Two isoforms of 17-kDa essential light chain of aorta media smooth muscle myosin

Aorta myosin contains two kinds of light chain, 20-kDa phosphorylatable light chain and 17-kDa essential light chain (LC17). Purified myosin from porcine aorta media showed 3 distinct light chain bands on polyacrylamide gel electrophoresis (PAGE) in the presence of urea (urea-PAGE). The mobilities of the faster two components did not change after incubation of the myosin with a myosin light chain kinase. Gel slices containing the faster two bands were separately subjected to PAGE in the presence of sodium dodecylsulfate. Both components showed identical mobility with that of LC17. The two components were designated as LC17a and LC17b in increasing order of mobility on urea-PAGE. They were isolated by DEAE-Toyopearl ion exchange column chromatography. The amino acid compositions of LC17a and LC17b were similar to each other, but the contents of Ser, Met, Ile, and His were distinctly different. These results suggest that the two components are isoforms. The ratio of the content of each isoform (LC17a: LC17b) in the purified porcine aorta myosin was 39:61, and essentially the same ratio was found with washed muscle homogenate of porcine aorta. Then washed aorta muscle homogenates of rabbit and rat were examined. Two bands having similar mobilities to those of porcine homogenate were also found in urea-PAGE. The ratios of the two components were 31:69 and 66:34, respectively, for rabbit and rat. Aorta smooth muscle thus may contain many types of isomyosin.     

Inhibitory effect of MgATP on the release of regulatory light chain from scallop myosin and light chain composition of scallop myosin hybridized with abalone light chain 2 at 30 degrees C

The experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.     

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.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

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.     

Temperature-dependent conformational transition in the head-rod junctional region of the myosin molecule

The effects of temperature, Mg2+, ATP, and actin on the conformation of the neck region of the myosin head were studied by limited proteolysis of heavy meromyosin (HMM) and subfragment 1 (S1) preparations obtained by papain digestion of myosin in the presence of Mg2+ (Mg-S1) or EDTA (EDTA-S1). The preparations were fluorescently labelled at the SH1 thiol group to enable identification of the COOH-terminal fragments of the head portion of the heavy chain where this group is located. The results indicate that the head-rod junctional region of the myosin heavy chain contains at least three different sites readily susceptible to trypsin at 25 degrees C if the light chain LC2 or its LC2' fragment are absent. The susceptibility of one of these sites dramatically decreases when the temperature is lowered to 0 degree C, indicating a temperature-dependent conformational transition in the head-rod junction. With the method used, this transition is detectable only in LC2/LC'2-deficient preparations since all three sites are protected, although to different extents, by LC2 and its LC'2 derivative. It is, however, most probable that the effect of the light chain is confined to steric hindrance of trypsin access and that the temperature-dependent structural transition in the head-rod junction can occur in the presence of intact LC2 as well and may contribute to the temperature sensitivity of force generation in muscle.     

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.     

Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin

Three-dimensional reconstructions of “barbed” and “blunted” arrowheads (Craig et al., 1980) show that these two forms arise from arrangement of scallop myosin subfragments (S1) that appear about 40 Å longer in the presence of the regulatory light chain than in its absence. A similar difference in apparent length is indicated by images of single myosin subfragments in partially decorated filaments. The extra mass is located at the end of the subfragment furthest from actin, and probably comprises part of the regulatory light chain as well as a segment of the myosin heavy chain. The fact that barbed arrowheads are also formed by myosin subfragments from vertebrate striated and smooth muscles implies that the homologous light chains in these myosins have locations similar to that of the scallop light chain.The scallop light chain probably does not extend into the actin-binding site on the myosin head, and is therefore unlikely to interfere physically with binding. Rather, regulation of actin-myosin interaction by light chains may involve Ca²⁺-dependent changes in the structure of a region near the head-tail junction of myosin.The reconstructions suggest locations for actin and tropomyosin relative to myosin that are similar to those proposed by Taylor & Amos (1981) and are consistent with a revised steric blocking model for regulation by tropomyosin. The identification of actin from these reconstructions is supported by images of partially decorated filaments that display the polarity of the actin helix relative to that of bound myosin subfragments.     

平滑肌肌球蛋白磷酸化与肌动球蛋白功能调节

在有Ｃａ２＋和钙调蛋白存在时,肌球蛋白轻链激酶催化肌球蛋白磷酸化,促使肌动蛋白激活的肌球蛋白（肌动球蛋白）Ｍｇ２＋－ＡＴＰ酶活性显著增加．然而,肌球蛋白磷酸化水平与Ｍｇ２＋－ＡＴＰ酶之间的关系是非线性的,原肌球蛋白可以进一步增加Ｍｇ２＋－ＡＴＰ酶的活性,但仍不改变它们之间的非线性关系．肌球蛋白轻链激酶的合成肽抑制剂抑制了肌球蛋白磷酸化和Ｍｇ２＋－ＡＴＰ酶活性,并导致平滑肌去膜肌纤维的等长收缩张力与速度的降低．结果提示肌球蛋白轻链激酶参与脊椎动物平滑肌收缩的调节过程,肌球蛋白轻链磷酸化作用会引起平滑肌收缩     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

The regulatory light chain is required for folding of smooth muscle myosin

Light chain phosphorylation causes the folded monomeric form of myosin to extend and assemble into filaments. This observation established the involvement of the 20-kDa regulatory light chain (LC20) in conformational transitions of smooth muscle myosin. To further assess the role of this subunit in the intramolecular folding of myosin, LC20 was removed from turkey gizzard myosin at elevated temperatures in the presence of EDTA through the use of an antibody affinity column. Metal-shadowed images showed that LC20-deficient myosin had a tendency to aggregate through the neck region. When MgATP was added to filaments formed from this myosin, less than 10% of the myosin was solubilized, indicating that myosin could not fold in the absence of light chain. Readdition of native regulatory light chain restored the myosin to its original solubility properties, thus establishing reversibility. Addition of foreign light chains from skeletal muscle myosin or a chymotryptic-cleaved gizzard light chain produced the same amount of monomeric myosin in high salt that was obtained by recombination with the homologous light chain. However, the ability of the hybrid myosins to assume the folded conformation was impaired, and only a partially folded species was obtained. Single-headed myosin, like rod and light chain-deficient myosin, remained filamentous in the presence of MgATP. These results are consistent with the hypothesis that the regulatory light chain in the neck region of myosin contributes to a binding site for the myosin tail.     

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.     

Role of the regulatory light chains in skeletal muscle actomyosin ATPase and in minifilament formation

Incubation of myosin with myopathic hamster protease results in substantial (more than 80%) removal of light chain 2 (LC2) with limited breakdown of the heavy chains. LC2-deficient myosin, purified by ion exchange chromatography, migrates as a single, monodisperse boundary in the analytical ultracentrifuge. The Ca2+- and EDTA-activated ATPases of LC2-deficient myosin are similar to those of the control and LC2-recombined myosins indicating that no denaturation occurred in its preparation. Double reciprocal plots for LC2-deficient, control, and LC2-recombined myosins reveal a biphasic behavior i.e. at actin concentrations above 11 microM, there is a sharp break in the 1/V versus 1/[actin] plots for all samples. The Vm values for LC2-deficient myosin are 50% lower (at low actin, Vm = 3.0 s-1, and at high actin, Vm = 4.2 s-1) than those for control myosin (Vm = 5.3 s-1 at low actin and 8.3 s-1 at high actin). Readdition of LC2 to LC2-deficient myosin restores the actin-activated ATPase to control levels. Electron microscopy of shadow cast preparations reveals a subtle difference between LC2-deficient myosin, and control or recombined myosin. In control and recombined myosins, S1 heads appear "pear"-shaped, whereas in LC2-deficient myosin, the S1 heads are rounder and display a "thinning" of mass in the "neck" region, suggesting that LC2 binds at the S1/S2 junction. Furthermore, removal of LC2 apparently influences the assembly of myosin into minifilaments, as revealed to a certain degree, by an increase in the width of the bare zone, accompanied by a decrease in the stability of these minifilaments.     

Ca2+ binding to myosin regulatory light chain affects the conformation of the N-terminus of essential light chain and its binding to actin

We prepared a new type of skeletal myosin subfragment 1 (S1-MLC1F) containing both, the essential and the regulatory light chains, intact, by exchanging the essential light chains of papain S1 with bacterially expressed longer isoform (MLC1F) of this light chain. We then compared the enzymatic and structural properties of chymotryptic S1, papain S1, and S1-MLC1F in the presence and in the absence of Ca(2+) ions bound to the regulatory light chain. In the presence of Ca(2+), subfragment 1 containing both intact light chains exhibited lower V(max) and lower K(m) for actin activation of S1 ATPase. When S1-MLC1F was cross-linked to actin via the N-terminus of the essential light chain, the yield was much higher when Ca(2+) ions saturated the regulatory light chain. Limited proteolysis of the essential light chain in S1-MLC1F was significantly inhibited in the presence of calcium as compared to chymotryptic S1. We conclude that the effect of binding of Ca(2+) to the regulatory light chain is transmitted to the N-terminal extension of the longer isoform of the essential light chain. The resulting structure of the N-terminus is less susceptible to proteolytic digestion, binds tighter to actin, and has an inhibitory effect on actin-activated myosin ATPase. This new conformation of the N-terminus may be responsible for calcium induced myosin-linked modulation of striated muscle contraction.     

The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.     

Regulatory light chain-a myosin kinase (aMK) catalyzes phosphorylation of smooth muscle myosin heavy chains of scallop, Patinopecten yessoensis

Regulatory light chain-a myosin kinase (aMK), which phosphorylates one of the myosin regulatory light chains, RLC-a, contained in the catch muscle of scallop, was also found to phosphorylate heavy chains of scallop myosin. After incubation of myosin isolated from the opaque portion of scallop smooth muscle (opaque myosin) with aMK in the presence of [gamma-32P]ATP, about 2 mol of 32P was incorporated per mol of the myosin. The radioactivity was mostly found in the heavy chain at 0.26 M KCl. The pH-activity curve and MgCl2 requirement for the heavy chain phosphorylation were similar to those for RLC-a phosphorylation. In contrast, the dependency of activity on KCl concentration was different from that for RLC-a. The heavy chain phosphorylation activity decreased with increase in KCl concentration up to 0.06 M, and then increased at concentrations over 0.06 M to a maximum at around 0.26 M KCl. This complicated profile probably reflects the solubility of myosin, and the phosphorylation site may be located in the rod portion insoluble at low KCl concentrations. Phosphorylation of heavy chain did not change the solubility of the opaque myosin molecule at all. The acto-opaque myosin ATPase activity in the presence of Ca2+ was found to be decreased to less than one-fourth by the heavy chain phosphorylation.     

Conformational selection during weak binding at the actin and myosin interface

The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF(4), ADP-BeF(x), and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

Calcium sensitivity of abalone, Haliotis discus, myosin.

Superprecipitation was observed with abalone myosin and purified rabbit actin in the presence of calcium ions, but was not observed in the absence of calcium. The Mg-ATPase [EC 3.6.1.3] activity of abalone myosin and rabbit actin in the absence of calcium ions (EGTA present) showed about 60% inhibition as compared with values in the presence of calcium ions. The calcium sensitivity may be attributable to abalone myosin, as in the case of scallop myosin.     

Localization of an actin binding domain in smooth muscle myosin light chain kinase

Phosphorylation of the regulatory light chain of myosin II by myosinlight chain kinase is important for regulating many contractile processes.Smooth muscle myosin light chain kinase has been shown to be associated withboth actin and myosin filaments in vitro and in vivo. In this report wedefine an actin binding region by using molecular deletions to generaterecombinant mutant proteins that were analyzed by co-sedimentation withF-actin. An actin binding region restricted to residues 2-42 in the animoterminus of the rabbit smooth muscle myosin light chain kinase wasidentified.