The Changes of “Myosin B” during Storage of Rabbit Muscle

The irreversibility of the dissociation of “myosin B” stored in 0.6 m KCl at pH 5.7 and 3°C was attributed to the rapid denaturation of F-actin dissociated from “myosin B”

F-Actin was less stable than myosin A, in 0.18~0.60 m KCl at pH 5.7 and temperatures between 0 ~3°C.

The decrease in the ability of F-actin to bind with myosin A was slightly dependent on storage temperature, and there was no apparent relation with the decrease in the solubility.

A hypothetical scheme for F-actin denaturation was proposed.     

The Changes of “Myosin B” during Storage of Rabbit Muscle

The process of the denaturation of “myosin B” solution was studied by the measurement of ATPase activity, SH groups, sedimentation behaviour and flow birefringence. When “myosin B” solution was stored at lower temperature, lower pH or higher ionic strength, the interaction between myosin A and actin became less strong, and further storage brought about an irreversible dissociation.

The condition for measuring Mg-modified ATPase activity of “myosin B” at low ionic strength was explained in the relation with superprecipitation.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

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.     

Purification and characterization of myosin from bovine retina.

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal muscle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Plasma membrane ATPase of sugarbeet

A membrane fraction enriched with magnesium-dependent ATPase activity was isolated from sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI and sucrose density gradient centrifugation. This activity was inhibited by vanadate, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol, but was insensitive to molybdate, azide, oligomycin, ouabain, and nitrate, suggesting enrichment in plasma membrane ATPase. The enzyme was substrate specific for ATP, had a pH optimum of 7.0, but showed little stimulation by 50 mM KCl. The sugarbeet ATPase preparation contained endogenous protein kinase activity which could be reduced by extraction of the membranes with 0.1% (w/v) sodium deoxycholate. Reduction of protein kinase activity allowed the demonstration of a rapidly turning over phosphorylated intermediate on a M_r 105000 polypeptide, most likely representing the catalytic subunit of the ATPase. Phosphorylation was magnesium dependent, sensitive to diethylstilbestrol and vanadate but insensitive to oligomycin and azide. Neither the ATPase activity nor phosphoenzyme level were affected by combinations of sodium and potassium in the assay. These results argue against the presence of a synergistically stimulated NaK-ATPase at the plasma membrane of sugarbeet.     

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity.In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.     

Heavy meromyosin from skipjack tuna, Euthynus pelamis. Preparation and enzymic properties.

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin.     

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.     

Adenosine triphosphatase activity and "thick filament" formation of chicken gizzard myosin in low salt media.

The ATPase activity of chicken gizzard myosin was studied by varying the KCl concentration in the reaction medium. The following was thus found: (a) A sharp depression of the activity occurred when the KCl concentration was reduced to less than 0.3 M, showing the minimum activity around 0.15 M KCl. (b) The activity depression was removed by addition of urea or bay papain-digestion, but not by addition of p-chloromercuribenzoate. (c) In the KCl concentration where the activity depression occurred, the ATPase reaction proceeded in two distinct phases; the activity was relatively high in the early phase of the reaction and declined into the later phase where the steady state reaction took place. (d) In the KCl concentrations higher than that particular concentration or in the presence of urea, the ATPase reaction proceeded in one phase. (e) The temperature dependence of the ATPase activity in the early phase was of an ordinary magnitude being approximately equal to that of the ATPase activity in 0.6 M KCl. In contrast, the temperature dependence of the activity in the later phase was unusually small. Gizzard myosin in various concentrations of KCl was also examined by measuring the turbidity and the light-scattering intensity, and by observation under an electron microscope. The following was thus found: (a) In the KCl concentration where the activity depression occurred, there was a stagnation in the turbidity decrease as the KCl concentration was gradually increased and also the formation of "thick filaments," each of which was approximately 0.6-0.9 micron in length and 20-30 nm in diameter with no central "bare zone." (b) Addition of ATP induced dissociation of the thick filaments, and the dissociation occurred during the early phase of the ATPaseeaction. (c) Moreover, the temperature dependence of the ATP-induced dissociation rate was approximately equal to that of the ATPase activity in the early phase. On the basis of the findings mentioned above, it is concluded that the activity depression results from the ATP-induced dissociation of myosin filaments. Moreover, since high concentrations of KCl or urea also caused dissociation of myosin filaments and yet did not produce the activity depression, it was strongly suggested that gizzard myosin in the ATP-dissociated form must be different from that in the urea- or KCl-dissociated form, probably in the physical state of some myosin aggregates which were not detectable by the physical methods we used. As a side-observation, gizzard myosin filaments formed in the presence of ADP were found to be unusually long (longer than 2 micron), and they looked very similar to the particular filaments of skeletal myosin that were reported, by Moos, to be formed in the absence of the C protein.     

In vitro motility of skeletal muscle myosin and its proteolytic fragments

We have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KCl concentrations below a critical value (60 mM with myosin and HMM, 100 mM with S-1), and the sliding velocities were quite similar below the critical KCl concentration. However, at KCl concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were approximately 300 microM for S-1 and approximately 200 microM with HMM below the critical KCl concentration, and approximately 5,000 microM above the critical KCl concentration. This increase in KATPase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KCl concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.     

Myosin : Subunits and their interactions

There is fairly general agreement that myosin isolated from rabbit skeletal muscle has a molecular weight of about 500,000. The higher values that have been reported apparently reflect protein aggregation related to the method of preparation. On the basis of present evidence, the myosin molecule has an elongate helical core of two f subunits (average weight about 215,000) that extend into a globular head region containing three g subunits (average weight about 20,000). Myosin may be dissociated into subunits by a number of methods. In 5 M guanidine, the myosin molecule is dissociated into f and g subunits, while at pH above 10, the g subunits are dissociated from the intact fibrous core of myosin. The dissociation of g subunits at pH 10 is accompanied by the loss of both ATPase activity and actin-binding capacity; however, the exact biological significance of the g subunits is presently uncertain. In preliminary studies, the f subunits appear to contain the sulfhydryl residues currently implicated in myosin ATPase, and there is some indication of allosteric regulation of enzymic activity.     

ELECTRON MICROSCOPE OBSERVATIONS ON MYOSIN FROM PHYSARUM POLYCEPHALUM

Myosin has been separated from Physarum polycephalum actomyosin in confirmation of the results of Hatano and Tazawa. In an intermediate step, myosin-enriched actomyosin has also been obtained. The mean yield of free myosin was 4.4 mg from 100 g of mold. It was obtained as water-clear solutions at µ = 0.055 with calcium ATPase activity of up to 0.5 µM P_i/min per mg. Negatively stained preparations were examined by electron microscopy. Physarum myosin in 0.5 M KCl interacted with actin from rabbit skeletal muscle to form polarized arrowhead complexes similar to but less regular than those of natural actomyosin from muscle or myosin-enriched Physarum actomyosin. The Physarum myosin-enriched actomyosin at low ionic strength displayed evidence of head-to-tail and tail-to-tail aggregation attributable to the myosin component. Yet Physarum myosin alone did not produce detectable filaments at µ = 0.055 at pH 7, 6.5, or 5.8, nor when dialyzed against 0.01 M ammonium acetate, nor when the dielectric constant of the medium was reduced. However, aggregation approaching the extent of ‘thick filaments’ up to 0.3 µ long was found in some preparations of myosin-enriched actomyosin put into solutions containing adenosine triphosphate. Myosin alone in such solutions did not form filaments. The results are compatible with the idea that head-to-tail aggregations are favored by actin-myosin interactions in Physarum, possibly due to alignment of the extended or tail portions of this myosin molecule.     

Velocity of myosin-based actin sliding depends on attachment and detachment kinetics and reaches a maximum when myosin-binding sites on actin saturate

Molecular motors such as kinesin and myosin often work in groups to generate the directed movements and forces critical for many biological processes. Although much is known about how individual motors generate force and movement, surprisingly, little is known about the mechanisms underlying the macroscopic mechanics generated by multiple motors. For example, the observation that a saturating number, N, of myosin heads move an actin filament at a rate that is influenced by actin–myosin attachment and detachment kinetics is accounted for neither experimentally nor theoretically. To better understand the emergent mechanics of actin–myosin mechanochemistry, we use an in vitro motility assay to measure and correlate the N-dependence of actin sliding velocities, actin-activated ATPase activity, force generation against a mechanical load, and the calcium sensitivity of thin filament velocities. Our results show that both velocity and ATPase activity are strain dependent and that velocity becomes maximized with the saturation of myosin-binding sites on actin at a value that is 40% dependent on attachment kinetics and 60% dependent on detachment kinetics. These results support a chemical thermodynamic model for ensemble motor mechanochemistry and imply molecularly explicit mechanisms within this framework, challenging the assumption of independent force generation.     

Prevention of freeze denaturation of carp actomyosin by sodium glutamate.

1) Denaturation of carp actomyosin during storage at -20 degrees was studied with particular interest in the cryoprotective effect of sodium glutamate, the most cryoprotective of the compounds tested previously. 2) Storage with glutamate prevented the rapid decrease in solubility, viscosity, and ATPase (EC 3.6.1.3)activity of actomyosin during storage. Ultracentrifugal studies suggested that aggregation occurred in the frozen state without glutamate, but that added glutamate prevented aggregation or denaturation. 3) Electron microscopy showed that the original actomyosin consisted of long filaments with typical "arrowhead" structures, and that these decomposed into small fragments and sticked with globular portions, forming loosely packed aggregates during storage without glutamate. On storage with glutamate, the filaments were well preserved, and their fine structure was clearer than that of the original sample. 4) Preparations of actomyosin extracted with 10 mM glutamate were of better quality and their ultrastructure and physicochemical and biochemical properties showed increased stability on freezing. 5) Freeze-denaturation seems to involve complicated aggregation with transconformation of proteins besides the side-to-side aggregation discussed previously.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

Ultraslow Myosin Molecular Motors of Placental Contractile Stem Villi in Humans

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n = 20) and electrical tetanic stimulation (n = 20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 10³ times lower whereas myosin ATPase activity was 10⁵ times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue.     

Cell motility as an entangled quantum coherence.

Cell motility underlying muscle contraction is imputed to a macroscopic quantum mechanical coherence actualized locally in the body of a biological organism. Actin-activated myosin ATPase activity functions as a heat sink operating effectively at an extremely low temperature. Extraction of heat energy from the actin filament can help condensing the atomic degrees of freedom constituting the filament into a macroscopic quantum state carrying a nonvanishing linear momentum. Sliding movement of an actin filament on myosin molecules while hydrolyzing ATP molecules is a consequence of the quantum mechanical coherence due to an extremely slow release of energy stored in an ATP molecule.     

Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.     

Condensation Reaction Occurring during Plastein Formation by α-Chymotrypsin

An investigation was conducted on myosin and actin-activated heavy meromyosin (HMM) ATPase activities in normal porcine muscle stored for varying periods of time after death. Studies were also made on temperature dependent myosin ATPase, initial burst of ATPase and actin-activated HMM ATPase in normal and in pale, soft and exudative (PSE) porcine muscle. The maximum velocity of acto-HMM ATPase of normal muscle decreased considerably with postmortem time, while the apparent dissociation constant decreased slightly. The maximum velocity of acto-HMM ATPase of postmortem normal muscle was approximately two-times larger than that of the corresponding PSE muscle. However, almost no difference was found in the apparent dissociation constant. The size of the initial burst of phosphate-liberation of myosin prepared from normal muscle was approximately 1.2 mol/mol of myosin and from PSE muscle 0. It is assumed that the lack of contractility of PSE muscle was brought about by two basic myosin malfunctions: one, the irreversible binding of myosin to actin filament and the other, the functional damage of myosin ATPase, responsible for the formation of phosphorylated complex, even when dissociable.