Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Purification and Characterization of Myosin from Calf Brain

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Binding of connectin to myosin filaments

Binding of native connectin (2,100 kDa fragment of alpha-connectin) to myosin filaments was investigated using a sedimentation technique and densitometric estimations of the separated proteins. In the presence of 60 mM KCl and 5 mM phosphate buffer, pH 7.0, as much as 1.5 mol of connectin was bound to 1 mol of myosin, suggesting that some 150 connectin filaments bound to a single myosin filament of approximately 0.5 micron in length. This value was much more than the ratio found in muscle (12:1). It appeared that C protein did not affect the binding of connectin to myosin filaments.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.     

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.     

Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.     

Structure and function of chicken gizzard myosin.

In our previous study (Onishi, H., Susuki, H., Nakamura, k., and Watanabe, S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosin that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amount of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosin in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filaments of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filaments of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confer ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) Unphosphorylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2 M KCl-dissociated gizzard myosin in monomeric form is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15 M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATP-resistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.     

LC2 involvement in the assembly of skeletal myosin filaments

The assembly of LC2-deficient myosin was studied under conditions where control and LC2-reassociated myosin assemble around the native length of about 1.5 microns. The aim of this work was to determine how loss of LC2 affects the assembly characteristics. The findings of this study can be summarized as follows: (a) LC2-deficient myosin assembles into two populations of filaments, one around 0.5 micron in length and the other around 1 micron in length. This suggests that loss of the LC2 perturbs the length-determining mechanism. (b) The population of filaments around 0.5 micron has a diameter around 14 nm and that around 1 micron a diameter around 22 nm. Neither diameter corresponds to the 18 nm obtained with the control and LC2-reassociated myosins, suggesting that the presence of LC2 may have a role in regulating the side-to-side assembly of the myosin rods. (c) Filaments assembled from LC2-deficient myosin tend to aggregate side-by-side, but not those assembled from control and LC2-reassociated myosin. (d) The presence of MgATP has no effect on the length distribution of LC2-deficient myosin filaments in contrast to the sharpening of the distribution observed with control and reassociated myosin.     

Unidirectional sliding of myosin filaments along the bundle of F-actin filaments spontaneously formed during superprecipitation

I reported previously (Higashi-Fujime, S., 1982, Cold Spring Harbor Symp. Quant. Biol., 46:69-75) that active movements of fibrils composed of F-actin and myosin filaments occurred after superprecipitation in the presence of ATP at low ionic strengths. When the concentration of MgCl2 in the medium used in the above experiment was raised to 20-26 mM, bundles of F-actin filaments, in addition to large precipitates, were formed spontaneously both during and after superprecipitation. Along these bundles, many myosin filaments were observed to slide unidirectionally and successively through the bundle, from one end to the other. The sliding of myosin filaments continued for approximately 1 h at room temperature at a mean rate of 6.0 micron/s, as long as ATP remained in the medium. By electron microscopy, it was found that most F-actin filaments decorated with heavy meromyosin pointed to the same direction in the bundle. Myosin filaments moved actively not only along the F-actin bundle but also in the medium. Such movement probably occurred along F-actin filaments that did not form the bundle but were dispersed in the medium, although dispersed F-actin filaments were not visible under the microscope. In this case, myosin filament could have moved in a reverse direction, changing from one F-actin filament to the other. These results suggested that the direction of movement of myosin filament, which has a bipolar structure and the potentiality to move in both directions, was determined by the polarity of F-actin filament in action.     

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.     

ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.Pi.

In vitro and at physiological ionic strength, unphosphorylated smooth muscle myosin filaments dissolve on addition of ATP, forming folded (10S) myosin monomers. By following the fate of ATP and the time course of filament disassembly we have established details of the mechanism of this process. Myosin filaments first bind and hydrolyse 2.0 mol/mol of ATP before significant filament dissolution occurs. Following dissolution, the hydrolysis products ADP.Pi are retained on the heads of the folded myosin monomers, and are released so slowly (half time approximately 100 min at 100 mM KCl) as to be effectively trapped. The straight (6S) conformation of myosin, stable at greater than 225 mM KCl, did not exhibit this product trapping, and neither did myosin filaments held under conditions which disfavour ATP-induced disassembly. The implications of these results for filament stability in vivo are discussed in terms of a simple, testable model for smooth muscle myosin self-assembly.     

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.     

ATP-dependent movement of myosin in vitro: characterization of a quantitative assay

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP- dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP- dependent movement of myosin molecules on actin.     

The myosin filament: XI. Filament assembly

The critical parameters required for the assembly of myosin filaments with a length distribution comparable to that for native myosin filaments were examined. It was found that: Two steps are required in the dilution of a myosin solution from 0.6M KCl to 0.15M KCl. In Step I the KCl concentration is reduced from 0.6 to 0.3M KCl and in Step II from 0.3 to 0.15M KCl. The rate of change of KCl required for Step I is different than that required for Step II. Increasing the total time of dilution in either Step I or II alone leads to an increase in length and a broadening of the length distribution. In Step I assembly of myosin molecules into nonsedimentable units occurs. These may be the basic units from which the filaments are assembled in Step II. Rapid dilution in Step I alone has no effect on the length distribution obtained at 0.15M KCl, but rapid dilution in Step II alone leads to short filaments (about 0.6 micron). Increasing the time of dilution in Step II alone to 3 hrs or 6 hrs gives a bimodal distribution in lengths with one peak at about 0.8 micron and the other at about 2.2 microns. The length distribution obtained at 0.15M KCl is not critically dependent on information contained in the portion of the filament previously assembled in Step II, but is critically dependent on the rate of change of KCl concentration during the assembly of the rest of the filament.     

Heart mitochondria in physiological salt solution: not ionic strength but salt composition is important for association of creatine kinase with the inner membrane surface

In physiological salt solution (PSS) which mimicks the cardiac cells cytoplasm and contains 120 mM K-MES, 10 mM NaCl, 20 mM imidazole, pH 7.2, 20 mM taurine, 15 mM creatine, 15 mM Na2phosphocreatine, 5 mM Na2ATP, 8 mM MgCl2, 5 mM K2HPO4, 3 mM glutamate, 3 mM malate, 0.5 mM dithiothreitol and 10 mg/ml of bovine serum albumine both isolated mitochondria and intracellular structures in skinned fibers stay intact. In PSS mitochondrial creatine kinase remains firmly attached to the inner membrane surface. CKmi-mi is extracted from cardiac mitoplasts in 0.125 M KCl solution, but addition of 10 mM sodium borate to this KCl solution completely inhibits dissociation of CKmi-mi. Therefore, not ionic strength but ion composition is important for association of CKmi-mi with mitochondrial membrane. Functional and structural studies using antibodies against CKmi-mi showed that in PSS CKmi-mi is bound to the inner mitochondrial membrane in spatially close relationship to adenine nucleotide translocase (ANT). Thus, under physiological conditions CKmi-mi is structurally and functionally coupled to ANT in cardiac mitochondria and functions to catalyze almost complete utilization of mitochondrial ATP for aerobic phosphocreatine synthesis.     

Dense precipitate of brain tubulin with skeletal muscle myosin

Purified tubulin from porcine brain formed a dense precipitate at 37 degrees C with muscle myosin filaments from rabbit skeletal muscle; this effect was greater than that with partially purified tubulin. ATP or GTP, which prevented the myosin filaments from precipitating, inhibited the formation of the dense precipitate, but did not dissociate the dense precipitate once formed. The dense precipitate was found by thin-section electron microscopy to be composed to side-by-side aggregates of myosin filaments whose projections might be decorated by tubulin. The decoration was also seen by negative-stain electron microscopy. The binding of tubulin to myosin filaments decreased the Mg2+- and Ca2+-GTPase activity of the myosin by about half, but did not affect either Mg2+- or Ca2+-ATPase activity. The binding ratio of tubulin to myosin in the presence of 5 mM MgCl2 was 2.2 mol/mol using purified tubulin and 1.8 mol/mol using partially purified tubulin. Five mM ATP and GTP in the presence of 5 mM MgCl2 decreased the tubulin binding by 1.6-2.0 and 1.1-1.3 mol/mol, respectively, when added before an encounter of tubulin with myosin filaments, but did not cause any decrease when added after such an encounter.     

Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.     

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.