Calcium-activated ATPase Activity Research Articles

Maximal ATPase Activity and Calcium Sensitivity of Reconstituted Myofilaments are Unaltered by the Fetal Troponin T Re-expressed During Human Heart Failure

J. R. Torrealba, E. Lozano, M. Griffin, S. Stoker, K. Mcdonald, M. Greaser and M. R. Wolff. Maximal ATPase Activity and Calcium Sensitivity of Reconstituted Myofilaments are unaltered by the Fetal Troponin T Re-expressed During Human Heart Failure. Journal of Molecular and Cellular Cardiology (2002) 34, 797–805. Re-expression of a fetal isoform of troponin T (TnT4) has been demonstrated in failing human ventricular myocardium and associated with a decrease in myofibrillar ATPase activity. In order to elucidate the regulatory role of the re-expressed TnT4 in the failing human heart, we measured ATPase activity in reconstituted cardiac myofilaments prepared with recombinant human TnT4 or the adult human isoform of troponin T (TnT3). Neither the maximal calcium-activated ATPase activity nor the calcium sensitivity of this biochemical assay was significantly different between reconstituted myofilaments containing adult TnT3 or fetal TnT4. Our results suggest that the re-expressed fetal TnT4 is not responsible for the depressed ATPase activity of failing ventricular myofibrils. The increased expression of the fetal isoform of this thin filament regulatory protein in the failing ventricle may be a consequence of a programmed change in gene expression occurring in response to hemodynamic stress, but probably does not contribute to depressed ventricular function characteristic of dilated cardiomyopathies.

Correlation of calcium-activated ATPase activity, lipid peroxidation, and the contractile response of rabbit corporal smooth muscle treated with in vitro ischemia

Oxygen and glucose are critical to support the survival and integrity of all smooth muscles. Hypoxia alone has been demonstrated to suppress the contractile response of corporal smooth muscle, and one might expect simultaneous deprivation of oxygen and glucose (in vitro model of ischemia) to exert more serious damage to corporal smooth muscle contraction. The effect of in vitro ischemia on the pharmacological responses of isolated rabbit corporal smooth muscle was correlated with the level of tissue lipid peroxidation. The effects of in vitro ischemia were as follows: (1) In vitro ischemia resulted in an 85% reduction in the contractile response to phenylephrine; (2) more than a 50% reduction in the activity of thapsigargin-sensitive calcium-activated ATPase activity of the microsomes (sarcoplasmic reticulum [SR]); (3) more than a fourfold increase in the tissue concentration of thiobarbituric acid reactive substances (TBARS) (level of lipid peroxidation). In conclusion, stimulation of lipid peroxidation in part may be responsible for the decrease in thapsigargin-sensitive calcium-activated ATPase activity of the SR (SERCA), and the correlated decrease in the contractile response to phenylephrine in response to ischemia.

The dictyostelium essential light chain is required for myosin function

A Dictyostelium mutant (7–11) that expresses less than 0.5% of wild-type levels of the myosin essential light chain (EMLC) has been created by overexpression of antisense RNA. Cells from 7–11 contain wild-type levels of the myosin heavy chain (MHC) and regulatory light chain (RMLC). Myosin isolated from 7–11 cells consists of the MHC with the RMLC associated in reduced stoichiometry, and binds to purified actin in an ATP-sensitive fashion. Purified 7–11 myosin displays calcium-activated ATPase activity with a V max about 15%–25% of that of wild type, and a Km for ATP of 27 ± 5 μM versus 83 ± 30 μM for wild type. At actin concentrations as high as 17 μM, 7–11 myosin displays greatly reduced actin-activated ATPase activity. Phenotypically, 7–11 cells resemble MHC mutants, growing poorly in suspension and becoming large and multinucleate. When starved for multicellular development, 7–11 cells take several hours longer than wild-type cells to aggregate. Although multicellular aggregates eventually form, they fail to develop further. The cells are also unable to cap receptors in response to Con A treatment. Since cells expressing the EMLC are phenotypically similar to MHC null mutants, the EMLC appears necessary for myosin function, at least in part because it is required for normal actin-activated ATPase activity.

Shortening velocity and myosin and myofibrillar ATPase activity related to myosin isoenzyme composition during postnatal development in rat myocardium.

The relation between functional properties of the contractile apparatus, such as shortening velocity and ATPase activity, and myosin isoenzyme composition was studied in ventricular myocardium of adult (60-90-day-old) rats and of newborn (3-day-old) and young (10- and 20-day-old) rats. In adult animals, variations of isomyosin pattern were produced by reducing food intake and by changing the thyroid state. Hyperthyroidism was induced with triiodothyronine daily injection for 15 days; hypothyroidism was induced with iodine-free diet and KClO4 in drinking water for 50-60 days. The following parameters were studied: 1) calcium-magnesium-activated and magnesium-activated ATPase activity of washed and purified myofibrils, 2) calcium-activated ATPase activity of purified myosin, 3) isomyosin composition and relative content of alpha-myosin heavy chains (alpha-MHCs), and 4) force-velocity curve of left and right ventricle papillary muscles. To take into account the difference in excitation-contraction coupling between newborn and adult myocardium, the determination of the force-velocity curve was repeated in Krebs' solution with normal [CaCl2] (2.5 mM) and in Krebs' solution with high [CaCl2] (10 mM). During postnatal growth, the relative content of alpha-MHC increased and reached a maximum at about 20 days. Pronounced increases of myofibrillar and myosin ATPase activity and in shortening velocity occurred during the same period. In adult hyperthyroid rats, alpha-MHC content as well as enzymatic activity and shortening velocity were higher than in control adult animals. Hypothyroidism and food deprivation caused a decrease of alpha-MHC content and a reduction of both enzymatic activities and shortening velocity. The study of the relations between alpha-MHC relative content and functional parameters showed that 1) in ventricular myocardium of adult rats a linear relation existed between alpha-MHC content and myosin and myofibrillar ATPase activity and shortening velocity, and 2) in newborn and young rat ventricular myocardium, both enzymatic activities and shortening velocity were lower than would have been expected on the basis of the linear relation described above. This latter observation could be accounted for by a variation in specific activity of myosin during postnatal development or by the presence of peculiar isomyosins that cannot be detected with usual electrophoretic techniques.

Plasma membrane vesicles prepared from unadhered monocytes: Characterization of calcium transport and the calcium ATPase

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 × 10 8 monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/μg vesicle protein/ min and a Km for calcium of 0.53 μM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 μM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/μg vesicle protein/min and a Km for calcium of 0.60 μM. Calcium-activated ATPase activity was absent if Mg was omitted or if (γ - 32P) GTP replaced (γ - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.

Relaxin alters rat uterine myosin light chain phosphorylation and related enzymatic activities.

Uteri from estrogen-primed rats were suspended isometrically, contracted by 0.28 microM prostaglandin F2 alpha, and exposed to 1 microgram/ml purified porcine relaxin for 1 and 10 min. Relaxin treatment for 10 min, but not for 1 min, resulted in a significant decrease in the activity of myosin light chain kinase (MLCKase). Calcium-activated ATPase activity of a crude actomyosin fraction was also decreased by treatment with relaxin for 10 min. Relaxin treatment (10 min) also decreased the relative amount of phosphorylated myosin 20,000 dalton light chains. These effects were consistent with the degree of inhibition of uterine contractions by relaxin. The data suggest that relaxin may inhibit uterine contractile activity by decreasing MLCKase activity and, in turn, myosin phosphorylation and ATPase activity.

Contractile behavior of rat myocardium after reversal of hypertensive hypertrophy.

The effects of renovascular hypertension and its reversal on the contractile performance of papillary muscles from rats has been examined. Hypertension of 10 wk duration caused a 48% increase in heart weight and significant prolongations of isometric time to peak tension (TPT), time to half relaxation, and time to peak shortening (TPS). A significant depression in the velocity of shortening was observed in the 10-wk group. However, muscles from hypertensive rats were still able to maintain normal levels of peak isometric developed tension and peak shortening; this may be due to the observed prolongation of TPT and TPS, respectively. In addition, calcium-activated actomyosin ATPase activity was depressed in hearts of hypertensive animals. Reversal of hypertension was studied at 20 wk after the onset of hypertension (10 wk of hypertension followed by 10 wk of normotension). Contractile and biochemical alterations observed in hypertensive animals were reversed in rats undergoing this regime. Thus reversal of a gradually applied pressure overload resulted in the regression of mechanical and biochemical abnormalities associated with the pressure overload myocardial hypertrophy.

Embryonic and foetal myosins in human skeletal muscle: The presence of foetal myosins in duchenne muscular dystrophy and infantile spinal muscular atrophy

Recently described techniques for separating myosin isoenzymes have been adapted for analysis of myosins from diseased and developing human skeletal muscle. The method is highly suitable for analysis of human myosins because only 2–3 mg of muscle are required for routine analyses. Human embryonic/foetal myosins are electrophoretically distinct from mature skeletal myosins, and are not normally detected beyond the first month of post-natal life, except in premature infants. They have a high alkaline calcium-activated ATPase activity. This would account for the histochemical classification of foetal fibres as “Type II”, although physiological differences between adult fast-twitch muscle and foetal muscle are well recognized. Foetal myosins are also synthesized in human skeletal muscle under certain pathological circumstances. Their presence in Duchenne dystrophy probably reflects the associated marked muscle regeneration, with immaturity of some muscle cells. The large amounts of foetal myosin present in many cases of infantile spinal muscular atrophy is evidence that innervation is necessary for the normal cessation of foetal myosin synthesis.

Branchial ATPases and ionic transport in the eel Anguila japonica—II. Ca2+-ATPase

Abstract 1. 1. Calcium-activated ATPase activity was demonstrated in the gills of both seawater and freshwater Japanese eels, Anguila japonica. The specific enzyme activity was highest in the heavy microsomal fraction. 2. 2. The enzyme characteristics in the microsomal fraction of the gills from freshwater and seawater adapted fish were studied and they showed similarities in the following aspects: (a) the enzyme was activated by either Ca2+ or Mg2+, maximal activation occurred at divalent ion concentration of 3 mM, and Mg2+ was less effective than Ca2+ in activating the enzyme; (b) the K m of the enzyme for ATP was 0.2–0.3 mM; (c) maximal enzyme activity was noted at 25–35°C and at pH 8.1–8.3 and (d) mersalyl acid and EDTA (ethylenediamine tetra-acetic acid) inhibited the enzyme while ouabain had no effect. 3. 3. When freshwater fish were transferred to seawater, serum Ca2+ level rose for the first 4 days and then returned to control value. Gill Ca2+-ATPase activity started to increase on day 4 after transfer and maintained at higher level throughout sea water adaptation. 4. 4. Enzyme isolated from the gills of seawater fish showed higher V max and K m values. These changes in enzyme activity and kinetics were suggested to be related to the calcium regulation in the fish.

Action of caffeine on calcium transport by isolated fractions of myofibrils, mitochondria, and sarcoplasmic reticulum from rabbit heart.

We studied the effects of caffeine on calcium transport by subcellular organelles isolated from rabbit myocardium. Caffeine increased myofibrillar basic and calcium-activated ATPase activity at 20 mM but not at lower concentrations. Mitochondrial and sarcoplasmic reticulum (SR) calcium accumulation was measured both by dual wavelength spectrophotometry with the calcium-sensitive dye, murexide, and by Millipore filtration with 45Ca. In mitochondria, caffeine impaired phosphate-assisted calcium transport but did not alter the closely related parameters of oxygen uptake, P/O ratio (nmol adenosine diphosphate consumed/n ats oxygen consumed, state 3 respiration) or limited calcium loading. In SR, caffeine impaired calcium accumulation. New methods were used to characterize calcium accumulation in the absence of oxalate according to first order reaction kinetics. Caffeine increased the rate constant while decreasing the calcium accumulated. It also increased the associated calcium-activated ATPase activity at low (30 mM) but not high (240 micrometer) external calcium concentration. In the presence of oxalate, caffeine decreased the rate of calcium accumulation, more with low than high calcium concentration. Net efflux of 45Ca from preloaded SR also was increased by caffeine. The findings indicate that caffeine impairs active calcium accumulation by making SR vesicle membranes more permeable to calcium.

Effects of lanthanum ion on calcium transport by guinea-pig, rat and canine cardiac sarcoplasmic reticulum

The trivalent earth metal lanthanum has been shown to inhibit calcium uptake and the calcium-activated ATPase activity of isolated guinea-pig and canine cardiac sarcoplasmic reticulum, at concentrations of lanthanum of 10 −5 and 10 −4 m. Calcium uptake, in the presence of 2.5 to 5.0 m m K-oxalate, and the calcium activated ATPase activity of isolated rat cardiac sarcoplasmic reticulum is less sensitive to lanthanum than guinea-pig or canine sarcoplasmic reticulum. Calcium storage, in the absence of precipitating anions, by the cardiac sarcoplasmic reticulum of all species studied is not affected by lanthanum at concentrations of 10 −5 m and 10 −4 m.

The relationship of mechanicalV max to myosin ATPase activity in rabbit and marmot ventricular muscle

Papillary muscle mechanics and ventricular myosin calcium-activated ATPase activity were measured in the same heart as a function of temperature (8--28 degrees) in rabbits and marmots, in order to examine further the hypothesis that the velocity of cardiac muscle shortening at zero load (Vmax) is correlated with myosin ATPase activity. There was a similar Q10 for Vmax in each muscle type, as measured with isotonic afterloaded quick-releases at 30--33% time-to-peak tension; the calcium activated ATPase of myosin in the two muscle types also was similar. The least squares linear regression of rabbit Vmax on calcium-activated myosin ATPase activity was the same as in the marmot, so all the data were pooled to yield a linear regression (Y = 0.47 +/- 3.82X) with a high correlation between the two variables [r = 0.95, P less than 0.01 (ANOV)]. Furthermore, the correlation proved to be predictive of cardiac Vmax and myosin ATPase activity levels in other experiments where these two measurements decreased below normal as a result of hypertrophic growth. Consequently, the quantitative relationship between Vmax and myosin ATPase defined here may prove to be predictive of the ability of cardiac muscle to release bond energy.

Proceedings of the British Cardiac Society

Caffeine partly influences mechanical performance of heart muscle by affecting internal membrane function. In skeletal and heart muscle caffeine alters calcium ac- cumulation by isolated sarcoplasmic reticulum fractions but the manner of its action is uncertain. Sarcoplasmic reticulum fractions were isolated from rabbit heart by differential centrifugation. Calcium accumulation, with and without oxalate, was followed using dual-wavelength spectrophotometry with murexide at 35°C. The calcium accumulation data were analysed according to first order kinetics. ATPase activity (basic and calcium activated) was also measured. Calcium accumulation both with and without oxalate was inhibited in a dose related manner by caffeine. At low but not high concentrations calcium activated ATPase activity was increased by caffeine but in the presence of oxalate it was not altered by caffeine at any calcium concentration. Caffeine did not alter the basic ATPase activity. The findings support the con- clusion that caffeine reduced calcium accumulation by altering the passive permeability of the sarcoplasmic recticulum vesicles to calcium.

Structural and functional properties of myosin associated with the compensatory cardiac hypertrophy in the rabbit

Right ventricular hypertrophy was produced in rabbits by pulmonary artery constriction. Measurement of intraventricular pressure, ventricular wet and dry weights and histologic examination of the liver demonstrated substantial hypertrophy with no signs of congestive failure 3 to 6 weeks following constriction. Right ventricular papillary muscle from these hearts exhibited a depressed force-velocity relationship and an increased time to peak tension but otherwise were normal. Myosin prepared from control and hypertrophied hearts was of comparable purity and yield. Quantitative densitometry of SDS polyacrylamide gels showed the light chains of hypertrophy myosin were unchanged in number, molecular weight and stoichiometry. Total protein sulfhydryl content was not different for control and hypertrophy myosins. Calcium-activated ATPase activity of hypertrophy myosin was significantly decreased shen measured at varying ATP concentration. By Lineweaver-Burk analysis, V max of hypertrophy myosin was depressed by 50%, while the apparent K m was 10% lower. When sulfhydryl-dependent ATPase activity was measured as a function of p-chloromercuribenzoate (PCMB) concentration, hypertrophy myosin exhibited a normal activation profile. The ATPase activity of hypertrophy myosin was initially depressed by 50% and was significantly reduced at low PCMB concentrations. However the difference was absent at peak activation, suggesting that the SH 1 sulfhydryls were capable of activation by PCMB to the same level as for control myosin. These data were consistent with a subtle conformational change at or near the active site, rather than with a change in the composition of major subunits of myosin from hypertrophied hearts.

Reconstitution of an active calcium pump in sarcoplasmic reticulum.

Recovery of calcium transport and calcium-activated ATPase activity was studied in relation to the retention of protein components in sarcoplasmic reticulum reconstituted after solubilization with deoxycholate and centrifugation, followed by removal of the detergent from the supernatant by dialysis. Control sarcoplasmic reticulum was similarly treated except for omission of deoxycholate. Maximum capacity for oxalate- and phosphate-supported calcium uptake was increased 2- to 3-fold in reconstituted sarcoplasmic reticulum compared to original and control. Calcium uptake velocity of the reconstituted sarcoplasmic reticulum was approximately 80% that of original and 90% of control sarcoplasmic reticulum. Calcium uptake/ATP hydrolysis ratio was approximately 2 in the original sarcoplasmic reticulum and decreased to approximately 1 in the control and reconstituted sarcoplasmic reticulum. Calcium storage in the absence of calcium-precipitating anion was approximately 85% in control and 70% in reconstituted sarcoplasmic reticulum, compared to the original sarcoplasmic reticulum. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-induced calcium release after phosphate-supported calcium uptake was slower in reconstituted sarcoplasmic reticulum than in original or control sarcoplasmic reticulum. Polyacrylamide gel electrophoresis of original and control sarcoplasmic reticulum showed similar amounts of protein components of approximately 93,000, 59,000, 50,000, 30,000 to 37,000, and 20,000 to 26,000 daltons. Reconstituted sarcoplasmic reticulum, however, lost over 85% of the 50,000- and 20,000- to 26,000-dalton proteins while retaining most of its calcium transport functions.

Calcium-activated ATPase activity in a plasma membrane-rich preparation of rat pancreas

1. Calcium-stimulated ATPase activity was studied in a plasma membrane rich fraction of rat pancreas homogenate. 2. The enzyme is stimulated to the same maximum rate of ATP hydrolysis by either calcium or magnesium, but the apparent requirement for calcium is five-fold lower than for magnesium. 3. Maximum hydrolytic activity of the enzyme is not increased when additional magnesium is added to the optimal amount of calcium. 4. The enzyme does not require Na+ or K+ for its activation by Ca2+ and is not inhibited by ouabain or 2,4-dinitrophenol. 5. Pancreozymin, in concentrations which evoke secretion of zymogen protein, inhibits the calcium-stimulated ATPase. 6. Carbachol and dibutyryl cyclic AMP, in concentrations which increase the release of digestive proteins, do not alter the activity of the calcium-stimulated enzyme. 7. It is suggested that the plasma membrane calcium-activated enzyme, is not involved in the active calcium extrusion, previously reported to occur with use of the various pancreatic secretagogues tested.

Structural and functional comparison of myosin from the rabbit Oryctolagus cuniculus and the marmot marmota monax

1. 1. Cardiac myosin calcium-activated ATPase activity and subunit composition of the rabbit, Oryctolagus cuniculus, and woodchuck, Marmota monax, were compared. 2. 2. M. monax myosin had a higher ATPase activity from 2 to 35°C. 3. 3. Myosin ATPase from both species had the same energy of activation (15·6 kcal mol −1) from 2 to 25°C. 4. 4. Myosin from hibernating and nonhibernating M. monax had the same ATPase activity from 2 to 20°C. 5. 5. Myosin subunit structure from both species was the same by two distinct electrophoretic criteria. 6. 6. Results indicate that differences in myosin structure cannot explain the low-temperature differences in cardiac muscle mechanical behaviour of the two species.

Kinetic analysis of axoneme and dynein ATPase from sea urchin sperm

The behavior of the ATPase of axoneme (detergent-treated flagellum) and dynein from sea urchin sperm was investigated. The activation of the ATPase by divalent cations was attributed to formation of a complex of ATP and the divalent cation; the metal-ATP complex is an effective substrate. However, free ATP is a modifier of the ATPase. Free ATP markedly changes the affinity of the metal-ATP complex to the enzymes. Calcium-activated ATPase activity of axoneme decreased at high concentration of CaCl 2, but that of dynein did not decrease.

Allosteric Inhibition by Alkali Ions of the Ca2+ Uptake and Adenosine Triphosphatase Activity of Skeletal Muscle Microsomes

Abstract The Ca2+ uptake and the calcium-activated adenosine triphosphatase activity of skeletal muscle microsomes are inhibited by alkali ions in assay media containing ATP concentrations ranging from 10-6 to 10-5 m. No inhibition is observed at ATP concentrations higher than 10-4 m. The pattern of inhibition of Ca2+ uptake by different alkali ions varies depending on the ATP concentration. For 2 µm ATP, the sequence of inhibition is Li+ g Na+ g K+ and for 10 µm ATP is Na+ g Li+ ≥ K+. The calcium-activated ATPase is inhibited according to the same pattern, Na+ g Li+ ≥ K+, at both 2 and 10 µm ATP. The dependence of the initial rate of Ca2+ uptake on the ATP concentration displays hyperbolic saturation curves in assay media containing 20 mm KCl, 120 mm KCl, or 120 mm NaCl. Na+ and K+ inhibit both the Ca2+ uptake and the calcium-activated ATP-ase activity to the same extent. In contrast, sigmoidal saturation curves for Ca2+ uptake and hyperbolic saturation curves for the calcium-activated ATPase are obtained in media containing 120 mm LiCl. When the free Ca2+ concentration is varied from 1.0 to 7.1 µm in media containing 10 µm ATP and 20 mm KCl, 120 mm KCl, or 120 mm NaCl, both the initial rate of Ca2+ uptake and the calcium-activated ATPase activity increase hyperbolically. Lowering the ATP concentration to 2 µm results in sigmoidal saturation curves for Ca2+ uptake and hyperbolic saturation curves for the calcium-activated ATPase activity. The Ca2+ uptake in media containing 120 mm LiCl is a sigmoidal function of the Ca2+ concentration both at ATP concentrations of 2 µm and 10 µm. However, the calcium-activated ATPase always shows a hyperbolic dependence on the Ca2+ concentration. The effect of alkali ions on Ca2+ influx and efflux through the skeletal muscle microsomes membrane was studied with the use of 2 µm ATP. Both the rate of Ca2+ efflux and of active transport are inhibited by Na+ and Li+. In media containing 120 mm of either NaCl or LiCl, the equilibrium between active transport and Ca2+ efflux is reached at Ca2+ concentrations higher than 0.5 µm. The rate of Ca2+ exchange between the interior of the vesicles and the assay medium is essentially the same in the presence or absence of 120 mm of either NaCl or LiCl. The data presented suggest that: (a) acetyl phosphate and ATP are both substrates of a common active site for the Ca2+ transport; and (b) the binding of ATP to the Ca2+ carrier system promotes conformational changes which result in modification of its affinity for Ca2+.

Pyrophosphate Binding to and Adenosine Triphosphatase Activity of Myosin and Its Proteolytic Fragments: IMPLICATIONS FOR THE SUBSTRUCTURE OF MYOSIN

The binding of pyrophosphate to myosin, heavy meromyosin, and Subfragment 1 was studied by an equilibrium dialysis technique. Myosin binds approximately 2 (1.82) moles/5 × 105 g of protein, K = 2.07 × 106. Identical results were obtained with myosin prepared by the ammonium sulfate precipitation procedure with or without LiCl and the dilution method. Red and white skeletal muscle myosin bound the same number of moles of pyrophosphate, and variation of the ionic strength up to 0.6 did not affect either the binding number or the binding constant. No selective blocking of any of the sites could be achieved with N-ethylmaleimide or p-chloromercuribenzoate. Heavy meromyosin bound 1.83 moles of pyrophosphate per 3.6 × 105 g, and Subfragment 1 with a molecular weight of 9.74 × 104 to 1.19 × 105 bound 0.85 mole/105 g, K = 3.67 × 106. Natural actomyosin bound only 1 mole of pyrophosphate per 5 × 105 g of myosin. The addition of actin to myosin reduced both the number of binding sites and the affinity constant. The effect of actin on the pyrophosphate binding of heavy meromyosin was greater than on myosin and led to essentially complete suppression of binding. When heavy meromyosin was digested with trypsin (heavy meromyosin to trypsin, 16:1) at 23°, there was no alteration in the potassium-ethylenediaminetetraacetate activation or the calcium-activated ATPase activity. Isolated Subfragment 1 had a specific potassium-EDTA-activated ATPase activity which was 1.86 times higher than that of heavy meromyosin and a calcium-activated ATPase activity which was 2.60 times higher. After a 10-min tryptic digestion of heavy meromyosin, 25 to 45% of the total nitrogen was nonprecipitable with 6% trichloracetic acid. Disc electrophoresis of Subfragment 1 isolated by gel filtration on Sephadex G-200 contained two major bands and several minor ones in the leading edge of the peak, while the trailing edge contained only one major band and a few minor bands. The simplest explanation of these results is that tryptic digestion of heavy meromyosin produces two subfragments, each of which binds pyrophosphate, hydrolyzes ATP, and has the ability to combine with actin.