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Journal of Muscle Research and Cell... Dec 2019Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in...
Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.
Topics: Actomyosin; Adenosine Triphosphate; Animals; Myosins; Rabbits
PubMed: 31556008
DOI: 10.1007/s10974-019-09556-4 -
Journal of Cell Science Apr 2021The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not...
The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formin from different species. Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and found that SMIFH2 inhibits activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions. This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper.
Topics: Actin Cytoskeleton; Actins; Animals; Cattle; Formins; Myosins
PubMed: 33589498
DOI: 10.1242/jcs.253708 -
Biophysical Journal Sep 1992The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro...
The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.
Topics: Actins; Adenosine Triphosphatases; Animals; Cation Transport Proteins; Dithionitrobenzoic Acid; Kinetics; Models, Biological; Muscle Contraction; Muscle Relaxation; Muscles; Myofibrils; Myosins; Rabbits
PubMed: 1420910
DOI: 10.1016/S0006-3495(92)81646-6 -
Cell Structure and Function Oct 1996The Acanthamoeba myosin-IA and myosin-IB molecular motors bind to membranes, so they may produce the force to move organelles and membranes along actin filaments. We... (Review)
Review
The Acanthamoeba myosin-IA and myosin-IB molecular motors bind to membranes, so they may produce the force to move organelles and membranes along actin filaments. We have determined the rate constants for the actin-activated myosin-I ATPase by pre-steady state kinetic analysis. ATP binds rapidly to myosin-I and dissociates the enzyme from actin filaments at a rate > 500 s-1. Myosin-I hydrolyzes ATP to ADP and inorganic phosphate (Pi) at 20-50 s-1. Phosphate dissociation is the rate limiting step in the ATPase cycle, 0.01 s-1 for myosin-I alone and at 10 s-1 when myosin-I is bound to actin filaments. ADP dissociation is rapid. Phosphorylation controls the ATPase cycle by increasing the rate of phosphate release from myosin-I bound to actin. At steady state the major species are myosin-ATP and myosin-ADP-Pi, which rapidly bind to and dissociate from actin filaments. During the ATPase cycle myosin-I binds so weakly to actin filaments that it cannot support processive movement like kinesin, unless several motors cluster together on a membrane or actin filament. These properties of the enzyme emphasize the importance of characterizing mechanisms that promote the self-association of myosin-I isoforms at specific binding sites in cells.
Topics: Acanthamoeba; Actins; Animals; Cell Movement; Evolution, Molecular; Myosin Heavy Chains; Myosins; Phosphorylation
PubMed: 9118240
DOI: 10.1247/csf.21.351 -
Archives of Biochemistry and Biophysics Feb 2019The effects of Mg on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional...
The effects of Mg on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional and spectroscopic methods. Mg inhibited striated muscle myosin ATPase in the presence of F-actin. Significant effects of Mg were observed in both rate constants of NOE build-up and maximal intensities in WaterLOGSY NMR experiments as F-actin concentration increased. In the absence of F-actin, myosin S1 with Mg bound to a fluorescent ADP analog about five-times tighter than without Mg. In the presence of F-actin, the affinity of myosin S1 toward the ADP analog significantly decreased both with and without Mg. The equilibrium titration of myosin-S1 into F-actin revealed that in the presence of ADP the apparent dissociation constant (K) without Mg was more than five-fold smaller than with Mg. Further, we examined effects of F-actin, ADP and Mg binding to myosin on the tertiary structure of myosin-S1 using near UV circular dichroism (CD) spectroscopy. Both in the presence and absence of ADP, there was a Mg-dependent difference in the near UV CD spectra of actomyosin. Our results show that Mg affects myosin-ADP and actin-myosin interactions which may be reflected in myosin ATPase activity.
Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphatases; Animals; Magnesium; Muscle, Striated; Myosins; Protein Binding
PubMed: 30529103
DOI: 10.1016/j.abb.2018.12.004 -
The Journal of Cell Biology Jun 1978Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA...
Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA Pharmaceuticals, New York) was essential in the isolation of pituitary myosin. Secretory tissue myosins were very similar to smooth muscle myosin, having a heavy chain of 200,000 daltons and light chains of 14,000 and 19,000 daltons. Salivary gland myosin cross-reacted with antibodies directed toward both smooth muscle myosin and fibroblast myosin, but not with antiskeletal muscel myosin serum. The specific myosin ATPase activity measured in 0.6 M KCl was present. Tissues associated with secretion of hormone granules contained substantial amounts of this ATPase, rat pancreatic islets having 4.5 times that of rat liver. Activation of low ionic strength myosin ATPase by actin could not be demonstrated despite adequate binding of the myosin to muscle actin and elution by MgATP. The myosins were located primarily in the cytoplasm as determined by cell fractionation and were quite soluble in buffers of low ionic strength.
Topics: Actins; Adenosine Triphosphatases; Animals; Aprotinin; Enzyme Activation; Fishes; Islets of Langerhans; Myosins; Pituitary Gland; Salivary Glands; Swine
PubMed: 150427
DOI: 10.1083/jcb.77.3.827 -
FEBS Letters Aug 1986Reversible phosphorylation of myosin subunits is observed in almost all eukaryotic cells. The data concerning sites and effects of phosphorylation on actin-activated... (Review)
Review
Reversible phosphorylation of myosin subunits is observed in almost all eukaryotic cells. The data concerning sites and effects of phosphorylation on actin-activated ATPase activity of myosin and on its filament formation are described. These observations are discussed in terms of possible evolutionary trends and rules which may govern the process of myosin phosphorylation.
Topics: Adenosine Triphosphatases; Amoeba; Animals; Biological Evolution; Dictyostelium; Eukaryota; Horseshoe Crabs; Muscle, Smooth; Myosins; Phosphorylation; Physarum
PubMed: 2942420
DOI: 10.1016/0014-5793(86)80806-7 -
The Journal of Biological Chemistry Jun 2018Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor...
Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state.
Topics: Actins; Adenosine Triphosphate; Amino Acid Sequence; Animals; Cardiac Myosins; Cardiomyopathy, Dilated; Cell Line; Humans; Kinetics; Mice; Models, Molecular; Myosin Heavy Chains; Point Mutation; Protein Domains
PubMed: 29666183
DOI: 10.1074/jbc.RA118.001938 -
The Journal of General Physiology May 2019Moss and Solaro recall Bárány’s landmark study that identified myosin ATPase as the fundamental driver of contraction speed. (Review)
Review
Moss and Solaro recall Bárány’s landmark study that identified myosin ATPase as the fundamental driver of contraction speed.
Topics: Animals; Humans; Muscle Contraction; Muscle, Skeletal; Myosins
PubMed: 30890556
DOI: 10.1085/jgp.201912323 -
Molecular Brain Aug 2018The alpha1 (α1) subunit of the sodium/potassium ATPase (i.e., Na/K-ATPase α1), the prototypical sodium pump, is expressed in each eukaryotic cell. They pump out three...
The alpha1 (α1) subunit of the sodium/potassium ATPase (i.e., Na/K-ATPase α1), the prototypical sodium pump, is expressed in each eukaryotic cell. They pump out three sodium ions in exchange for two extracellular potassium ions to establish a cellular electrochemical gradient important for firing of neuronal and cardiac action potentials. We hypothesized that myosin (myo or myh) motor proteins might interact with Na/K-ATPase α1 subunits in order for them to play an important role in the transport and trafficking of sodium pump. To this end immunoassays were performed to determine whether class II non-muscle myosins (i.e., NMHC-IIA/myh9, NMHC-IIB/myh10 or NMHC-IIC/myh14), myosin Va (myoVa) and myosin VI (myoVI) would interact with Na/K-ATPase α1 subunits. Immunoprecipitation of myh9, myh10, myh14, myoVa and myoVI from rat brain tissues led to the co-immunoprecipitation of Na/K-ATPase α1 subunits expressed there. Heterologous expression studies using HEK293 cells indicated that recombinant myh9, myh10, myh14 and myoVI interact with Na/K-ATPase α1 subunits expressed in HEK293 cells. Additional results indicated that loss of tail regions in recombinant myh9, myh10, myh14 and myoVI did not affect their interaction with Na/K-ATPase α1 subunits. However, recombinant myh9, myh10 and myh14 mutants having reduced or no actin binding ability, as a result of loss of their actin binding sites, displayed greatly reduced or null interaction with Na/K-ATPase α1 subunits. These results suggested the involvement of the actin binding site, but not tail regions, of NMHC-IIs in their interaction with Na/K-ATPase α1 subunits. Overall these results suggest a role for these diverse myosins in the trafficking and transport of sodium pump in neuronal and non-neuronal tissues.
Topics: Actins; Amino Acid Sequence; Animals; Antibodies; Binding Sites; Brain; HEK293 Cells; Humans; Mice; Myosins; Protein Binding; Protein Subunits; Rats; Recombinant Proteins; Sodium-Potassium-Exchanging ATPase
PubMed: 30086768
DOI: 10.1186/s13041-018-0388-1