Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.     

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.     

Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin

1. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 microM purealin. 2. The ATPase activity of myosin B was also elevated to 160% of the control value by 10 microM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaCl2 and 0.5 M KCl (Ca2+-ATPase), and the concentration for the half inhibition was 4 microM. 3. On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA-ATPase). The maximum activation by 10 microM purealin was 160% of the control value. 4. Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment-1 instead of myosin. 5. These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA-ATPase and actomyosin ATPase of myosin B.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

The nucleoside triphosphate (NTP)-induced superprecipitation and NTPase reaction of chicken gizzard actomyosin as a function of the NTP concentration

The ATPase or ITPase reaction and ATP- or ITP-induced superprecipitation were studied as a function of the ATP or ITP concentration with suspensions of chicken gizzard "native" myosin B or "reconstituted" myosin B (a combination of actin, myosin, and native tropomyosin). The specific aim of the study was to answer the following questions: i) Is the superprecipitation or the ATPase reaction sensitive to calcium ions even at very low concentrations of ATP? ii) Is tropomyosin required for calcium sensitivity? iii) Does "native" myosin B from gizzard muscle behave differently from "reconstituted" myosin B? iv) Does the troponin-tropomyosin complex of rabbit skeletal muscle act as a regulatory protein for the contractile activity of acto-phosphorylated myosin? Considering the overall time course of reaction rather than single values of activity, we found that the answers to the first three questions were negative, while that to the last question was positive. These results favor the kinase-phosphatase mechanism of calcium regulation rather than the leiotonin mechanism.     

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.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

Myosin from abdominal flexor muscle in a crayfish, Procambarus clarki Girard.

1. Crayfish (Procambarus clarki) myosin was obtained from abdominal flexor muscle. The Ca2+-ATPase activity of crayfish myosin was much lower than that of rabbit skeletal myosin. However, F-actin-activated Mg2+-ATPase of crayfish and its superprecipitation closely resembled those of rabbit skeletal myosin. This fact suggests that the ability of crayfish myosin to combine with F-actin is essentially the same as that of skeletal myosin, although the chemical structures of both the myosin molecules when involved in their Ca2+-ATPast activity must be different from each other. 2. Crayfish and rabbit skeletal myosins were subjected to SDS-polyacrylamide gel electrophoresis. Crayfish myosin was found to have one heavy chain and two distinct light chain components (CF-gl and CF-g2), which have molecular weights of 18,000 and 16,000, respectively. These light chains correspond in molecular weight to the light chains (SK-g2 and SK-g3) in rabbit skeletal myosin. 3. CF-g1 could be liberated from the crayfish myosin molecule reacting with 5,5'-dithio-bis (2-nitrobenzoic acid), (Nbs2), without recovery of ATPase activity by the addition of DTT. These properties are equivalent to those of SK-g2 in rabbit skeletal myosin, although Nbs2-treated crayfish myosin did not recover its ATPase activity at all.     

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.     

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.     

Identification of myosin in Nitella flexilis.

A myosin B-like protein was extracted from the alga Nitella flexilis. SDS-polyacrylamide gel electrophoresis revealed the presence of myosin heavy chain and actin as the main components. At high ionic strength, its ATPase [EC 3.6.1.3] reaction was activated by EDTA or Ca2+ and inhibited by Mg2+. At low ionic strength, superprecipitation was induced by the addition of ATP. Myosin was purified from Nitella myosin B. The molecular weight of the heavy chain of Nitella myosin, estimated by SDS-gel electrophoresis, was slightly higher than that of skeletal muscle myosin. At low ionic strength, Nitella myosin aggregated to form bipolar filaments about 0.2 micron long. At high ionic strength, its ATPase reaction was activated by EDTA or Ca2+, and inhibited by Mg2+. The Mg2+-ATPase reaction of Nitella myosin was activated by skeletal muscle F-actin.     

平滑肌肌球蛋白B的超沉淀与肌球蛋白轻链磷酸化

本文报导了牛胃肌球蛋白B(天然肌动球蛋白)的超沉淀性质。当钙离子、钙调蛋白和ATP存在时,肌球蛋白B出现超沉淀,在pH6.8和7.5处,有两个峰值。Ca~(2+)(PCa值8-4)对超沉淀影响的浓度-反应曲线呈典型的S形,表明当Ca~(2+)浓度处于微摩尔水平时产生超沉淀。伴随超沉淀发生了肌球蛋白调节轻链磷酸化。这说明肌球蛋白轻链的Ca~(2+)-CaM依赖性磷酸化可能包含在脊椎动物平滑肌收缩活动的调节机制中。     

Reversible superprecipitation and bundle formation of plasmodium actomyosin.

Synthetic actomyosin from plasmodium was found to undergo reversible superprecipitation upon addition of ATP. According to electronmicroscopic investigation upon clearing, short myosin filaments of about 0.2 micron in length appeared predominantly coexisting with actin filaments, and after superprecipitation, bundles of actin filaments were formed where short myosin filaments or myosin molecules were bound to the side of the bundle, making a whisk-like structure. The turbidity and the ATPase activity of actomyosin were measured at various ATP concentrations clamped by using an ATP-regenerating system. The turbidity was high below 1 . 10(-6) M ATP, corresponding to the state of superprecipitation, and with increasing ATP concentration it dropped in the range of 1 . 10(-6)--1 . 10(-5) M ATP. On the other hand, the ATPase activity was low below 1 . 10(-6) M ATP and increased above 1 . 10(-5) M after the turbidity dropped. Characteristic features of superprecipitation of plasmodium actomyosin observed here were discussed in relation to the mechanism of motility in vivo.     

Cytoplasmic and plasma membrane adenosine triphosphatase of polymorphonuclear neutrophils: comparison of their enzymatic properties and attempt for a direct determination of myosin ATPase activity using polymorphonuclear neutrophil extract

Enzymatic properties of the ATPase of the plasma membrane and cytoplasmic myosin B from guinea-pig polymorphonuclear neutrophils were compared. In the plasma membrane, Mg2+- and Ca2+-activated ATPases showed the same dependence pattern on KCl concentration and pH, i.e., both ATPases increased with decreasing KCl concentration and with rising pH until pH 9.0. The maximum activation of Mg2+-ATPase was observed at 1 . 10(-3) M Mg2+. On the other hand, EDTA-activated ATPase activity was so low that no clear dependence curve was obtained. In myosin B, Mg2+-ATPase activity was below one-tenth that of the plasma membrane ATPase with the maximum activation at 1 . 10(-2) M Mg2+ and pH 9.0 EDTA- and Ca2+-activated ATPase exhibited almost the same activity and the same KCl-dependence curve, i.e., both ATPases increased and increasing KCl concentration. With regard to pH-dependence, Ca2+-ATPase showed a U-shaped curve with the minimum at pH 7.0, wherease EDTA-activated ATPase indicated a bell-shaped curve with the maximum at pH 9.0. Based on the findings that the EDTA-activated ATPase activity was hardly detected in the plasma membrane but high in myosin B, the distribution of ATPase activity on subcellular fractions was studied and the results obtained that the myosin-ATPase activity could be directly measured using the polymorphonuclear neutrophil extract if the EDTA-activated ATPase activity was used as an enzymatic marker for myosin.     

Head-head and head-tail interaction: a general mechanism for switching off myosin II activity in cells

Intramolecular interaction between myosin heads, blocking key sites involved in actin-binding and ATPase activity, appears to be a critical mechanism for switching off vertebrate smooth-muscle myosin molecules, leading to relaxation. We have tested the hypothesis that this interaction is a general mechanism for switching off myosin II-based motile activity in both muscle and nonmuscle cells. Electron microscopic images of negatively stained myosin II molecules were analyzed by single particle image processing. Molecules from invertebrate striated muscles with phosphorylation-dependent regulation showed head-head interactions in the off-state similar to those in vertebrate smooth muscle. A similar structure was observed in nonmuscle myosin II (also phosphorylation-regulated). Surprisingly, myosins from vertebrate skeletal and cardiac muscle, which are not intrinsically regulated, undergo similar head-head interactions in relaxing conditions. In all of these myosins, we also observe conserved interactions between the 'blocked' myosin head and the myosin tail, which may contribute to the switched-off state. These results suggest that intramolecular head-head and head-tail interactions are a general mechanism both for inducing muscle relaxation and for switching off myosin II-based motile activity in nonmuscle cells. These interactions are broken when myosin is activated.     

Preparation and properties of smooth muscle myosin from horse esophagus

Myosin was prepared from smooth muscle of horse esophagus in good yield (about 150 mg/100 g tissue) and was designated myosin S. Its properties were compared with those of myosin A from skeletal muscle.

The ratio of the absorption of myosin S at 280 nm to that at 260 nm was about 1.8, and the amount of contaminating phosphorus was only 0.91 g/10⁵ g of myosin S, indicating that the latter is free of nucleic acid. The purity of this protein was examined by ultracentrifugation, gel filtration in the presence of 0.5 M KCl and 6 M urea and chromatography on DEAE-cellulose columns. These experiments all indicated that myosin S was homogeneous, like highly purified rabbit skeletal myosin A.

Amino acid analyses showed differences in the composition of smooth and skeletal myosins. Myosin S contained the same amount of sulfhydryl groups per 10⁵ g of protein as horse and rabbit skeletal myosin A (about 8 moles/10⁵ g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline.

Ca²⁺-ATPase of myosin S in the presence of 0.5 M KCl and Mg²⁺-ATPase in the presence of 0.05 M KCl at 37° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca²⁺-ATPase in the presence of 0.05 M KCl were much lower than those of skeletal myosin A. Lowering the temperature from 37 to 25°, the degree of decrease of the ATPase activities was much larger in myosin S than in skeletal myosin A. The reaction of N-ethylmaleimide with myosin S caused inhibition of the EDTA-ATPase but did not affect the Ca²⁺-ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca²⁺-ATPase during the course of the reaction of sulfhydryl groups of myosin with N-ethylmaleimide. These facts suggest that the structure of the active site of myosin S ATPase differs significantly from that of skeletal myosin A. These differences appear to influence the interaction of myosin with F-actin, so that the rate of superprecipitation found in an actomyosin reconstituted from myosin S and F-actin was only one fortieth of that found with skeletal myosin A.     

扇贝肌钙蛋白及其对肌球蛋白超沉淀的影响

分离了扇贝闭壳肌肌钙蛋白,其分子量为４６（ＩｎＩ）,４０（ＴｎＴ）,和２２（ＴｎＣ）ｋＤ．肌球蛋白Ｂ含有主要的收缩蛋白质与调节蛋白质,在有Ｃａ２＋和ＡＴＰ存在时,它会发生超沉淀作用．经低离子强度溶液反复沉淀处理,即失去Ｃａ２＋－敏感性,成为去敏肌球蛋白Ｂ．在Ｃａ２＋和ＡＴＰ作用下,它仍可发生超沉淀作用,但仅及最大活性的５０％．若加入肌钙蛋白,则反应活性可完全恢复．兔骨骼肌肌钙蛋白可替代扇贝闭壳肌肌钙蛋白．这表明扇贝闭壳肌兼有肌动蛋白相关调节和肌球蛋白相关调节．     

Purification of chicken gizzard myosin light-chain kinase, and its calcium and strontium sensitivities as compared with those of superprecipitation and ATPase activities of actomyosin

1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130,000 (130 K)-dalton subunit and 17,000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca2+ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region. 2. The Ca2+ and Sr2+ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of "reconstituted" myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of "reconstituted" myosin B were practically identical with "natural" myosin B in the Ca2+ and Sr2+ requirements for the three activities measured above. 3. Both the extent and the activity of superprecipitation, and both the limited and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32 P incorporation from [gamma-32P]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.