Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum

A myosin-like protein was purified from amoebae of the cellular slime mold Dictyostelium discoideum. The purification utilized newly discovered solubility properties of actomyosin in sucrose. The amoebae were extracted with a 30% sucrose solution containing 0.1 m-KCl, and actomyosin was selectively precipitated from this crude extract by removal of the sucrose. The myosin and actin were then solubilized in a buffer containing KI and separated by gel filtration.The purified Dictyostelium myosin bears a very close resemblance to muscle myosin. The amoeba protein contains two heavy chains, about 210,000 molecular weight each, and two classes of light chains, 16,000 and 18,000 molecular weight. Dictyostelium myosin is insoluble at low ionic strength and forms bipolar thick filaments. The myosin possesses ATPase activity that is activated by Ca²⁺ but not EDTA, and is inhibited by Mg²⁺; under optimal conditions the specific activity of the enzyme is 0.09 μmol P₁/min per mg myosin.Dictyostelium myosin interacts with Dictyostelium actin or muscle actin, as shown by electron microscopy and by measurements of enzymatic activity. The ATPase activity of Dictyostelium myosin, in the presence of Mg²⁺ at low ionic strength, exhibits an average ninefold activation when actin is added.     

Identification of the structural gene for a myosin heavy-chain in Caenorhabditis elegans

Mutants in the unc-54 gene of Caenorhabditis elegans have been characterized by cyanylation and sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the total myosin present in each mutant. In the recessive mutants lacking a major fraction of the total myosin, the high molecular weight doublet of 15 × 10⁴ and 14 × 10⁴ which dominates the cyanylation pattern of the total wild-type myosin is absent. In the mutant E675, which possesses a novel heavy-chain with a molecular weight of 2 × 10⁵, each component of the cyanylation doublet is reduced by 10⁴ daltons, indicating that the doublet is derived from partial cleavage of a single polypeptide chain. This suggests that unc-54 is the structural gene for a myosin heavy-chain present in a major fraction of the total nematode myosin.     

Calcium regulation of the ATPase activity of Physarum and scallop myosins using hybrid smooth muscle myosin: The role of the essential light chain

To examine the role of two light chains (LCs) of the myosin II on Ca²⁺ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca²⁺ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca²⁺ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function.     

Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS.

The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca²⁺-ATPase activity and inhibition of the K⁺-EDTA-ATPase activity of myosin. The stoichiometry of the [³H]1,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH₁” groups responsible for the catalytic properties of myosin. Moreover, it has been unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with [³H]1,5 IAEDANS or iodo[1-¹⁴C]acetamide indicates strongly that the identical thiol was labeled by both reagents.     

Force-Generating Capacity and Contractile Protein Content of Arterial Smooth Muscle

After correction for extracellular space (40%) determined from electron micrographs, the maximum isometric force developed by strips prepared from the media of the hog carotid artery (2.2 x 10⁶ dyn/cm²) can be extrapolated to give a value of 3.7 x 10⁶ dyn/cm² for the smooth muscle component of the strip. Three independent estimates of the myosin content of the smooth muscle cells were made based on (a) exhaustive extraction and purification with estimates of preparative losses, (b) the myosin catalyzed ATPase activity of media homogenates, and (c) quantitative densitometry of the peaks containing myosin, actin, and tropomyosin after disk electrophoresis of sodium dodecyl sulfate-treated media homogenates. The results were consistent and gave a myosin content of 5–10 mg/g media, or 8–17 mg/g cell. Method (c) gave myosin:actin:tropomyosin weight ratios of 1:3.2:0.8. Although measured force developed by the smooth muscle cell exceeds that of mammalian striated muscle, the myosin content in smooth muscle is about five times lower. The actin content of smooth muscle is relatively high. The actin and myosin contents are consistent with thick and thin filament ratios observed in electron micrographs of vascular smooth muscle.     

The nonmuscle myosin phosphatase PP1beta (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1β (PP1β). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw¹, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw¹ and puc^A251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw¹ enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.     

The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin

Evidence is presented that both myosin and actomyosin in presence of Mg²⁺ and KCl catalyze an incorporation of ³²P_i into ATP. The rate with actomyosin is about $\text{1}\text{500}$ the rate of ATP hydrolysis; the rate with myosin is less than $\text{1}\text{100}$ of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of ³²P_i incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [³²P]-ATP present and the rate constant, k_?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔG^o for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔG^o for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage.     

The behavior of fetal canine cardiac cells in culture: synthesis and phosphorylation of myosin.

This study describes the first in vitro culturing of canine cardiac cells. Canine cardiac myosin which was synthesized in a 14-day tissue culture, based on l-[³H]leucine incorporation, was precipitated with goat γG antimyosin (cardiac-specific) and analyzed on dodecylsulfate gels; the specific activity of the highly purified myosin chains was determined. Incorporation of ³²PO₄ was similarly analyzed. The comparative degree of synthesis and phosphorylation of myosin chains, occurring in culture, was the same as that obtained in vivo. Both l-[³H]leucine and ³²PO₄ incorporation were inhibited by addition of cycloheximide to the culture medium. Removal of ³²PO₄ from myosin heavy chains with base treatment indicated the presence of phosphoserine and/or phosphothreonine in canine cardiac myosin heavy chains. Myosins from fetal and adult canine cardiac tissue were immunologically identical with each other and with the cultured fetal tissue; all had similar myosin ATPase activity and the degree of heavy chain phosphorylation was similar. The tissue and techniques used here gave a high yield of cardiac myocytes based principally on synthesis of cardiac-specific myosin.     

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile⁵⁰⁸, Asn⁵⁰⁹, and Asp⁵¹¹) in communicating with converter domain residue Arg⁷⁵⁹. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Mechanism of the Ca-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca²⁺ binds to the EF2 domain of S100A4 with micromolar affinity and that the K_d value for Ca²⁺ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K_d results from a reduced dissociation rate constant (from 16 s^− 1 to 0.3 s^− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca²⁺]. However, for Ca²⁺ transients to be effective in further promoting dissociation, the elevated Ca²⁺ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.     

Actin and Myosin in pea tendrils

We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K⁺-EDTA and Ca²⁺ and is inhibited by Mg²⁺. At low ionic strength, the Mg²⁺-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril.     

Crystal Structure of Calcium Binding Protein-5 from Entamoeba histolytica and Its Involvement in Initiation of Phagocytosis of Human Erythrocytes

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca²⁺ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Å resolution in the Ca²⁺-bound state, which shows an unconventional mode of Ca²⁺ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca²⁺ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role.     

Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a^−/−Myo3b^−/− mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b^−/− mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a^−/−Myo3b^−/− cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a^−/−Myo3b^−/− stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.     

Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

Evidence for a role for cardiac myosin in regulating the contractile response.

Dinitrophenylated bovine cardiac myosin incorporates 1.3 mol of 1-fluoro-2,4-dinitro-benzene per 5 × 10⁵ g of protein. Concomitantly there was an activation of the Ca²⁺-ATPase activity and an inhibition of the K⁺(EDTA)-ATPase activity. The dinitrophenyl group is located in the smallest active proteolytic fragment, subfragment 1. Virtually all of the labeling occurs in the region containing the heavy chains of cardiac myosin as judged by dissociation experiments in sodium dodecyl sulfate. Dinitrophenylated myosin failed to form calcium-sensitive actomyosin when tested in an ATPase assay system containing actin, tropomyosin, troponin and ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid. Thiolysis of the dinitrophenyl group from myosin with 2-mercaptoethanol restored its ability to form a calcium-sensitive actomyosin. The Ca²⁺ and K⁺(EDTA)-ATPase activities were also restored to control values. These results indicate that cardiac myosin participates in the regulation of the interaction between the contractile proteins.     

Magnesium Modulates Actin Binding and ADP Release in Myosin Motors

We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg²⁺-dependent manner (0.3–9.0 mm free Mg²⁺) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg²⁺ in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg²⁺ in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg²⁺ coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg²⁺ concentrations, demonstrating that the ADP release rate constant is slowed by Mg²⁺ in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg²⁺ reduces actin affinity in the M·ADP·P_i state, although it does not change the rate of phosphate release. Therefore, the Mg²⁺ inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg²⁺-dependent alterations in actin binding. Overall, our results suggest that Mg²⁺ reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.     

Cardiomyopathy Mutations Reveal Variable Region of Myosin Converter as Major Element of Cross-Bridge Compliance

The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the β-myosin heavy chain, 723Arg → Gly (R723G) and 736Ile → Thr (I736T), in comparison to 719Arg → Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S^∗_{rigor R723G} = 0.84 pN/nm S^∗_{rigor WT} = 0.29 pN/nm); and ii), a significant increase in force per head (F^∗_10°C, 1.99 pN vs. 1.49 pN = 1.3-fold increase; F^∗_20°C, 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S^∗_active10°C, 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S^∗_rigor; 1.8-fold increase in F^∗_10°C, 1.6-fold in F^∗_20°C; twofold increase in S^∗_active10°C). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous findings that for the slow myosin isoform S^∗ and F^∗ are significantly lower than for fast myosin e.g., of rabbit psoas muscle. The data indicate that two mutations, R723G and R719W, are associated with an increase in resistance to elastic distortion of the individual mutated myosin heads whereas mutation I736T has essentially no effect. The data strongly support the notion that major elastic distortion occurs within the converter itself. Apparently, the compliance depends on specific residues, e.g., R719 and R723, presumably located at strategic positions near the long α-helix of the light chain binding domain. Because amino acids 719 and 723 are nonconserved residues, cross-bridge stiffness may well be specifically tuned for different myosins.     

Molecular mechanism regulating myosin and cardiac functions by ELC

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM^hVLC-1) or E56G-mutated hVLC-1 (hVLC-1^E56G; TgM^E56G). hVLC-1 or hVLC-1^E56G expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM^hVLC-1 (1.67 pN/nm and 2.3 μm/s, respectively) were significantly higher than myosin with hVLC-1^E56G prepared from TgM^E56G (1.25 pN/nm and 1.7 μm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 μm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM^hVLC-1 (80.0 mmHg) were significantly higher than hearts from TgM^E56G (66.2 mmHg) or C57/BL6 (59.3 ± 3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1 > hVLC-1^E56G ≈ mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.