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.     

Regulatory light chains in myosins.

Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity.Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA.There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.     

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.     

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.     

Regulatory properties of single-headed fragments of scallop myosin.

Calcium control was studied in single-headed myosin and subfragment-1 (S1) preparations obtained by papain digestion of scallop myosin. Single-headed myosin, containing light chains in stoichiometric amounts, was calcium regulated; in contrast, the actin-activated Mg-ATPase of all S1 species lacked calcium sensitivity. Both regulatory and essential light chains were retained by S1 and single-headed myosin preparations provided divalent cations were present during papain digestion, although a peptide amounting to 10% of the mass was removed from regulatory light chains. The modified regulatory light chain retained its ability to confer calcium binding and restore calcium sensitivity to the ATPase of desensitized myofibrils. Regulatory light chains protected the essential light chains from fragmentation by papain. S1 bound regulatory light chains with a uniformly high affinity and appeared to consist of a single species. The results demonstrate that head to head interactions are not obligatory for calcium control, although they may occur in the intact myosin molecule, and suggest a role for the subfragment-2 region in calcium regulation of myosin.     

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.     

Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.     

Locking regulatory myosin in the off-state with trifluoperazine

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 microM trifluoperazine, a concentration 10-fold less than that required for light-chain 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 alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.     

Regulatory light chains and scallop myosin. Form of light chain removal or reuptake is dependent on the presence of divalent cations

Readdition of regulatory light chains to regulatory light chain denuded scallop myofibrils, in the presence of magnesium, results in a negatively co-operative restoration of calcium sensitivity as a function of regulatory light chain content. The form of the stoichiometry curves obtained in the presence of 10 mM-EDTA, by light chain removal from scallop myofibrils at various temperatures, are parabolic in shape, consistent with a random removal process. However, in the presence of EDTA at low temperatures, regulatory light chains are removed in a biphasic manner, indicating that the binding constants of the light chains for each myosin head are not equivalent under these conditions. It is shown here that as the temperature is raised, light chain removal by EDTA approaches that of a random process. The stoichiometry curves obtained in the presence of 10 mM-EDTA may therefore be seen as a composite of both a biphasic removal process (temperatures below 20 degrees C) and a random removal process (temperatures above 20 degrees C), there being a temperature-dependent switch in the myosin molecule between 17 and 23 degrees C that governs the mode of light chain removal. These results indicate that both myosin heads must contain light chains for calcium sensitivity and are consistent with our earlier proposals for head-head co-operativity within the scallop myosin molecule.     

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.     

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.     

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.     

An immunological approach to myosin light-chain function in thick filament linked regulation. 2. Effects of anti-scallop myosin light-chain antibodies. Possible regulatory role for the essential light chain

Specific antibodies directed against the regulatory light chains (R-LC) or essential light chains (SH-LC) of scallop myosin abolished calcium regulation in myofibrils, myosin, and heavy meromyosin by elevating the actin-activated Mg2+-ATPase activity in the absence of calcium. Calcium dependence was completely eliminated at molar ratios of 2.5-3 antibodies bound per myosin. Monovalent anti-R-LC Fab and anti-SH-LC Fab fragments also desensitized myofibrils fully. High Ca2+-ATPase activity remained unaffected by the antibodies. Anti-SH-LC IgG reduced to about one-half the actin-activated Mg2+-ATPase in the presence of calcium and the potassium-activated ethylenediaminetetraacetic acid (EDTA)-ATPase activities. Anti-SH-LC Fab, however, desensitized without inhibiting the actin-activated Mg2+-ATPase. The desensitizing effect of both antibodies was abolished by prior absorption with the homologous myosin light chain. Calcium binding and R-LC and anti-SH-LC IgG's and by anti-SH-LC Fab. The anti-R-LC Fab fragment induced a significant (70%) dissociation of R-LC from myofibrils and myosins with concomitant losses in calcium binding. In contrast, anti-R-LC IgG prevented the dissociation of R-LC from myosin by EDTA. Binding of anti-R-LC IgG to myofibrils was proportional to thier R-LC content. Increased amounts of anti-SH-LC IgG were bound by myofibrils devoid of R-LC. Bound anti-SH-LC antibody significantly inhibited the reuptake of R-LC by EDTA-treated myofibrils as well as the full binding of anti-R-LC antibody. Certain rabbits produced a population of anti-SH-LC antibodies which were specific for this light chain and bound extensively to myosin but failed to desensitize it (nondesensitizing anti-SH-LC antibody). The desensitizing and nondesensitizing anti-SH-LC populations bound to different regions of the SH-LC on the myosin, and the binding of the two types of antibody to the SH-LC was nearly additive. The nondesensitizing SH-antibody inhibited the reuptake of R-LC less, and its binding to myofibrils was not influenced by the absence of R-LC. These studies indicate a direct or indirect involvement of the SH-LC's in myosin-linked regulation, raise the possibility of an interaction between the R-LC and SH-LC, and confirm the regulatory function of the scallop R-LC. A model for a relative location of the two types of light chains and the involvement of the subfragment-2 region of myosin linked regulation is discussed.     

Investigation of immunological relationships among myosin light chains and troponin C.

Two classes of myosin light chains can be distinguished functionally: those that restore calcium regulation to "desensitized" scallop myofibrils, and those that do not (Kendrick-Jones, J., et al. (1976), J. Mol. Biol. 104, 747--775). Despite this functional classification, chemical analyses reveal few patterns unique to regulatory light chains, and, indeed, sequence comparisons suggest structural similarities between both classes of myosin subunits (Collins, J. H. (1977), Nature (London) 259, 699--700; Kendrick-Jones, J., and Jakes, R. (1977), in International Symposium on Myocardial Failure at Tegernsee, Riecker, G., and Boehringer, Ed., Munich, West Germany, Springer-Verlag, pp. 28--40). Immunological assays using antisera to regulatory and to nonregulatory light chains showed no correlation between antigenic activity and the presence or absence of regulatory function. Weak cross-reactivity was observed, however, among myosin light chains and troponin C, consistent with the suggestion made on the basis of sequence homologies that these subunits contain similar structural domains (Weeds, A. G., and McLachlan, A. D. (1974), Nature (London) 252, 646--649). Unexpectedly, the strongest cross-reactivity observed was that between the vertebrate myosin alkali 1 and DTNB light chains.     

Conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction. Polarized microfluorometry investigations

Results of studies on the modulation of skeletal muscle contraction by phosphorylation of myosin regulatory light chains and by exchange of magnesium for calcium in myosin heads were reviewed. The polarized fluorescence method was used in these studies, and conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction were investigated. It was found that both the exchange of bound magnesium for calcium on myosin heads and the phosphorylation of myosin regulatory light chains control the ability of myosin heads to induce, upon binding to actin, conformational changes of thin filament leading to decrease or increase of its flexibility. The changes in actin filament flexibility may be caused by alteration of both the inter- and the intramonomer structural organization.     

Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin

Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified either with 1,5-IAEDANS (N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine), as energy transfer donor, or with IAF (5-(iodoacetamido)fluorescein) or DABMI (4-dimethylaminophenylazophenyl-4'-maleimide), as energy transfer acceptor. The sulfhydryl groups on these light chains are located at different positions within the regulatory light-chain primary sequence; this enables one to probe a variety of locations, with respect to regulatory light-chain topology, on each myosin head. These independently modified regulatory light chains were added back to desensitized scallop myosin under a variety of conditions, including biphasic re-addition, the aim being to maximize the number of interhead energy transfer couples present. The efficiency of energy transfer was determined on the same samples by both steady-state and time-decay techniques. Results obtained by these two techniques were in good agreement with each other and indicated that the efficiency of energy transfer did not exceed 20% in any of the hybrids studied. Transfer efficiencies were invariant, irrespective of the presence or absence of MgATP, calcium or actin, either separately or in combination. Results using heavy meromyosin at low ionic strength were identical. It is shown that these results, in conjunction with the results of recent crosslinking studies performed on comparable myosin hybrids, may place certain restrictions on the configurations of the two heads of myosin.     

Segmental flexibility and head-head interaction in scallop myosin. A study using saturation transfer electron paramagnetic resonance spectroscopy

Saturation transfer electron paramagnetic resonance spectroscopy was used to investigate the rotational motion of the head domains of native and desensitized scallop myosin and its proteolytic subfragments. Scallop myosin was spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, which reacted with a heavy chain residue in the subfragment 1 domain. As previously shown for rabbit skeletal muscle myosin (Thomas et al., 1975), the two head domains of native scallop myosin appear to have independent motion (rotational correlation time, pi, = 0.8 X 10(-7) s for subfragment 1; 1.4 X 10(-7) s for myosin). However, removal of a regulatory light chain, to effect desensitization of the actin-activated ATPase, was associated with an increase in pi for myosin to a value of 2.4 X 10(-6) s. The Ca2+ sensitivity and initial correlation time were restored on recombination of the regulatory light chain in the presence of Mg2+. Sedimentation velocity profiles in an analytical ultracentrifuge indicated that the desensitized myosin preparations were largely monomeric and therefore the change in pi appears to reflect an intramolecular event. Addition of EDTA to spin-labelled scallop heavy meromyosin caused an immediate 2.5 to 4-fold increase in pi and a partial desensitization of the ATPase activity. Comparable experiments with subfragment 1 yielded a barely detectable increase in pi (1.5-fold) in the first ten minutes. The restricted rotational motion observed in desensitized myosin and heavy meromyosin could arise by a conformational change in the subfragment 1-subfragment 2 hinge region or by an association of one head with its partner. The latter mechanism, involving the exposed light chain binding site, would also explain the preferential release of one regulatory light chain from scallop myosin, and might account for some other co-operative effects observed in this molecule (Bagshaw, 1980).     

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.     

Amino acid sequences of the two kinds of regulatory light chains of adductor smooth muscle myosin from Patinopecten yessoensis

Smooth muscle myosin from scallop (Patinopecten yessoensis) adductor muscle contains two kinds of regulatory light chains (regulatory light chains a and b), and myosin having regulatory light chain a is suggested to be suitable for inducing "catch contraction" rather than myosin having regulatory light chain b (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673-681). The amino acid sequences of these two light chains were determined and compared. Regulatory light chain a consists of 161 amino acid residues, while regulatory light chain b consist of 156 amino acid residues. Amino acid substitutions and insertions were found only in the N-terminal regions of the sequences. The structural difference between the two light chains may contribute to the functional difference between myosins having regulatory light chains a and b.