Distinct interactions between actin and essential myosin light chain isoforms

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein–protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin^ala3) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (K_D = 575 nM) was significantly (p < 0.01) lower compared with the affinity of hVLC-1 to α-actin (K_D = 186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p < 0.01) lower association rate (k_on: 1018 M⁻¹ s⁻¹) compared with k_on of the hVLC-1/α-actin complex interaction (2908 M⁻¹ s⁻¹). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.     

Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin

A low-speed centrifugation assay has been used to examine the binding of myosin filaments to F-action and to regulated actin in the presence of MgATP. While the cross-linking of F-actin by myosin was Ca2+ insensitive, much less regulated actin was cross-linked by myosin in the absence of Ca2+ than in its presence. Removal of the 19000-dalton, phosphorylatable light chain from myosin resulted in the loss of this Ca2+ sensitivity. Readdition of this light chain partially restored the Ca2+-sensitive cross-linking of regulated actin by myosin. Urea gel electrophoresis has been used to distinguish that fraction of heavy meromyosin which contains intact phosphorylatable light chain from that which contains a 17000-dalton fragment of this light chain. In the absence of Ca2+, heavy meromyosin which contained digested light chain bound to regulated actin in MgATP about 10-fold more tightly than did heavy meromyosin which contained intact light chain. The regulated actin-activated ATPases of heavy meromyosin also showed that cleavage of this light chain causes a substantial increase in the affinity of heavy meromyosin for regulated actin in the absence of Ca2+. Thus, the binding of both myosin and heavy meromyosin to regulated actin is Ca2+ sensitive, and this sensitivity is dependent on the phosphorylatable light chain.     

Influence of the regulatory light chain of fast skeletal muscle myosin on its interaction with actin in the presence and absence of ATP

The effect of myosin LC2 modifications (phosphorylation or selective proteolytic removal of a seven-residue N-terminal peptide) and partial or complete removal of the whole LC2 was studied under various conditions. (1) Actin binding in the absence of ATP is not influenced by the nature of the myosin species (phosphorylated, dephosphorylated or devoid of LC2). (2) A 50% inhibition of K+/EDTA-ATPase was obtained with actin concentrations hardly different when phosphorylated and dephosphorylated myosins were compared (of the order of 5 microM), whereas both myosin devoid of LC2 and myosin in which the LC2 N-terminal peptide has been removed required significantly higher concentrations of actin (13.0 +/- 2 and 12.0 +/- 2.0 microM, respectively). (3) Dissociation of the actomyosin complex at high ionic strength with nucleotides is not influenced by phosphorylation. (4) Actin activation of Mg2+-ATPase is enhanced when LC2 is phosphorylated; no activation enhancement is observed with myosin devoid of LC2. (5) Translational diffusion coefficient measurements of myosin in high-ionic-strength solutions indicate a tendency for LC2-deprived myosin to form autoassociation oligomers. It thus appears that a structural modification (partial cleavage or removal of LC2) induces important structural changes in myosin, pointing to a role for LC2 in the intrinsic conformation of the molecule and its interaction potentialities. Effects of LC2 removal at high ionic strength are best explained by interactions bearing no relationship to physiological functions. A physiologically significant effect of LC2 phosphorylation requires a minimum degree of organization (actomyosin complex) to be expressed in which LC2 could play the role of a return-spring in the cross-bridge mechanism.     

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.     

Role of 17-kDa essential light chain isoforms of aorta smooth muscle myosin.

Aorta smooth muscle myosin contains two kinds of 17-kDa essential light chain, LC17nm (nonmuscle-type) and LC17gi (gizzard-type) [Hasegawa, Y., Ueda, Y., Watanabe, M., & Morita, F. (1992) J. Biochem. 111, 798-803]. The LC17 isoforms were released from porcine aorta myosin by incubation at 46 degrees C. The rate of release was 1.5 to 2 times higher with LC17gi than with LC17nm. Aorta myosins containing the two LC17 isoforms in various ratios could be reconstituted. The actin-activated ATPase activity was measured as a function of LC17nm content. The Vm value was lower with myosin which contained more LC17nm. The apparent dissociation constant for F-actin, Km, was 20-fold less with myosin which contained 81% LC17nm than myosin which contained 23% LC17nm. A similar difference in the dissociation constants of myosin for F-actin was observed in the presence of adenylyl imidodiphosphate. The role of LC17nm appears to be to make aorta myosin suitable for maintaining the muscle tension with a low expenditure of energy. The isoform-dependent difference in the F-actin-binding affinities of myosin seems partly due to the difference in the affinities of LC17 isoforms themselves for F-actin. We found that the isolated LC17nm itself could bind with F-actin with a dissociation constant of 64 microM, but LC17gi could not.(ABSTRACT TRUNCATED AT 250 WORDS)     

A sensitive SDS-PAGE method separating myosin heavy chain isoforms of rat skeletal muscles reveals the heterogeneous nature of the embryonic myosin

Myosin isoforms are used as markers of heterogeneity and plasticity of skeletal muscle fibers and motor units. Tedious and time-consuming methods, needing microgram or milligram amounts of myosin are widely used to characterize the heavy subunits. We here describe a sensitive method that separates in nanogram or microgram amounts the heavy chains of immature, fast and slow adult rat muscles in complex mixtures of myosins. Though the method is assembled from published procedures (SDS-PAGE, peptide mapping in the presence of SDS, silver stain) for the logical extensions introduced the end-product is a powerful tool to separate and characterize these high molecular weight biopolymers until now inseparable from complex mixtures. The method reveals the heterogeneous nature of the embryonic myosin heavy chains.     

Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.     

Phosphorylation of myosin light chain modulates the in vitro movement of fibrils composed of actin and myosin filaments from skeletal muscle

In vitro movement of fibrils composed of actin and myosin filaments purified from skeletal muscle was observed by dark field microscopy during superprecipitation at low ionic strengths at room temperature. The movement was activated by phosphorylation of light chain (LC2) of myosin. The activity of the movement was evaluated in terms of the spreading of the area where the fibrils were moving. Adenosine triphosphatase activity of actomyosin was also enhanced by phosphorylation of LC2 and was correlated with the activity of the in vitro movement.     

Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.     

The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.     

Kinetic effects of myosin regulatory light chain phosphorylation on skeletal muscle contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca(2+) regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2(fast), 2(slow), 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2(slow). In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca(2+) levels on isometric tension and rate of force redevelopment (k(TR)).     

Mechanisms underlying the asynchronous replacement of myosin light chain isoforms during stimulation-induced fibre-type transformation of skeletal muscle

During the fibre-type transformation induced by chronic electrical stimulation of rabbit fast-twitch muscle, replacement of the fast forms of the two classes of myosin light chain by their slow isoforms occurs asynchronously. Studies of total cellular myosin light chains and of the slow-to-fast transition now justify the conclusion that the asynchrony is due to switching between the expression of fast and slow genes for the two light chain classes at sequential stages of the transformation process.     

The correlation between the synthesis of skeletal muscle actin,myosin heavy chain,and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis

Cloned cDNA probes were used to measure the accumulation of myosin heavy chain, myosin light chain 2, and actin mRNA during differentiation of rat skeletal muscle cell cultures. This was compared with the changes in the rate of synthesis of the corresponding proteins. Accumulation of those mRNA sequences was detectable a few hours before the onset of the phase of cell fusion; however, the main increase in hybridizable RNA occurred during the phase of rapid cell fusion. A close correlation was found between the amounts of mRNAs coding for these proteins and the rate of synthesis of the proteins. The results suggest that the activation of stored mRNA is not a major mechanism for controlling the time at which these proteins are synthesized.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.