The kinetics of bivalent metal ion dissociation from myosin subfragments.

Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.     

Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains

The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

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.     

Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease

Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.     

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.     

Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

Both heads of tissue-derived smooth muscle heavy meromyosin bind to actin in the presence of ADP

The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240-23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states.     

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.     

A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity.

Myosin from the striated adductor muscle of the scallop Pecten maximus is shown to fold into a compact 10 S conformer under relaxing conditions, as has been characterized for smooth and non-muscle myosins. The folding transition is accompanied by the trapping of nucleotide at the active site to give a species with a half-life of about an hour at 20 degrees C. Ca2+ binding to the specific, regulatory sites on a myosin head promotes unfolding to the extended 6 S conformer and activates product release by 60-fold. The unfolding transition, however, remains much slower than the contraction-relaxation cycle of scallop striated muscle and could not play a role in the regulation of these events. The dissociation of products from myosin heads in native thick filaments is Ca2(+)-regulated, but under relaxing conditions the nucleotide is released at least an order of magnitude faster than from the 10 S monomeric myosin, at a rate similar to that observed with heavy meromyosin. Thus, there is no evidence for any intermolecular interaction between neighbouring molecules in the filament analogous to the head-neck intramolecular interaction in the 10 S conformer. It is possible that the 10 S myosin state represents an inert form involved in the control of filament assembly during muscle growth and development. Removal of regulatory light chains or labelling the reactive heavy chain thiol of myosin prevents, or at least disfavours, formation of the folded 10 S conformer and allows separation of the modified protein from the native molecules.     

Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.

The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.     

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.     

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 disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.     

Reversible dissociation of dog cardiac myosin regulatory light chain 2 and its influence on ATP hydrolysis

The regulatory light chains of dog heart myosin were removed by digestion with myopathic hamster neutral protease. The heavy chains were also cleaved to an extent of 15%, but a homogeneous, rod-free LC2-deficient myosin was obtained by ion-exchange chromatography. A similar approach was used to prepare LC2-deficient heavy meromyosin. Neither Ca2+- nor K+-EDTA-activated ATPases were affected by LC2 removal. The Lineweaver-Burk plots for actin-activated ATPase in 25 mM KCl were biphasic giving a Vmax of 1.54 s-1 for control and LC2-recombined myosins and 1.08 s-1 for LC2-deficient myosin at low actin concentrations. At high actin concentrations, the Vmax for control and recombined myosins was 2.33 s-1 and 1.39 s-1 for LC2-deficient myosin. Increasing the KCl concentration in the reaction mixtures resulted in more linear plots without suppressing the 35-45% decrease in Vmax that accompanied LC2 removal. The results from assays with control and LC2-deficient heavy meromyosin performed in the absence of KCl, paralleled those obtained with myosin. The latter was also assayed in the presence of equimolar concentrations of C-protein in 50 mM KCl: C-protein induced a significant increase in the actin-activated ATPase of both control and LC2-recombined myosins, with no effect on LC2-deficient myosin. The Vmax for actin-activation in the presence of C-protein was 2.38 s-1, 0.83 s-1, and 1.71 s-1 for control, LC2-deficient, and recombined myosins, respectively. The enhancement of actin-activation in both the control and LC2-recombined myosins represents a possible role for C-protein in a LC2-mediated potentiation of actomyosin ATPase.     

Localization of a light-chain binding site on smooth muscle myosin revealed by light-chain overlay of sodium dodecyl sulfate-polyacrylamide electrophoretic gels

The interactions of smooth muscle myosin and its light chains have been examined by incubating sodium dodecyl sulfate-polyacrylamide gels of myosin with radioactively labeled regulatory or essential light chains. The technique involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fixation with methanol and acetic acid followed by an extensive series of washes. The gel is incubated overnight with labeled light chains in the presence of bovine serum albumin and then washed extensively to remove unbound protein. Following staining and destaining, the gel is autoradiographed to reveal which protein bands have bound light chain. The myosin heavy chain was able to rebind labeled regulatory or essential light chains despite the harsh procedure described above. By fragmenting the myosin heavy chain proteolytically, we were able to determine the binding site for both types of light chains to be within the 26,000-Da COOH-terminal segment of smooth muscle subfragment 1 (S-1) or the 20,000-Da COOH-terminal segment of skeletal muscle S-1. The extent of binding was 0.1-0.4 mol of light chain/mol of S-1 heavy chain. No binding was observed to portions of the myosin molecule which do not contain this segment such as myosin rod, light meromyosin, S-2, or the NH2-terminal 75,000-Da segment of S-1.     

Effects of actin and calcium ion on chymotryptic digestion of skeletal myosin and their implications to the function of light chains

Experiments have been carried out to assess the involvement of the myosin light chains [obtained by treatment of myosin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2)] in the control of cross-bridge movement and actomyosin interactions. Chymotryptic digestions of myosin, actomyosin, and myofibrils do not detect any Ca2+-induced change in the subfragment 2 region of myosin. Actin, like Ca2+, protects the in situ Nbs2 light chains from proteolysis and causes a partial switch in the digestion product of myosin from subfragment 1 to heavy meromyosin. This effect is independent of the state of aggregation of myosin, and it persists in acto heavy meromyosin and in actinomyosin in 0.6 M NaCl. Digestions and sedimentation studies indicate that there is no direct acto light chain interaction. Proteolysis of myosin shows a gradual transition from production of heavy meromyosin to subfragment 1 with lowering of the salt level. In the presence of Ca2+ heavy meromyosin is generated both in digestions of polymeric and of monomeric myosin. These results are explained in terms of localized changes within the Nbs2 light chains and subfragment 1. Subunit interactions in the myosin head lead to a Ca2+-induced reduction in the affinity of heavy meromyosin for actin in the presence of MgATP. The resulting Ca2+ inhibition of the actin-activated ATPase of myosin can be detected at high salt concentrations(75 mM KCl).     

A spin-label study of phosphatidylcholine exchange protein. Regulation of the activity by phosphatidylserine and calcium ion.

The effects of the divalent cations Mg2+, Mn2+ and Ca2+ on the Brownian rotational motion of fluorescently labeled myosin, heavy meromyosin and myosin subfragment-1 were measured by the method of time-resolved fluorescence depolarization. When Mg2+ was added to solutions of myosin or heavy meromyosin and EDTA, their rotational mobility increased. Ca2+ had no effect. Mn2+ increased the mobility of heavy meromyosin but decreased that of myosin. None of these divalent cations effected the mobility of subfragment-1. The binding of heavy meromyosin to actin was affected very little by Mg2+ or EDTA over a wide range of conditions. Divalent cations appear to change the swivel about which the heads of myosin rotate, presumably by binding to light chain 2 (also called DTNB light chain). However, the heads are still able to bind actin in nearly the same way whether Mg2+ is present or not. The concentration of free Mg2+ for the mid-point of the change in heavy meromyosin mobility is in good agreement with that for EDTA activation of ATPase activity. This suggests that EDTA activation is due to removal of Mg2+ bound to myosin itself.     

Regulation of asymmetric smooth muscle myosin II molecules

The emerging view of smooth/nonmuscle myosin regulation suggests that the attainment of the completely inhibited state requires numerous weak interactions between components of the two heads and the myosin rod. To further examine the nature of the structural requirements for regulation, we engineered smooth muscle heavy meromyosin molecules that contained one complete head and truncations of the second head. These truncations eliminated the motor domain but retained two, one, or no light chains. All constructs contained 37 heptads of rod sequence. None of the truncated constructs displayed complete regulation of both ATPase and motility, reinforcing the idea that interactions between motor domains are necessary for complete regulation. Surprisingly, the rate of ADP release was slowed by regulatory light chain dephosphorylation of the truncated construct that contained all four light chains and one motor domain. These data suggest that there is a second step (ADP release) in the smooth muscle myosin-actin-activated ATPase cycle that is modulated by regulatory light chain phosphorylation. This may be part of the mechanism underlying "latch" in smooth muscle.     

Two different preparations of subfragment-1 from scallop adductor myosin

Chymotryptic digestion of scallop myosin yielded two different preparations of subfragment-1, having the following features. The major product from chymotryptic digestion of scallop myosin was subfragment-1 (S1) either in Ca-medium or in EDTA-medium. However, the S1 preparations obtained from the digestion in Ca-medium, abbreviated as Ca-S1(CT), had both types of light chain subunits (regulatory light chains (R-LC) and essential light chains (SH-LC], and 100 Kdaltons (Kd) heavy chain subfragments (HCs), whereas the S1 preparations obtained from the digestion in EDTA-medium, ED-S1(CT), had no R-LC, partially fragmented SH-LC (SH-LC), and 90 Kd HCs. On the other hand, Ca-S1(CT) and ED-S1(CT) were practically identical with each other in ATPase activity and in actin-binding ability. The two S1 preparations were also identical in that the Mg-ATPase activity of both S1 and acto-S1 was insensitive to calcium ions. Ca-S1(CT), which contained both R-LC and SH-LC in a stoichiometric amount, was further digested with trypsin, which is known to cleave rabbit skeletal myosin not only at the head-tail junction but also in the head. The tryptic digestion of Ca-S1(CT) appeared, in terms of the SDS-gel electrophoretic pattern, to occur at a much faster rate in Ca-medium than in EDTA-medium, and with a different digestion profile. It is therefore suggested that association of R-LC induces changes in the heavy chain conformation which result in an increase in the proteolytic digestibility of heavy chains and in an alteration of the site of proteolytic cleavage on heavy chains.