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.     

Visualization of myosin in living cells

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.     

Association of microinjected myosin and its subfragments with myofibrils in living muscle cells

Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10- 15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.     

Fast skeletal muscle isoforms exhibit the highest incorporation level into myofibrils and stress fibers among members of myosin alkali light chain isoform family

Isoproteins of myosin alkali light chain (LC) were co-expressed in cultured chicken cardiomyocytes and fibroblasts and their incorporation levels into myofibrils and stress fibers were compared among members of the LC isoform family. In order to distinguish each isoform from the other, cDNAs of LC isoforms were tagged with different epitopes. Expressed LCs were detected with antibodies to the tags and their distribution was analyzed by confocal microscopy. In cardiomyocytes, the incorporation level of LC into myofibrils was shown to increase in the order from nonmuscle isoform (LC3nm), to slow skeletal muscle isoform (LC1sa), to slow skeletal/ventricular muscle isoform (LC1sb), and to fast skeletal muscle isoforms (LC1f and LC3f). Thus, the hierarchal order of the LC affinity for the cardiac myosin heavy chain (MHC) is identical to that obtained in the rat (Komiyama et al., 1996. J. Cell Sci., 109: 2089-2099), suggesting that this order may be common for taxonomic animal classes. In fibroblasts, the affinity of LC for the nonmuscle MHC in stress fibers was found to increase in the order from LC3nm, to LC1sb, to LC1sa, and to LC1f and LC3f. This order for the nonmuscle MHC is partly different from that for the cardiac MHC. This indicates that the order of the affinity of LC isoproteins for MHC varies depending on the MHC isoform. Further, for both the cardiac and nonmuscle MHCs, the fast skeletal muscle LCs exhibited the highest affinity. This suggests that the fast skeletal muscle LCs may be evolved isoforms possessing the ability to associate tightly with a variety of MHC isoforms.     

Single actomyosin motor interactions in skeletal muscle

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:10⁵. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~ 9 s during muscle contraction.     

Volume occupation, environment, and accessibility in proteins. Environment and molecular area of RNase-S

The myosin light chains of cultured muscle cells and embryonic muscle tissue have been examined by two-dimensional gel electrophoresis. Myosin purified from primary cultures of rat muscle cells or the myogenic cell line L6 contain not only the light chains corresponding to those of fast twitch muscle but also another protein, differing slightly in molecular weight and isoelectric point from the adult LC1 protein. By a number of criteria this additional protein is shown to be a myosin light chain: (1) it is found in highly purified myosin preparations; (2) in L6 myosin it replaces the other LC1-type light chains in stoichiometric amounts; (3) it is part of the subfragment-1 complex of myosin produced by chymotrypsin. as expected for an LC1-type light chain. Total extracts of fused cultured muscle cells, when analyzed by two-dimensional electrophoresis, contain substantial amounts of this additional LC1-type protein, strongly suggesting that it is not a proteolytic fragment produced during myosin isolation. Unfused cultures do not synthesize detectable amounts of the adult light chains or the additional LC1-type light chain. This additional LC1 protein can be detected in embryonic or newborn muscle tissue but it is not present in adult myosin or myofibrils. These results indicate that a novel form of myosin light chain, referred to as an embryonic LC1 or LC1_emb, is characteristic of the early stages of muscle development.     

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch,slow-twitch,and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s⁻¹) and super-relaxed states (SRX, 0.005 s⁻¹) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969–1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C–30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.     

Analysis of the metal-induced conformational change in myosin with a monoclonal antibody to light chain two

A monoclonal antibody capable of detecting a conformational change in myosin light chain two (LC2) was characterized in detail. The antibody was shown to bind only to myosin LC2 when tested against fast skeletal myosin (chicken pectoralis muscle). With cardiac or slow muscle myosins, the antibody exclusively recognized their first light chains (LC1). Staining of myofibrils by the monoclonal antibody could be observed only after their irreversible denaturation by acetone or ethanol, or after incubation of the myofibrils in divalent metal chelators. This latter effect was shown to be fully reversible. The metal effect was independent of ionic strength although the affinity of the antibody for myosin was depressed at high salt concentrations. Similar metal effects were detected in the binding of antibody to cardiac or slow myosins. Neither the metal nor the ionic strength-related inhibition of antibody binding were detected with denatured myosin. The antibody binding site overlaps one of the alpha-chymotryptic sites in LC2 protected by divalent metals. Electron microscopic observations of myosin-antibody complexes demonstrated that the antibody binding site is located near the head-rod junction of myosin. Since the binding site of this monoclonal antibody has been mapped by recombinant DNA methods to the junction of the first alpha-helical domain with the calcium binding site of LC2, the location of the calcium binding site must also be located near the head-tail junction of myosin. A model for conformational changes at the myosin head-tail junction is proposed to account for the metal-induced blockage of antibody binding and the inhibition of alpha-chymotryptic digestion of LC2.     

E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.     

Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species

Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.     

Coexistence of cardiac-type and fast skeletal-type myosin light chains in embryonic chicken cardiac muscle

Myosin from embryonic chicken ventricle contained a light chain component which comigrated with fast skeletal myosin light chain 1 (Lf1) on two dimensional electrophoresis in addition to cardiac type light chains (Lc1 and Lc2). Immunoblot analysis showed that this minor light chain band reacted with anti-Lf1 antibody. Antigens binding with anti-Lc1 and anti-Lf1 antibodies were located on myofibrils in embryonic cardiac muscle cells in vivo and in vitro. From these observations, we conclude that a small amount of Lf1 exists in embryonic chicken cardiac muscle.     

The power stroke causes changes in the orientation and mobility of the termini of essential light chain 1 of myosin

Binding of ATP to the catalytic domain of myosin induces a local conformational change which is believed to cause a major rotation of an 8.5 nm alpha-helix that is stabilized by the regulatory and essential light chains. Here we attempt to follow this rotation by measuring the mobility and orientation of a fluorescent probe attached near the C- or N-terminus of essential light chain 1 (LC1). Cysteine 178 of wild-type LC1, or Cys engineered near the N-terminus of mutant LC1, was labeled with tetramethylrhodamine and exchanged into skeletal subfragment-1 (S1) or into striated muscle fibers. In the absence of ATP, the fluorescence anisotropy (r) and the rotational correlation time (rho) of S1 reconstituted with LC1 labeled near the C-terminus were 0.195 and 66.6 ns, respectively. In the presence of ATP, r and rho increased to 0.233 and 233 ns, indicating considerable immobilization of the probe. A related parameter indicating the degree of order of cross-bridges in muscle fibers, Deltar, was small in rigor fibers (-0.009) and increased in relaxed fibers (0.030). For S1 reconstituted with LC1 labeled near the N-terminus, the steady-state anisotropy was 0.168 in rigor, and increased to 0.223 in relaxed state. In fibers, the difference in rigor was large (Deltar = 0.080), because of binding to the thin filaments, and decreased to 0.037 in relaxed fibers. These results suggest that before the power stroke, in the presence of ATP or its products of hydrolysis, the termini of LC1 are immobilized and ordered, and after the stroke, they become more mobile and partially disordered. The results are consistent with crystallographic structures that show that the level of putative stabilizing interactions of LC1 with the heavy chain of S1 in the transition state is reduced as the regulatory domain rotates to its post-power stroke position.     

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.     

Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils.

Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.     

Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.     

Association of melittin with the isolated myosin light chains

Melittin is a 26-residue peptide which undergoes high-affinity calcium-dependent binding by calmodulin [Barnette, M.S., Daly, R., & Weiss, B. (1983) Biochem. Pharmacol. 32, 2929; Comte, M., Maulet, Y., & Cox, J.A. (1983) Biochem. J. 209, 269; Anderson, S.R., & Malencik, D.A. (1986) Calcium Cell Funct. 6, 1]. The results in this paper show that three different types of myosin light chain--the smooth muscle regulatory light chain, the smooth muscle essential light chain, and the skeletal muscle regulatory 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chain--also associate with melittin. The resulting complexes have dissociation constants ranging from 1.1 to 2.5 microM in the presence of 0.10 M NaCl and from approximately 50 to approximately 130 nM in solutions of 20 mM 3-(N-morpholino)propanesulfonic acid alone. The regulatory smooth muscle myosin light chain exhibits two equivalent melittin binding sites while each of the others displays only one. The myosin light chains evidently contain elements of structure related to the macromolecular interaction sites present in calmodulin and troponin C but not in parvalbumin. The association of melittin and other peptides with the light chains requires consideration whenever assays of the calmodulin-dependent activity of myosin light chain kinase are used to determine peptide binding by calmodulin. The binding measurements performed on the DTNB light chain and melittin necessitated derivation of the equation relating complex formation to the observed fluorescence anisotropy of a solution containing three fluorescent components. This analysis is generally applicable to equilibria involving the association of two fluorescent molecules emitting in the same wavelength range.     

Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Tryptic digestion of rabbit skeletal myofibrils at physiological ionic strength and pH results in cleavage of the myosin heavy chain at one site giving two bands (M_r = 200,000 and 26,000) on sodium dodecyl sulfate/polyacrylamide gels. Following addition of sodium pyrophosphate (to 1 mm) to dissociate the myosin heads from actin, tryptic proteolysis results in production of three bands, 160K², 51K and 26K, with a 74K band appearing as a precursor of the 51K and 26K species. Under these conditions, there is insignificant cleavage of heavy chain to the heavy and light meromyosins. Trypsin-digested myofibrils yield the same amount of rod as native myofibrils when digested with papain. These results indicate that actin blocks tryptic cleavage of the myosin heavy chain at a site 74K from the N terminus. From measurements of the amount of 51K species formed by digestion of rigor fibers at various sarcomere lengths, we estimate that at least 95% of the myosin heads are bound to actin at 100% overlap of thick and thin filaments. Hence all myosin molecules can bind to actin, and consequently both heads of a myosin molecule can interact simultaneously with actin filaments under rigor conditions.     

Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.