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Haruo Sugi - One of the best experts on this subject based on the ideXlab platform.
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tension recovery following ramp shaped release in high ca and low ca rigor Muscle Fibers evidence for the dynamic state of amadp myosin heads in the absence of atp
PLOS ONE, 2016Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Maki Yamaguchi, Tetsuo Ohno, Hiroshi OkuyamaAbstract:During Muscle Contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned Muscle Fiber Contraction, though it covers the actin-binding sites on myosin. It follows from this that, during Muscle Contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in Muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas Muscle Fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The Fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor Fibers, while it tended to decrease quickly in low-Ca rigor Fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor Fibers, while it completely eliminated the tension recovery in low-Ca rigor Fibers. These results suggest that the AMADP myosin heads in rigor Muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in Muscle is discussed in connection with the X-ray diffraction pattern from contracting Muscle, which is intermediate between resting and rigor Muscles.
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Evidence against the Swinging Lever Arm Mechanism in MuscleContraction Based on the Effect of Antibodies to Myosin Head
Journal of Nanomedicine & Nanotechnology, 2016Co-Authors: Haruo Sugi, Shigeru ChaenAbstract:During Muscle Contraction, myosin heads extending from myosin filaments first attach to actin filaments, perform power stroke producing force and motion in Muscle, and then detach from actin filaments. A myosin head (or myosin subfragment-1, S-1) consists of catalytic and lever arm domains (CAD and LD), which are connected via converter domain (COD). It is widely believed that the myosin head power stroke is caused by swinging lever arm mechanism, which assumes active rotation of the LD around the COD, caused by structural changes in and around the COD. The lever arm mechanism is, however, constructed from nucleotide-dependent structural changes of crystals of truncated myosin head, consisting only of the CAD and the COD, and therefore overlooks possible role of the LD and myosin subfragment-2 (S-2), connecting myosin heads to myosin filament backbone. In this article, we present evidence against the lever arm hypothesis based on the following results: (1) antibody to reactive lysine residue (Lys83) located in the COD (anti-RLR antibody) has no effect on Ca2+-activated Muscle Fiber Contraction; (2) using the gas environmental chamber attached to electron microscope, we record ATP-induced power stroke of myosin heads, position-marked with anti-RLR antibody; (3) antibodies to myosin head LD (anti-LD antibody) and to myosin S-2 (anti-S-2 antibody) inhibit Ca2+-activated Contraction without changing MgATPase activity. The absence of inhibitory effect of anti-RLR antibody, attaching to RLR in the COD, makes the lever arm mechanism unlikely, since attachment of bulky antibody (IgG) to RLR in the COD is expected to inhibit structural changes in and around the COD. Meanwhile, inhibitory effect of anti-LD and anti-S-2 antibodies indicate essential role of the LD and the S-2 in Muscle Contraction.
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definite differences between in vitro actin myosin sliding and Muscle Contraction as revealed using antibodies to myosin head
PLOS ONE, 2014Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle Contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in Muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated Contraction of Muscle Fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor Muscle Fiber Contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on Muscle Contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in Muscle Fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and Muscle Contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Suguru Tanokura, Yoshiki Ohnuki, Haruo SugiAbstract:Mechanism of myosin head power stroke, responsible for Muscle Contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over myosin fixed on a glass surface, it is not clear whether the in vitro actin-myosin sliding takes place by a mechanism similar to Contraction in Muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the myosin head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the myosin head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- myosin sliding and Contraction of skinned rabbit psoas Muscle Fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-myosin sliding without changing actin-activated myosin head ATPase activity, while it showed no effect on Ca2+-activated Contraction of Muscle Fibers; (2) anti-LD antibody had no effect on in vitro actin-myosin sliding, but suppressed Ca2+-activated Muscle Fiber Contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-myosin sliding and Muscle Contraction.
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Definite Difference Between In Vitro Actin-Myosin Sliding and Muscle Contraction Revealed by the Effect of Antibody to Myosin Head Converter Domain
Biophysical Journal, 2012Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Hiroki MinodaAbstract:Myofilament sliding in Muscle is believed to result from rotation of the myosin head catalytic domain (CAD) around the converter domain (CD). To explore the validity of this mechanism, we compared the effect of antibody to myosin head converter domain (IgG, anti-CD antibody) between in vitro actin-myosin sliding and Muscle Fiber Contraction. In agreement with the expectation that binding of massive antibody to the CD impairs rotation of the CAD around the CD, the ATP-dependent sliding of actin filaments over myosin heads on a glass surface was inhibited by the antibody (0.14mg/ml). Meanwhile, the antibody (up to 1.5mg/ml) showed no appreciable effect on the actin-activated myosin head ATPase activity, indicating that the antibody has no effect on the ATPase activity in the CAD. Unexpectedly, the antibody (up to 3mg/ml) showed no appreciable effect on the maximum Ca2+-activated isometric force, the maximum shortening velocity, and the Mg-activated ATPase activity in glycerol-extracted rabbit psoas Muscle Fibers. The possibility that the antibody does not diffuse into Muscle Fibers can be excluded by our published results that other antibodies readily inhibit Muscle Fiber Contraction. These findings therefore suggest that the antibody binding to the myosin head CD does not impair performance of myosin heads producing force and motion in Muscle Fibers.
Takakazu Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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tension recovery following ramp shaped release in high ca and low ca rigor Muscle Fibers evidence for the dynamic state of amadp myosin heads in the absence of atp
PLOS ONE, 2016Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Maki Yamaguchi, Tetsuo Ohno, Hiroshi OkuyamaAbstract:During Muscle Contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned Muscle Fiber Contraction, though it covers the actin-binding sites on myosin. It follows from this that, during Muscle Contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in Muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas Muscle Fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The Fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor Fibers, while it tended to decrease quickly in low-Ca rigor Fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor Fibers, while it completely eliminated the tension recovery in low-Ca rigor Fibers. These results suggest that the AMADP myosin heads in rigor Muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in Muscle is discussed in connection with the X-ray diffraction pattern from contracting Muscle, which is intermediate between resting and rigor Muscles.
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definite differences between in vitro actin myosin sliding and Muscle Contraction as revealed using antibodies to myosin head
PLOS ONE, 2014Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle Contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in Muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated Contraction of Muscle Fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor Muscle Fiber Contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on Muscle Contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in Muscle Fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and Muscle Contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Suguru Tanokura, Yoshiki Ohnuki, Haruo SugiAbstract:Mechanism of myosin head power stroke, responsible for Muscle Contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over myosin fixed on a glass surface, it is not clear whether the in vitro actin-myosin sliding takes place by a mechanism similar to Contraction in Muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the myosin head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the myosin head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- myosin sliding and Contraction of skinned rabbit psoas Muscle Fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-myosin sliding without changing actin-activated myosin head ATPase activity, while it showed no effect on Ca2+-activated Contraction of Muscle Fibers; (2) anti-LD antibody had no effect on in vitro actin-myosin sliding, but suppressed Ca2+-activated Muscle Fiber Contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-myosin sliding and Muscle Contraction.
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Definite Difference Between In Vitro Actin-Myosin Sliding and Muscle Contraction Revealed by the Effect of Antibody to Myosin Head Converter Domain
Biophysical Journal, 2012Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Hiroki MinodaAbstract:Myofilament sliding in Muscle is believed to result from rotation of the myosin head catalytic domain (CAD) around the converter domain (CD). To explore the validity of this mechanism, we compared the effect of antibody to myosin head converter domain (IgG, anti-CD antibody) between in vitro actin-myosin sliding and Muscle Fiber Contraction. In agreement with the expectation that binding of massive antibody to the CD impairs rotation of the CAD around the CD, the ATP-dependent sliding of actin filaments over myosin heads on a glass surface was inhibited by the antibody (0.14mg/ml). Meanwhile, the antibody (up to 1.5mg/ml) showed no appreciable effect on the actin-activated myosin head ATPase activity, indicating that the antibody has no effect on the ATPase activity in the CAD. Unexpectedly, the antibody (up to 3mg/ml) showed no appreciable effect on the maximum Ca2+-activated isometric force, the maximum shortening velocity, and the Mg-activated ATPase activity in glycerol-extracted rabbit psoas Muscle Fibers. The possibility that the antibody does not diffuse into Muscle Fibers can be excluded by our published results that other antibodies readily inhibit Muscle Fiber Contraction. These findings therefore suggest that the antibody binding to the myosin head CD does not impair performance of myosin heads producing force and motion in Muscle Fibers.
Shigeru Chaen - One of the best experts on this subject based on the ideXlab platform.
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tension recovery following ramp shaped release in high ca and low ca rigor Muscle Fibers evidence for the dynamic state of amadp myosin heads in the absence of atp
PLOS ONE, 2016Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Maki Yamaguchi, Tetsuo Ohno, Hiroshi OkuyamaAbstract:During Muscle Contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned Muscle Fiber Contraction, though it covers the actin-binding sites on myosin. It follows from this that, during Muscle Contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in Muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas Muscle Fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The Fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor Fibers, while it tended to decrease quickly in low-Ca rigor Fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor Fibers, while it completely eliminated the tension recovery in low-Ca rigor Fibers. These results suggest that the AMADP myosin heads in rigor Muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in Muscle is discussed in connection with the X-ray diffraction pattern from contracting Muscle, which is intermediate between resting and rigor Muscles.
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Evidence against the Swinging Lever Arm Mechanism in MuscleContraction Based on the Effect of Antibodies to Myosin Head
Journal of Nanomedicine & Nanotechnology, 2016Co-Authors: Haruo Sugi, Shigeru ChaenAbstract:During Muscle Contraction, myosin heads extending from myosin filaments first attach to actin filaments, perform power stroke producing force and motion in Muscle, and then detach from actin filaments. A myosin head (or myosin subfragment-1, S-1) consists of catalytic and lever arm domains (CAD and LD), which are connected via converter domain (COD). It is widely believed that the myosin head power stroke is caused by swinging lever arm mechanism, which assumes active rotation of the LD around the COD, caused by structural changes in and around the COD. The lever arm mechanism is, however, constructed from nucleotide-dependent structural changes of crystals of truncated myosin head, consisting only of the CAD and the COD, and therefore overlooks possible role of the LD and myosin subfragment-2 (S-2), connecting myosin heads to myosin filament backbone. In this article, we present evidence against the lever arm hypothesis based on the following results: (1) antibody to reactive lysine residue (Lys83) located in the COD (anti-RLR antibody) has no effect on Ca2+-activated Muscle Fiber Contraction; (2) using the gas environmental chamber attached to electron microscope, we record ATP-induced power stroke of myosin heads, position-marked with anti-RLR antibody; (3) antibodies to myosin head LD (anti-LD antibody) and to myosin S-2 (anti-S-2 antibody) inhibit Ca2+-activated Contraction without changing MgATPase activity. The absence of inhibitory effect of anti-RLR antibody, attaching to RLR in the COD, makes the lever arm mechanism unlikely, since attachment of bulky antibody (IgG) to RLR in the COD is expected to inhibit structural changes in and around the COD. Meanwhile, inhibitory effect of anti-LD and anti-S-2 antibodies indicate essential role of the LD and the S-2 in Muscle Contraction.
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definite differences between in vitro actin myosin sliding and Muscle Contraction as revealed using antibodies to myosin head
PLOS ONE, 2014Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle Contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in Muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated Contraction of Muscle Fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor Muscle Fiber Contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on Muscle Contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in Muscle Fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and Muscle Contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Suguru Tanokura, Yoshiki Ohnuki, Haruo SugiAbstract:Mechanism of myosin head power stroke, responsible for Muscle Contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over myosin fixed on a glass surface, it is not clear whether the in vitro actin-myosin sliding takes place by a mechanism similar to Contraction in Muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the myosin head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the myosin head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- myosin sliding and Contraction of skinned rabbit psoas Muscle Fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-myosin sliding without changing actin-activated myosin head ATPase activity, while it showed no effect on Ca2+-activated Contraction of Muscle Fibers; (2) anti-LD antibody had no effect on in vitro actin-myosin sliding, but suppressed Ca2+-activated Muscle Fiber Contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-myosin sliding and Muscle Contraction.
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Definite Difference Between In Vitro Actin-Myosin Sliding and Muscle Contraction Revealed by the Effect of Antibody to Myosin Head Converter Domain
Biophysical Journal, 2012Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Hiroki MinodaAbstract:Myofilament sliding in Muscle is believed to result from rotation of the myosin head catalytic domain (CAD) around the converter domain (CD). To explore the validity of this mechanism, we compared the effect of antibody to myosin head converter domain (IgG, anti-CD antibody) between in vitro actin-myosin sliding and Muscle Fiber Contraction. In agreement with the expectation that binding of massive antibody to the CD impairs rotation of the CAD around the CD, the ATP-dependent sliding of actin filaments over myosin heads on a glass surface was inhibited by the antibody (0.14mg/ml). Meanwhile, the antibody (up to 1.5mg/ml) showed no appreciable effect on the actin-activated myosin head ATPase activity, indicating that the antibody has no effect on the ATPase activity in the CAD. Unexpectedly, the antibody (up to 3mg/ml) showed no appreciable effect on the maximum Ca2+-activated isometric force, the maximum shortening velocity, and the Mg-activated ATPase activity in glycerol-extracted rabbit psoas Muscle Fibers. The possibility that the antibody does not diffuse into Muscle Fibers can be excluded by our published results that other antibodies readily inhibit Muscle Fiber Contraction. These findings therefore suggest that the antibody binding to the myosin head CD does not impair performance of myosin heads producing force and motion in Muscle Fibers.
Takuya Miyakawa - One of the best experts on this subject based on the ideXlab platform.
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definite differences between in vitro actin myosin sliding and Muscle Contraction as revealed using antibodies to myosin head
PLOS ONE, 2014Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle Contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in Muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated Contraction of Muscle Fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor Muscle Fiber Contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on Muscle Contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in Muscle Fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and Muscle Contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Suguru Tanokura, Yoshiki Ohnuki, Haruo SugiAbstract:Mechanism of myosin head power stroke, responsible for Muscle Contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over myosin fixed on a glass surface, it is not clear whether the in vitro actin-myosin sliding takes place by a mechanism similar to Contraction in Muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the myosin head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the myosin head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- myosin sliding and Contraction of skinned rabbit psoas Muscle Fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-myosin sliding without changing actin-activated myosin head ATPase activity, while it showed no effect on Ca2+-activated Contraction of Muscle Fibers; (2) anti-LD antibody had no effect on in vitro actin-myosin sliding, but suppressed Ca2+-activated Muscle Fiber Contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-myosin sliding and Muscle Contraction.
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Definite Difference Between In Vitro Actin-Myosin Sliding and Muscle Contraction Revealed by the Effect of Antibody to Myosin Head Converter Domain
Biophysical Journal, 2012Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Hiroki MinodaAbstract:Myofilament sliding in Muscle is believed to result from rotation of the myosin head catalytic domain (CAD) around the converter domain (CD). To explore the validity of this mechanism, we compared the effect of antibody to myosin head converter domain (IgG, anti-CD antibody) between in vitro actin-myosin sliding and Muscle Fiber Contraction. In agreement with the expectation that binding of massive antibody to the CD impairs rotation of the CAD around the CD, the ATP-dependent sliding of actin filaments over myosin heads on a glass surface was inhibited by the antibody (0.14mg/ml). Meanwhile, the antibody (up to 1.5mg/ml) showed no appreciable effect on the actin-activated myosin head ATPase activity, indicating that the antibody has no effect on the ATPase activity in the CAD. Unexpectedly, the antibody (up to 3mg/ml) showed no appreciable effect on the maximum Ca2+-activated isometric force, the maximum shortening velocity, and the Mg-activated ATPase activity in glycerol-extracted rabbit psoas Muscle Fibers. The possibility that the antibody does not diffuse into Muscle Fibers can be excluded by our published results that other antibodies readily inhibit Muscle Fiber Contraction. These findings therefore suggest that the antibody binding to the myosin head CD does not impair performance of myosin heads producing force and motion in Muscle Fibers.
Takahiro Abe - One of the best experts on this subject based on the ideXlab platform.
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definite differences between in vitro actin myosin sliding and Muscle Contraction as revealed using antibodies to myosin head
PLOS ONE, 2014Co-Authors: Haruo Sugi, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle Contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in Muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated Contraction of Muscle Fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor Muscle Fiber Contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on Muscle Contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in Muscle Fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and Muscle Contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takakazu Kobayashi, Shigeru Chaen, Takuya Miyakawa, Suguru Tanokura, Yoshiki Ohnuki, Haruo SugiAbstract:Mechanism of myosin head power stroke, responsible for Muscle Contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over myosin fixed on a glass surface, it is not clear whether the in vitro actin-myosin sliding takes place by a mechanism similar to Contraction in Muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the myosin head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the myosin head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- myosin sliding and Contraction of skinned rabbit psoas Muscle Fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-myosin sliding without changing actin-activated myosin head ATPase activity, while it showed no effect on Ca2+-activated Contraction of Muscle Fibers; (2) anti-LD antibody had no effect on in vitro actin-myosin sliding, but suppressed Ca2+-activated Muscle Fiber Contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-myosin sliding and Muscle Contraction.
