Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

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.     

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.     

A solid-liquid biphasic model for characterization of properties of muscle and platelet contractile proteins.

Actomyosin, myosin, and actin from different sources are adsorbed, apparently as a monolayer, by polystyrene particles teins for 1 mg of Lytron were about 10-7 liters mol-1, while heterogeneity indices (alpha) varied from 0.70 to 1.0 presumably as a function of spontaneous aggregation in the liquid phase. Adsorption was irreversible. Orientation of absorbed molecules permitted association of bound muscle actin with platelet or muscle myosin. The association constant of the former reaction was 2.78 times 10-6 liters mol-1. Enzymatic properties of adsorbed actomyosin, Mg2+ATPase activity was abolished, but association of myosin with bound actin, or association of actin with bound myosin was accompanied by restoration of Mg2+ATPase activity. Every subunit of F-actin strands, unless F-actin had been fully depolymerized to G-actin, could bind myosin and activate Mg2+ATPase activity. Immunogenic characteristics of muscle myosin were enhanced by Lytron adsorption. Elicited antibodies showed selective specificity for an antigenic determinant located near or at the actin combining site of muscle myosin. Antibodies did not react with actomyosin. Antibodies prevented association of actin with muscle myosin because they inhibited both superprecipitation and development of Mg2+ATPase activity.     

Competitive and uncompetitive effects of 2,4-dinitrophenol on ATPase activities of rabbit skeletal actomyosin and myosin.

A kinetic study of the ATPase reactions catalyzed by myosin and actomyosin was carried out by varying the concentrations of ATP and 2,4-dinitrophenol (DNP). Mg-ATPase of myosin in the initial burst and that of actomyosin were both inhibited competitively by DNP. The dissociation contants for the DNP-myosin interaction (Ki) were estimated to be very similar, that is, 4.2 mM in the initial burst of ATP splitting, and 3.3 mM for the actomyosin ATPase. It is therefore suggested that DNP acts at the same site when it inhibits the burst splitting of ATP and the actomyosin ATPase. In contrast, Mg,-Ca-, and EDTA-ATPase activities of myosin in the steady state were all affected uncompetitively by DNP. Moreover, the Ki value for Mg-ATPase of myosin in the steady state was found to be 31 mM, which is much higher than those mentioned above for the initial burst and actomyosin ATPase. It is therefore suggested that the site at which DNP acts to inhibit the burst splitting of ATP is different from the site at which DNP acts to affect Mg-, Ca-, and EDTA-ATPases in the steady state.     

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.     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.     

Non-correlation of phosphorylation of the P-light chain and the actin activation of the ATPase of chicken gizzard myosin.

1. The enzymic properties of myosin isolated from chicken gizzard by three different methods have been compared. 2. Although the specific Ca2+-stimulated ATPases of all preparations were similar and high, there were significant differences in the specific activities of the Mg2+-stimulated actomyosin ATPases. 3. There was no direct correlation between the Mg2+-stimulated actomyosin ATPase activity and the extent of P-light-chain phosphorylation in any of the three myosin preparations. 4. A fraction that activates the Mg2+-stimulated actomyosin ATPase of gizzard muscle has been isolated from a gizzard muscle filament preparation. 5. The activator was specific for the Mg2+-activated actomyosin ATPase of smooth muscle. 6. The activator required the addition of calmodulin for full effect.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium/calmodulin regulation of ATPase activity and endogenous phosphorylation of mammalian brain actomyosin

Rabbit brain actomyosin showed several fold stimulation of the MgATPase activity by Ca2+ alone and by Ca2+/calmodulin. The calmodulin-binding drug, fluphenazine, abolished the stimulated activity. In the presence of Ca2+, exogenous calmodulin had a biphasic effect on ATPase activity at low concentrations (less than 0.15 microM) and activated the ATPase activity by 60-70% at about 1 microM. Tropomyosin-troponin complex from skeletal muscle did not stimulate the ATPase activity of brain actomyosin, but conferred Ca2+ sensitivity to a skeletal muscle myosin/brain actomyosin mixture. These results indicate the presence of myosin-linked, calmodulin-dependent, Ca2+-regulatory system for brain actomyosin. Heavy and light chains of brain myosin were found to be rapidly phosphorylated by endogenous Ca2+-dependent protein kinase(s). Ca2+-independent phosphorylation of one of the light chains was also observed.     

Studies on the initial phase of dynein ATPase activity.

Kinetic measurement of the reaction of dynein ATPase (ATP phosphohydrolase, EC 3.6.1.3) extracted from the gills of Mytilus edulis shows that in the presence of Mg2+ there is a very rapid initial liberation of Pi from the dynein-ATP system, followed by a slower liberation in the steady state. In view of following results, we have confirmed that this phenomenon is not due to the accumulation of end products, a fall in substrate concentration, nor to the presence of labile impurities in ATP but is due to the catalytic activity of dynein ATPase. 1. The replacement of native dynein by heat denatured dynein or other kinds of Mg2+-ATPase could not produce such a burst phenomenon under the same condition. 2. Both the rate of initial burst and that of steady state were proportional to enzyme content over a wide range under our standard condition. 3. Initial burst was also observed under the constant ATP level by using a ATP generate system. 4. Preincubation of dynein with Pi prior to initiation of the reaction did not eliminate the initial burst. Some properties of the initial rapid liberation of dynein ATPase were also examined. These are shown below. 5. The free ADP liberation did not show any initial burst though the Pi liberation did in the initial phase and the rate of free ADP liberation was almost equal to that of Pi liberation of the steady state. 6. Mg2+ was more effective than Ca2+ for the appearance of the initial burst while the liberation of Pi in the steady state was activated more by Ca2+ than by Mg2+. The addition of K+ in the presence of Mg2+ resulted in a marked increase of Pi liberation in the steady state but not in the initial state. 7. The activation energy of the initial burst was 9.7 kcal, which is slightly smaller than that of myosin ATPase.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers.

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca2+ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca2+ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechano-chemical cycle are Ca2+ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

The control of energy release in extracted muscle fibers

Tension and P liberation were determined at the same time in glycerol-extracted muscle fibers suspended in ATP solutions. In the relaxed state, produced by ATP in rather fresh preparations, P liberation was low, but somewhat higher than in normal resting muscle. On addition of small amounts of CaCl₂ the fibers gave a strong contraction during which P liberation was on the average about 5 times higher than in the relaxed condition. In aged muscle fibers ATP always produced a strong contraction associated with a high ATPase activity which was not influenced by Ca. The P liberation during a sustained contraction was much smaller in extracted fibers than in normal muscle, but the former maintained tension much more economically than the latter, resembling smooth muscle in this respect. Also the removal of Mg caused a contraction associated with high ATPase activity. Mg, therefore, is inhibitory in relaxed fibers. In fibers activated by Ca or by aging, however, it caused enhancement. The effects of ions on ATPase activity of relaxed fibers are similar to those on myosin and dissociated actomyosin, whereas activated fibers resemble actomyosin at low salt concentration.     

A temporal dissociation of energy liberation and high energy phosphate splitting during shortening in frog skeletal muscles

Measurements of the time course of high energy phosphate splitting and energy liberation were performed on rapidly shortening Rana pipiens skeletal muscles. In muscles contracting 30 times against small loads (less the 0.02P), the ratio of explained heat + work (H + W) (calculated from the measured high energy phosphate splitting) to observed H + W (from myothermal and mechanical measurements) was 0.68 +/- 0.08 and is in agreement with results obtained in isometric tetani of R. pipiens skeletal muscle. In lightly afterloaded muscles which were tetanized for 0.6a and whose metabolism was arrested at 3.0 s after the beginning of stimulation, a similar ratio of explained H + W to observed H + W was obtained. However, in identical contractions in which metabolism was arrested at 0.5-0.75 s after the beginning of stimulation, the ratio of explained H + W to observed H + W declined significantly to values ranging from 0.15 to 0.40. These results suggest that rapid shortening at the beginning of contraction induces a delay between energy production and measurable high energy phosphate splitting. This interpretation was tested and confirmed in experiments in which one muscle of a pair contracted isometrically while the other contracted against a small afterload. The afterload and stimulus pattern were arranged so that at the time metabolism was arrested, 0.5 s after the beginning of stimulation, the total energy production by both muscles was the same. Chemical analysis revealed that the isotonically contracting muscle spilt only 25% as much high energy phosphate as did the isometrically contracting muscle.     

Inhibition of actomyosin ATPase by high concentrations of 5-hydroxytryptamine. Possible basis of lesion in 5HT-induced experimental myopathy.

The effect of 5-hydroxytryptamine (5HT) on the ATPase activity and sulphydryl group reactivity of mammalian skeletal muscle actomyosin has been studied. 5HT inhibited the Mg2+-activated but not the Ca2+-activated ATPase activity of actomyosin. It slightly activated myosin ATPase. The sulphydryl groups of actomyosin reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) were blocked by concentrations of 5HT which inhibited the Mg2+-activated ATPase. The significance of the results are discussed in relation to the muscle lesions in the experimental myopathy induced by 5HT and imipramine.     

Calcium-sensitivity of pig-carotid-actomyosin ATPase in relation to phosphorylation of the regulatory light chain

Ca2+-dependent phosphorylation of the 20000-Mr regulatory light chain was found to be a necessary condition for the Ca2+-sensitivity of the Mg2+-dependent ATPase activity and superprecipitation of pig carotid actomyosin. Actin-myosin interaction independent of phosphorylation and Ca2+ (ATPase activity and superprecipitation) were demonstrated in aged actomyosin preparations and in preparations from which the regulatory light chains were removed by papain digestion.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca²⁺ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca²⁺ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechanochemical cycle are Ca²⁺ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Interaction of actin with myosin A and heavy meromyosin.

Ca2+ "free" actomyosin suspensions as well as actin heavy meromyosin (HMM) solutions in the presence of Ca2+ showed no contractile response (superprecipitation) and had low steady-state Mg2+-ATPase activity. Under the same experimental conditions both the enzymatic activity increased and contractile response was restored if the solubility of the proteins was depressed by the addition of polyethylene glycol 4000 (PEG-4000). The stability of the enzymatically active actomyosin or actin HMM complexes was 10 times lower in cleared solutions than in the insoluble actomyosin or actin HMM suspensions. It was concluded that soluble actomyosin or actin HMM solutions are inadequate test tube models for studying muscular contraction.     

Actomyosin ATPase activity of middle ear muscles in the cat

Summary When incubated for histochemical demonstration of actomyosin ATPase both the tensor tympani and the stapedius were found to contain two types of muscle fibres, one with high actomyosin ATPase activity, indicating a high speed of contraction, and one with low actomyosin ATPase activity, indicating a low speed of contraction. In the tensor tympani 59% and 41% of the muscle fibres had a high and low ATPase activity, respectively. The corresponding numbers in the stapedius muscle were 82% and 18%. These findings are discussed with reference to previous physiological and morphological studies.