dithiobis-(2-nitrobenzoate) and with 2,4,6-trinitrobenzene sulfonate accompanying dissociation and association of regulatory light chain

1. The reactivities of scallop myosin with 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and with 2,4,6-trinitrobenzene sulfonate (TNBS) were found to be affected by dissociation and association of regulatory light chains (RLC) of myosin. 2. Approximately 4 mol of sulfhydryl groups of "desensitized" myosin (DM) were masked on association of DM with RLC. When these sulfhydryl groups were reacted with DTNB, the modified DM became incapable of associating with RLC, but when the modified DM was treated with 2-mercaptoethanol, the ability to associate with RLC was fully recovered. 3. The DTNB-reactivity of scallop myosin and its RLC content were measured as a function of calcium and magnesium concentrations. The results thus obtained could be explained in terms of our previous suggestion (J. Biochem. 94, 1061 (1983] that there are two different attachments between DM and RLC. 4. The relation between the TNBS-reactivity and the RLC content was not simple but complex. Not the extent, but the rate of trinitrophenylation of scallop myosin was affected by dissociation and association of DM with RLC; thus, the involved TNBS-reactive lysine residues did not seem to be in the regions on DM and RLC that would be physically covered upon DM-RLC association. 5. The amount of the involved lysine residues was estimated to be only 1 mol per mol of myosin. Modification of the specific lysine residues resulted in a partial decrease in the DM-RLC association.     

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.     

Signaling to myosin regulatory light chain in sarcomeres

Myosin regulatory light chain (RLC) phosphorylation in skeletal and cardiac muscles modulates Ca(2+)-dependent troponin regulation of contraction. RLC is phosphorylated by a dedicated Ca(2+)-dependent myosin light chain kinase in fast skeletal muscle, where biochemical properties of RLC kinase and phosphatase converge to provide a biochemical memory for RLC phosphorylation and post-activation potentiation of force development. The recent identification of cardiac-specific myosin light chain kinase necessary for basal RLC phosphorylation and another potential RLC kinase (zipper-interacting protein kinase) provides opportunities for new approaches to study signaling pathways related to the physiological function of RLC phosphorylation and its importance in cardiac muscle disease.     

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.     

Rotational dynamics of the regulatory light chain in scallop muscle detected by time-resolved phosphorescence anisotropy.

We have used time-resolved phosphorescence anisotropy (TPA) to study the rotational dynamics of chicken gizzard regulatory light chain (RLC) bound to scallop adductor muscle myofibrils in key physiological states. Native RLC from scallop myofibrils was extracted and replaced completely with gizzard RLC labeled specifically at Cys 108 with erythrosin iodoacetamide (ErIA). The calcium sensitivity of the ATPase activity of the labeled myofibril preparation was quite similar to that of the native sample, indicating that the ErIA-labeled RLC is functionally bound to the myosin head. In rigor (in the absence of ATP, when all the myosin heads are rigidly bound to the thin filament), a slight decay was observed in the first few microseconds, followed by no change in the anisotropy. This indicates small-amplitude restricted motions of the RLC or the entire LC domain of myosin. Addition of calcium to rigor restricts these motions further. Relaxation with ATP (no Ca) causes a large decay in the anisotropy, indicating large-amplitude rotational motion with correlation times of 5-50 micros. Further addition of calcium, to induce contraction, resulted in a decrease in the rate and amplitude of anisotropy decay. In particular, there is clear evidence for a slow rotational motion with a correlation time of approximately 300 micros, which is not present either in rigor or relaxation. This indicates rotational motion that specifically correlates with force generation. The changes in the rotational dynamics of the light-chain domain in rigor, relaxation, and contraction support earlier work based on probes of the catalytic domain that muscle contraction is accompanied by a disorder-to-order transition of the myosin head. However, the motions of the LC domain are different from those of the catalytic domain, which indicates rotation of the two domains relative to each other.     

Effect of regulatory light chain on chymotryptic digestion of scallop adductor myosin

Chymotryptic digestability of scallop myosin was studied by measuring (a) changes in the gel electrophoretic pattern and (b) production of the soluble fraction obtained by centrifugation. Chymotryptic digestion of essential light chain (SH-LC) was strongly inhibited by association of regulatory light chain (R-LC) with myosin. This is in agreement with the observation of Stafford et al. (Biochemistry 18, 5273 (1979]. SH-LC and R-LC were both more resistant to the chymotryptic digestion when R-LCs were associated with myosin in the presence of calcium than when they dissociated from myosin in the presence of EDTA. In contrast, heavy chains of scallop myosin were digested more quickly in the presence of calcium than EDTA. This suggests that association of R-LC induces reversible changes in the heavy chain conformation, which lead to an increase in the chymotryptic digestability of heavy chains. The chymotryptic digestability of scallop myosin increased in two distinct phases as the calcium concentration in the digestion medium was increased, but monophasically as the magnesium concentration was increased. The magnesium increased the digestability by approximately half as much as did calcium. These findings suggest two types of attachment between regulatory light chains and desensitized myosin: one mediated specifically by low concentrations of calcium ions, the second by higher concentrations of either calcium or magnesium.     

Calcium binding and conformation of regulatory light chains of smooth muscle myosin of scallop

Calcium binding was studied with two regulatory light chains (RLC-a and RLC-b) of smooth muscle myosin of scallop. With the equilibrium dialysis method, the binding of 0.98 mol Ca2+ per mol of RLC-b was observed with a dissociation constant of 2.3 X 10(-5) M. Similar values for RLC-b, 1.9 X 10(-5) M, and RLC-a, 1.5 X 10(-5) M, were obtained by measuring the difference absorption spectrum induced by Ca2+. The difference molar absorption coefficient at 288 nm was 159 and 209 M-1 X cm-1 for RLC-a and RLC-b, respectively, while it was -34 M-1 X cm-1 for the regulatory light chain of striated muscle myosin of scallop (RLC-st). Proton NMR spectra of the three light chains were very similar to each other and were broader than those of other Ca2+ binding proteins, parvalbumin and calmodulin. The regulatory light chains may be more rigid than in these Ca2+ binding proteins. CD spectra were measured for the three light chains, and the estimated helix contents were 27, 29, and 24%, respectively, for RLC-a, RLC-b, and RLC-st. All these results in comparison with the primary structures led us to suppose that the polypeptide of regulatory light chains is folded in such a way that domain 4 becomes near to the calcium binding site of domain 1. The decrease in intact light chains on trypsin digestion was determined for the gel electrophoretic patterns. RLC-a was 6 times more susceptible to the tryptic digestion than RLC-b.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.