Interaction of the N-terminal part of the A1 essential light chain with the myosin heavy chain.

The kinetics of actin-dependent MgATPase activity of skeletal muscle myosin subfragment 1 (S1) isoform containing the A1 essential light chain differ from those of the S1 isoform containing the A2 essential light chain. The differences are due to the presence of the extra N-terminal peptide comprising 42 amino acid residues in the A1 light chain. This peptide can interact with actin; heretofore, there have no been reports of the direct interaction between this peptide and the heavy chain of S1. Here, using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and S. aureus V8 protease, we show for the first time that the N-terminal part of the A1-light chain can interact with the 22-kDa fragment of the S1 heavy chain. No such interaction has been observed for the S1(A2) isoenzyme. Localization of residues which can possibly react with the cross-linker suggests that the interaction might involve the N-terminal residues of the A1 light chain and the converter region of the heavy chain.

Truncation of vertebrate striated muscle myosin light chains disturbs calcium-induced structural transitions in synthetic myosin filaments.

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca(2+)-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca(2+)-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca(2+)-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions.

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.

Calcium-induced structural changes in synthetic myosin filaments of vertebrate striated muscles.

Using negative staining, freeze-drying, and shadowing techniques in electron microscopy we have for the first time demonstrated Ca-induced reversible structural transitions in the synthetic filaments of dephosphorylated column-purified rabbit skeletal and cardiac muscle myosins formed by dialysis against solutions containing 120 mM KCl, 1 mM MgCl(2), 10 mM imidazole-HCl buffer (pH 7.0), and either 0.1 mM CaCl(2) or 1 mM EGTA. It has been revealed that the compact ordered structure of the filaments with myosin heads and subfragments-2 (S2) disposed close to the filament backbone with an axial periodicity of about 14.5 nm in the absence of Ca(2+) transforms into a spread disordered structure due to the movement of the heads and S2 away from the filament surface in the presence of Ca(2+). Increasing the pH from neutrality to pH 7.8 leads to a spread, disordered structure while decreasing the pH value to 6.5 returns the filaments to their compact, rather ordered state independent of the Ca(2+) concentrations used. The fact that the reversible structural transitions in synthetic filaments of myosin are observed in the absence of actin and actin- and myosin-associated proteins suggests that Ca(2+)-induced S2 movement is an intrinsic property of myosin itself. Ca(2+)-induced S2 mobility may reflect the existence of functionally significant communications between the myosin head domains and the tails of myosin molecules in thick filaments, and its disappearance can be an indicator of the impairment of these communications, for example, in acute ischemia and myocardial infarction.

Dual effect of actin on the accessibility of myosin essential light chain A1 to papain cleavage.

The influence of various amounts of actin on the proteolytic susceptibility of myosin essential light chain (ELC) A1, the binding of isolated A1 light chain and the N-peptide spanning N-terminal sequence of A1 to actin is studied to obtain more information on the role of the N-terminus of A1 light chain in the myosin-actin interaction. Low ratios of actin to myosin (1:1) lead to stimulate cleavage, whereas higher ratios (4:1) lead to protection of A1. Exposure of A1 by actin is especially seen in heavy meromyosin (HMM) and monomeric myosin and this is probably related to the full saturation of actin protomers with myosin heads. The protecting action of actin on A1 cleavage is more pronounced in myosin filaments. Conditions favoring the saturation of myosin regulatory light chain (RLC) with calcium ions instead of magnesium ions promotes the protection of A1. Cross-linking of HMM and actin results in higher yields of A1-actin product at high actin to myosin heads ratios. Isolated A1 light chain is pelleted by actin. A synthetic peptide spanning the N-terminal sequence of A1 can be cross-linked to actin. It is postulated that the protective action of actin on A1 papain cleavage is caused by the binding of the A1 N-terminus to actin. Changes in the RLC phosphorylation level and magnesium-for-calcium exchange in RLC may affect the probability of this interaction.

The shortening of the N-terminus of myosin essential light chain A1 influences the interaction of heavy meromyosin with actin.

The effects resulting from the removal of the N-terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC-phalloidin labelled actin in ghost fibers is used as a tool for sensing conformational changes of rigor complex of phosphorylated and dephosphorylated HMM with actin filament. The experiments were performed both under conditions assuring saturation of RLC with magnesium cation (4 mM EGTA) or calcium cation (0.1 mM CaCl2), and in constant presence of 1 mM magnesium chloride. HMM native and with A1 shortened from the N-terminus is used. As it was observed previously rigor complex of actin filament and native HMM shows sensitivity to the kind of cation saturating RLC and to the phosphorylation status of RLC. In particular, the sin2 theta parameter of actin bound rhodamine-phalloidin fluorescence polarization representing roughly the flexibility of actin filament HMM complex changes significantly with the changes of RLC phosphorylation and cation saturation. Removal of the N-terminus of A1 reduces this sensitivity to cation and phosphorylation both in the case of dephosphorylated and phosphorylated HMM. Our results suggest that the N-terminus of A1 plays significant role in the rigor interaction of myosin heads with actin and is involved in modulatory function of RLC in this interaction.

The possible role of myosin A1 light chain in the weakening of actin-myosin interaction.

The effects resulting from the removal of the N-terminus of myosin A1 by limited papain cleavage are investigated. The myosin and heavy meromyosin K+-ATPase and Ca2+-ATPase activities, and actin-activated ATPase activity of heavy meromyosin (HMM) and subfragment-1, are studied. Myosin and HMM preparations devoid of the A1 N-terminus exhibits lower Ca2+-ATPase activities at low ionic strength whereas no differences in K+- or Ca2+-ATPase activities are observed at high ionic strength. Direct binding of actin to monomeric myosin under K+-activated ATPase conditions is much more effective for myosin containing a shortened A1 light chain. The kinetic constants K(app) for actin and V(max) are calculated from actin-activation curves for HMM and subfragment-1. The kinetic constants for HMM are determined under conditions assuring saturation of regulatory light chains (RLC) either with Mg2+ or Ca2+. The removal of the A1 N-terminus influences the actin-myosin interaction in a Ca2+- and phosphorylation-dependent manner; in most cases, this leads to an increase in affinity. In the case of subfragment-1, the removal of the N-terminus of A1 led to a decrease in affinity. It is reasonable to assume that the intact A1 light chain may cause weakening of the actin-myosin interaction under certain conditions. This weakening may be regulated by RLC phosphorylation and RLC-bound calcium-for-magnesium exchange. Such an effect requires a structural minimum that is present in HMM but not in subfragment-1. Implications of such a role for the A1 N-terminus in the myosin-actin interaction are discussed.

Serine base exchange enzyme activity is modulated by sphingosine and other amphiphilic compounds: possible role of positive charge in increasing the synthesis of phosphatidylserine.

It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) have a stimulatory, and cholesterol 3-sulfate (an amphiphilic anion), an inhibitory, effect on [14C]serine incorporation into phosphatidylserine in glioma C6 and rat liver microsomes. In glioma intact cells sphingosine stimulates phosphatidylserine synthesis in a process independent of protein kinase C, but suppressed by thapsigargin. We suggest that the stimulation of the enzyme occurs by the interaction of amphiphilic cations with the membrane cosubstrate phospholipids, leading to a charge redistribution on their phosphate groups, and hence facilitating the enzyme action. A new hypothesis concerning the mechanism of the serine base exchange reaction is discussed.

The role of the skeletal muscle myosin light chains N-terminal fragments.

The myosin regulatory and essential light chains in skeletal muscle do not play a role as significant as in scallop or smooth muscle, however, there are some data suggesting that the skeletal myosin light chains and their N-terminal parts may have a modulatory function in the interaction of actin with myosin heads. In this paper four conformational states of the myosin head with respect to the regulatory light chain bound cation (magnesium or calcium) and phosphorylation are proposed. Communication between regulatory and essential light chains and putative binding of the N-terminus of A1 essential light chain to actin is discussed.

Significance of the N-terminal fragment of myosin regulatory light chain for myosin-actin interaction.

The influence of myosin regulatory light chains (LC2s) lacking the 2kD N-terminal portions of these chains, on internal organization of myosin heads and on actin-myosin interaction was studied with limited proteolysis and polarized fluorescence methods. For these studies heavy meromyosin (HMM) preparations were used: HMM containing intact LC2s (phosphorylated or dephosphorylated) and HMM containing LC2s lacking the 2kD N-terminal portions (including serine which can be phosphorylated). It was found that the susceptibility of the heavy chain cleavage site to trypsin and the alkali light chain (LC1) site to papain of myosin containing shortened regulatory LC2s, is not dependent on saturation of LC2s with Ca2+ or Mg2+ ions. This is in contrast to the myosin containing intact LC2s where Ca2+ or Mg2+ ion saturation does demonstrate a dependence. Similarly, in spectroscopic experiments, dephosphorylated HMM containing intact LC2s causes decrease or increase of actin filament flexibility depending on whether Mg2+ or Ca2+ are bound to LC2s. Correspondingly, HMM with shortened LC2s induces only increase of actin flexibility despite cations being bound. We conclude that the N-terminal fragment of LC2 is important for ensuring a proper Ca2+ dependent conformation of myosin head in the course of its actin-activated ATP hydrolysis.

Interaction of calponin with actin and its functional implications.

Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.

Skeletal muscle myosin regulatory light chains conformation affects the papain cleavage of A1 light chains.

In the present study, the influence of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (RLC) on accessibility of myosin and heavy meromyosin alkali light chains (A1) for papain digestion was investigated. The properties of native and papain treated myosin and heavy meromyosin were compared. Exchange of magnesium ions bound to RLCs for calcium ions accelerates the digestion of A1 in the presence of ATP in dephosphorylated myosin, heavy meromyosin, acto-myosin and the acto-heavy meromyosin complex. In the absence of ATP the exchange of magnesium ions bound to RLCs for calcium ions delays the digestion of A1 in the acto-myosin complex. Myosin and heavy meromyosin having shortened A1 by papain cleavage shows decreased K(+)-ATPase and increased actin binding ability in the presence and absence of ATP. The cooperation of RLC and A1 with heavy chains in the changes of structural organization of myosin head during muscle contraction is discussed.

The actin-activated ATPase of co-polymer filaments of myosin and myosin-rod.

The actin activated ATPase of myosin at low ionic strength shows a complex dependence on actin concentration, in contrast with the simple hyperbolic actin activation kinetics of heavy meromyosin and subfragment-1. To investigate how the aggregation of myosin influences the actomyosin ATPase kinetics, we have studied the actin-activated ATPase of mixed filaments in which the myosin molecules are separated from each other by copolymerization with myosin rod. Electron microscopy of copolymer filaments, alone and bound to actin, indicates that the myosin heads are distributed randomly along the co-polymer filaments. The actin-activated ATPase of myosin decreases with increasing rod, approaching a plateau of about 30% of the control at a rod/myosin molar ratio of 4:1. The decrease in ATPase persists even at Vmax, the extrapolated limit at infinite actin, indicating that it is not due merely to the loss of cooperative actin binding. Furthermore, the actin dependence of the ATPase still shows a biphasic character like that of control myosin, even at rod/myosin ratio of 12:1, so this complexity is not probably due solely to the structural proximity of myosin molecules, but may involve a non-equivalence of myosin heads or myosin molecules in the filament environment.

Regulatory light chain influences alterations of myosin head induced by actin.

The effect of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromyosin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.

Ca(2+)-dependent effects of C-protein on the actin-activated ATPase of phosphorylated and dephosphorylated skeletal muscle myosin.

The effects of C-protein on actin-activated myosin ATPase depending on Ca(2+)-level and LC2-phosphorylation were studied. Column-purified myosin and non-regulated actin were used. At ionic strength of 0.06 C-protein inhibits actomyosin ATPase activity both in the presence and in the absence of calcium, more effective in the case of dephosphorylated myosin. For this myosin, at mu = 0.12 C-protein activates actomyosin ATPase at pCa4, but slightly inhibits at pCa8. No such effects have been observed in the case of phosphorylated myosin. The possibility of coordinative action of LC2-chains and C-protein in regulatory mechanism of skeletal muscle contraction is discussed.

Contractile proteins in globally "stunned" rabbit myocardium.

The isolated working rabbit heart preparation was used to study whether the "contractile machinery" remains unchanged in globally stunned myocardium. The function of the heart has been measured in nonischemic and postischemic conditions. The effect of isoprenaline or calcium chloride administration in both conditions was also studied. Myocardial contractile function was significantly depressed after 20-min global ischemia and returned to normal after CaCl2 and supranormal values after isoprenaline administration. From hearts used in experiments myofibrils were prepared and their ATPase activity was determined. It was observed that myofibrils prepared from "stunned" myocardium showed about 50% increase in ATPase activity in the presence of CaCl2. Subjection of the heart to ischemia caused a decrease in calcium sensitivity of the myofibrillar ATPase. Myofibrils obtained from ischemic hearts but subjected to isoprenaline or CaCl2 administration exhibited increased calcium sensitivity over that of control heart. These effects were accompanied by changes in the extent of phosphorylation of troponin I (TNI) and myosin light chains. The modification of contractile apparatus in the postischemic period described in this paper may contribute to the overall mechanism of myocardial stunning.

Effects of skeletal muscle myosin light chain phosphorylation on synthetic actomyosin ATPase activity and superprecipitation.

In the present study the effect of phosphorylation of skeletal muscle myosin light chains on the interaction between myosin and actin has been investigated. The actomyosin ATPase activities were determined for synthetic actomyosins formed from either phosphorylated or non-phosphorylated myosin and pure actin, with the help of the luciferin-luciferase system. The contractile properties of our preparations were simultaneously studied by the superprecipitation model. Phosphorylated form of myosin had lower actin-activated ATPase activity at particular conditions studied. In agreement with this, superprecipitation of phosphorylated actomyosin was delayed. On the basis of these results one can expect that phosphorylation of myosin light chains modulates contractile properties of intact skeletal muscle.