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Akio Nakamura,
Ce Xie,
Yue Zhang,
Ying Gao,
Hong-Hui Wang,
Li-Hong Ye,
Hiroko Kishi, Tsuyoshi Okagaki,
Shinji Yoshiyama,
Kohichi Hayakawa,
Ryoki Ishikawa,
Kazuhiro Kohama
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ABSTRACT: Myosin light-chain kinase (MLCK) of smooth muscle consists of an actin-binding domain at the N-terminal, the catalytic domain in the central portion, and the myosin-binding domain at the C-terminal. The kinase activity is mediated by the catalytic domain that phosphorylates the myosin light-chain of 20kDa (MLC20), activating smooth muscle myosin to interact with actin. Although the regulatory role of the kinase activity is well established, the role of non-kinase activity derived from actin-binding and myosin-binding domains remains unknown. This review is dedicated to Dr. Setsuro Ebashi, who devoted himself to elucidating the non-kinase activity of MLCK after establishing calcium regulation through troponin in skeletal and cardiac muscles. He proposed that the actin-myosin interaction of smooth muscle could be activated by the non-kinase activity of MLCK, a mechanism that is quite independent of MLC20 phosphorylation. The authors will extend his proposal for the role of non-kinase activity. In this review, we express MLCK and its fragments as recombinant proteins to examine their effects on the actin-myosin interaction in vitro. We also down-regulate MLCK in the cultured smooth muscle cells, and propose that MLC20 phosphorylation is not obligatory for the smooth muscle to contract.
Biochemical and Biophysical Research Communications 05/2008; 369(1):135-43. · 2.48 Impact Factor
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ABSTRACT: The thermal stability of carp G-actin was investigated by monitoring loss of actin polymerization ability. To determine the amount of native actin remaining after heat treatment, actin was labeled with a fluorescence reagent, N-(1-pyrene)iodoacetamide. The loss of polymerization ability of carp actin during heat treatment, at between 45 and 55°C, occurred faster than that of chicken actin. The inactivation rate was influenced by concentrations of ATP and Ca2+ in solution. With the increase of Ca2+ concentration, the inactivation of carp actin was markedly suppressed. Furthermore, the activation energy of the inactivation of carp actin obtained from an Arrhenius plot was similar to that of chicken actin. These results indicated that the thermal instability of carp G-actin was due to the low affinites of ATP and Ca2+ for carp actin described in a previous report.
Fisheries Science 01/2008; 74(1):193 - 199. · 0.94 Impact Factor
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ABSTRACT: Chara myosin, two-headed plant myosin belonging to class XI, slides F-actin at maximally 60 microm s(-1). To elucidate the mechanism of this fast sliding, we extensively investigated its mechanochemical properties. The maximum actin activated ATPase activity, Vmax, was 21.3 s(-1) head(-1) in a solution, but when myosin was immobilized on the surface, its activity was 57.6 s(-1) head(-1) at 2 mg ml(-1) of F-actin. The sliding velocity and the actin activated ATPase activity were greatly inhibited by ADP, suggesting that ADP dissociation was the rate limiting step. With the extensive assay of motility by varying the surface density, the duty ratio of Chara myosin was found to be 0.49-0.44 from velocity measurements and 0.34 from the landing rate analysis. At the surface density of 10 molecules microm(-2), Chara myosin exhibited pivot movement under physiological conditions. Based on the results obtained, we will discuss the sliding mechanism of Chara myosin according to the working stroke model in terms of its physiological aspects. aspects.
Cell Motility and the Cytoskeleton 03/2007; 64(2):131-42. · 4.19 Impact Factor
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ABSTRACT: Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.
Journal of Biochemistry 10/2005; 138(3):255-62. · 2.37 Impact Factor
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ABSTRACT: Myosin light chain kinase (MLCK) has been purified from various muscles as an enzyme to phosphorylate myosin light chains. While the regulatory role of smooth muscle MLCK is well understood, the role of skeletal muscle MLCK in the regulation of contraction has not been fully characterized. Such characterization of skeletal muscle MLCK is difficult because skeletal muscle myosin interacts with actin whether or not the myosin is phosphorylated. Taking the hint from our recent finding that smooth muscle MLCK inhibits the actin-myosin interaction by binding to actin (Kohama et al., Biochem Biophys Res Commun 184: 1204-1211, 1992), we investigated the regulatory role of the actin-binding activity of MLCK from chicken breast muscle in the actin-myosin interaction. The amount of MLCK that bound to actin increased with increases in the concentration of MLCK. However, MLCK hardly bound to myosin. The actin-binding activity of MLCK was affected when Ca2+ and calmodulin (Ca2+-CaM) were present. The effect of MLCK on the actin-myosin interaction was examined by an in vitro motility assay; the movement of actin-filaments on a myosin-coated glass surface was inhibited by increasing the concentration of MLCK. When CaM was present, the inhibition was overcome in a Ca2+-dependent manner at M levels. The inhibition of the movement by MLCK and the recovery from the inhibition by Ca2+-CaM were not altered whether we use phosphorylated or unphosphorylated myosin for the assay, ruling out the involvement of the kinase activity of MLCK.
Molecular and Cellular Biochemistry 12/1998; 190(1):85-90. · 2.06 Impact Factor
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ABSTRACT: In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through
which it can regulate the actin-myosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa,
R., Shimmen, T., and Inoue, A. (1992) Biochem. Biophys. Res. Commun. 184, 1204–1211). In this study, we have analyzed the actin binding activity of MLCK and related it to its amino acid sequence
by producing native and recombinant fragments of MLCK. Parent MLCK exhibited both calcium ion (Ca2+) and calmodulin (Ca2+/CaM)-sensitive and Ca2+/CaM-insensitive binding to actin filaments. The native fragment, which consists of the Met1–Lys114 sequence (Kanoh, S., Ito, M., Niwa, E., Kawano, Y., and Hartshorne, D. J. (1993)Biochemistry 32, 8902–8907), and the recombinant NN fragment, which contains this 1–114 sequence, showed only Ca2+/CaM-sensitive binding. An inhibitory effect of the NN fragment on the actin-myosin interaction was observed by assayingin vitro motility and by measuring the actin-activated ATPase activity of myosin. The recombinant NN/41 fragment, which is constructed
without the Met1–Pro41 sequence of the NN fragment, lost both the actin binding activity and the inhibitory effect. We confirmed the importance
of the 1–41 sequence by using a few synthetic peptides to compete against the NN fragment in binding to actin filaments. The
experiments using recombinant fragments and synthetic peptides also revealed that the site for CaM-binding is the Pro26–Pro41 sequence. The site for the Ca2+/CaM-insensitive binding, which is shown to be localized between the Ca2+/CaM-sensitive site and the central kinase domain of MLCK, exerted no regulatory effects on the actin-myosin interaction.
Journal of Biological Chemistry 12/1997; 272(51):32182-32189. · 4.77 Impact Factor
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ABSTRACT: From the plasmodia of a lower eukaryote,Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca2+. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations ofPhysarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.
Journal of Muscle Research and Cell Motility 01/1992; 13(3):321-328. · 1.98 Impact Factor
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ABSTRACT: Actin-activated ATPase activity of myosin from Physarum polycephalum decreases when it binds Ca2+ and increases when it loses Ca2+. This Ca-inhibition is observed with phosphorylated myosin [Kohama, K. (1990) Trend. Pharmacol Sci . 11, 433–435]. The activity of dephosphorylated myosin remained at a low level both in the presence and .absence of Ca2+, although Ca2+-binding ability was much the same as that of the phosphorylated myosin. The effect of phosphorylation has been studied at a conventional actin concentration, which is comparable with that of myosin by weight. When the concentration of actin was increased by 10 times, the dephosphorylated myosin became actin-activatable in the absence of Ca2+, and Ca-inhibition was recovered. As actin exists quite abundantly in non-muscle cells of Physarum , myosin phosphorylation plays virtually no role in regulating actin-myosin-ATP interaction in vivo . Physiologically the interaction may be regulated by Ca2+ by binding to and subsequent release from myosin. Latex beads coated by either phosphorylated or dephosphorylated myosin moved ATP-dependently on the actin cables of Characeae cells to the same extent in the absence of Ca2+, but the movement was abolished by increasing Ca2+. When the interaction was examined by monitoring the movement of actin filaments on myosin fixed on a coverslip, the movement and Ca-inhibition of the movement were detected with phosphorylated, not dephosphorylated, myosin [Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1989) J. Biochem. 106, 955–957]. The discrepancy between the in vitro motility assays suggests that the former monitors the interaction at a high concentration of actin and the latter, that at a conventional, low concentration of actin. The former assay should be more useful as a monitor of the physiological interaction.
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ABSTRACT: Physarum myosin is uniquely under an inhibitory Ca2+-regulation in the ATP-dependent interaction with actin [Kohama (1990) Trends Pharmacol. Sci. 11, 433–435, for review]. Calcium-binding light chain (CaLc) has been suggested to be of primary importance to the control from its amino acid sequence [Kobayashi et al . (1988) J. BioL Chem . 263, 305–313]. To provide a biochemical basis for this suggestion, the Ca-binding capacity of CaLc and its K d for Ca2+ were measured. The Ca-binding properties of CaLc allowed those of Physarum myosin to be explained in terms of CaLc. However, the mode of Ca2x+-regulation by CaLc differs according to the enzyme upon which Ca-sensitivity is confered by CaLc, i.e., CaLc activated bovine phosphodiesterase activity and inhibited Physarum myosin ATPase activity, with the same K d in μM levels. Thus, CaLc appears to work as a mere Ca-receptive subunit in Physarum myosin, with the secret of the inhibition lying in other subunits. CaLc was also shown to belong to a family of alkali light chains (AlLc) by allowing it to bind skeletal myosin as a substitute for its AlLc. Therefore, present study is the first biochemical indication that the AlLc family is involved in regulating the myosin function.
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ABSTRACT: Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain. The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1–Pro-41 sequence of MLCK binds to actin to inhibit this interaction. However, it is not known whether the myosin-binding domain modifies the actin–myosin interaction. We designed MLCK⋅cDNA to overexpress the Asp-777–Glu-972 sequence in Escherichia coli. The purified Asp-777–Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (Vmax = 7.36 ± 0.44-fold, Km = 1.06 ± 0.20 μM, n = 4). When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816–Glu-972, lost the stimulatory activity. We synthesized the Ala-777–Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (Vmax = 3.03 ± 0.22-fold, Km = 6.93 ± 1.61 μM, n = 3). When this peptide was further divided into Asp-777–Met-795 and Ala-796–Ser-815 peptides, the stimulatory activity was found in the latter. We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides. Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active.
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ABSTRACT: Myosin was isolated from two types of muscle, ordinary and dark muscles, of three species of fish living in sea water. The compositions of light chains were visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the mechanochemical activity was examined by in vitro motility and ATPase assays. Ordinary muscle myosin of either species had three species of light chain, whereas dark muscle myosin had another two species of light chain judged by SDS-PAGE. Sliding velocity of ordinary muscle myosin was in the range of 4.92-6.89 μm/s, whereas that of dark muscle myosin was in the range of 3.07-4.25 μm/s. Therefore, ordinary muscle myosin showed 1.26-1.95 times higher sliding velocity than dark muscle myosin in either species. The ratios of Vmax of actin-activated Mg2+ -ATPase activity of ordinary to dark muscle myosins were correlated quite well to the ratios of sliding velocity. Activity of ordinary muscle myosin was comparable to that mammalian fast muscle myosin, but that of dark muscle myosin was twice of that of mammalian slow muscle myosin. These results may reflect the essential role of fish dark muscle myosin always used in slow cruising.
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ABSTRACT: C-protein is a myosin-associated protein of vertebrate striated muscle, and its function and properties have been extensively examined. However, there has been no report of C-protein of fish skeletal muscle so far. C-protein was identified in carp skeletal muscle by immunoassay using antibody against chicken C-protein, and the muscle-type specific C-protein was purified from carp ordinary and dark muscles for the first time. Although C-protein could be prepared from crude myosin by the reported procedure, C-protein degraded appreciably during the purification steps. Accordingly, C-protein was selectively extracted from the muscle with 0.15 M K-phosphate buffer (pH 5.8), and purified by ammonium sulfate fractionation, followed by AF-blue chromatography. Myosin free from the accessory proteins was obtained by diethylaminoethyl(DEAE) chromatography and used to assay the binding of C-protein with myosin. Ordinary muscle C-protein bound to ordinary muscle myosin in a saturable manner, but its maximum amount of binding was approximately twice that of dark muscle myosin. Similarly, dark muscle C-protein bound to dark muscle myosin much more than to ordinary muscle myosin. These results suggest that C-protein isoforms specifically bound with myosin isoforms originated from the same type of muscle.
