Cross-linking between translationally equivalent sites on the two heads of myosin. Relationship to energy transfer results between the same pair of sites

Mercenaria myosin and scallop pure hybrid myosin possessing Mercenaria regulatory light chains were reacted with various concentrations of 4-4'-dimaleimidylstilbene-2-2'-disulfonic acid (DMSDS). Regulatory light chain homodimers are formed with great efficiency (20-50%). Dimers incorporating essential light chains were not formed upon reaction of DMSDS with Mercenaria myosin but some (less than 5%) essential light chain homodimers were obtained in the case of scallop hybrid myosin, probably occurring through relatively specific intermolecular associations within small myosin aggregates. Results were invariant, irrespective of the presence or absence of calcium and/or ATP. No radioactivity is incorporated into regulatory light chain homodimers upon post-labeling DMSDS-reacted myosin with 14C-labeled N-ethylmaleimide, irrespective of the original labeling ratio of DMSDS to myosin heads. This indicates the absence of free sulfhydryl groups in the regulatory light chain homodimer and suggests, therefore, that DMSDS links the two light chains together between translationally equivalent residues (Cys-50 of the Mercenaria regulatory light chain). These results imply that translationally equivalent sites on the two heads of myosin can come within 18 A of each other, the span of reacted DMSDS. Because energy transfer results between identical pairs of translationally equivalent sites on hybrid myosins indicated a low efficiency of energy transfer between these sites (Chantler, P.D., and Tao, T. (1986) J. Mol. Biol. 192, 87-99), it would appear that even though the two cysteines can come within 18 A of each other, their mean separation is much greater than this distance (greater than 50 A), a result consistent with a considerable flexibility of the two myosin heads with respect to each other.     

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.     

Electron microscopy of cross-linked scallop myosin

The N-terminal regions of the regulatory light chains on the two heads of scallop myosin can be cross-linked to one another. Electron microscopy of cross-linked myosin molecules, and of dimers of myosin subfragment-1 produced by digesting them with papain, shows that the site of cross-linking is very close to the head-rod junction.     

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.     

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.     

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.     

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.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

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.     

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.     

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.     

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.     

Phosphorylation-dependent regulation of Limulus myosin

Myosin from Limulus, the horseshoe crab, is shown to be regulated by a calcium-calmodulin-dependent phosphorylation of its regulatory light chains. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of a Limulus myosin preparation reveals three light chain bands. Two of these light chains have been termed regulatory light chains based on their ability to bind to light chain-denuded scallop myofibrils (Sellers, J. R., Chantler, P. D., and Szent-Gy?rgyi, A. G. (1980) J. Mol. Biol. 144, 223-245). Ths other light chain does not bind to these myofibrils and is thus termed the essential light chain. Both Limulus regulatory light chains can be phosphorylated with a highly purified turkey gizzard myosin light chain kinase or with a partially purified myosin light chain kinase which can be isolated from Limulus muscle by affinity chromatography on a calmodulin-Sepharose column. Phosphorylation with both of these enzymes requires calcium and calmodulin. Limulus myosin is isolated in an unphosphorylated form. The MgATPase of this unphosphorylated myosin is only slightly activated by rabbit skeletal muscle actin plus tropomyosin. The calcium-dependent phosphorylation of the myosin results in an increase in the actin-activated MgATPase rate. Once phosphorylated, the actin-activated MgATPase rate is only slightly modified by calcium. This suggests that calcium operates mainly at the level of the myosin kinase-calmodulin system.     

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.     

Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.     

Fluorescence intensity and UV absorption changes accompanying dissociation and association of regulatory light chain of scallop adductor myosin

Dissociation and association of regulatory light chains of scallop myosin were found to be accompanied by changes in the fluorescence intensity and in the UV absorption spectrum. The changes in the two optical properties of scallop myosin and the dissociation and association of regulatory light chains were studied as a function of the magnesium and calcium concentrations. The results thus obtained suggested that there are two different types of attachment between regulatory light chains and "desensitized" myosin; one type is a calcium-specific attachment, and the other type of attachment can be mediated by either calcium or magnesium ions. These changes in the optical properties of scallop myosin were distinguishable from those induced by Mg-ATP; for example, with "desensitized" scallop myosin, the former changes were not observed but the latter were.     

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.     

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

Summary Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10^–8 M Ca⁺⁺ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.     

Electron microscopy of negatively stained scallop myosin molecules. Effect of regulatory light chain removal on head structure

The heads of myosin molecules from the striated adductor muscle of scallop have been studied by electron microscopy after negative staining. In common with vertebrate skeletal muscle myosin visualized by this method, the scallop myosin heads were pear-shaped and often showed pronounced curvature. Staining suggestive of two or, more frequently, three domains could often be observed. Removal of regulatory light chains (R-LCs) resulted in a reduction in the length of the heads of about 2.6 nm, with no significant change in maximum width. In desensitized preparations a majority of heads displayed anticlockwise curvature, whereas intact heads were usually seen curved clockwise. Analysis of the head curvature in both intact and desensitized molecules was consistent with an ability of each head to rotate about its long axis. Desensitization resulted in an increased incidence of heads showing two domains. It seems likely that the reduction in length upon removal of the R-LC is due to the two small domains located in the neck region of the head collapsing into one.     

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.