Effects of phosphorylation, MgATP, and ionic strength on the rates of papain degradation of heavy and light chains of smooth muscle heavy meromyosin at the S1-S2 junction

The effects of ionic strength, MgATP, and phosphorylation on the degradation rates of heavy meromyosin (HMM) by papain have been compared to their effects on the sedimentation coefficient (s20,w) to determine the relationship of the degradation rate to the equilibrium between the flexed and the extended forms (Suzuki, H., Stafford, W. F., Slayter, H. S., and Seidel, J. C. (1985) J. Biol. Chem. 260, 14810-14817). At 0.025 M NaCl, where HMM is predominantly in the flexed form, MgATP, Mg-adenylyl imidodiphosphate or MgADP reduce kH by 80-90%. MgATP exerts its optimal effect at this ionic strength, where at least 70% of HMM is flexed in the presence or absence of MgATP, suggesting that nucleotides reduce kH by decreasing the proteolytic susceptibility of the flexed form. At 0.5 M NaCl, where HMM is in the extended form, MgATP has no effect on kH. At low ionic strengths phosphorylation decreases kH but increases it in the presence of MgATP. Plots of kH against s20,w determined at various ionic strengths are linear, the data for phosphorylated and dephosphorylated HMM falling on the same line. Thus, raising the ionic strength or phosphorylating the 20-kDa light chain appears to alter kH by increasing the fraction of HMM in the extended form. The degradation rate of the 20-kDa light chain (kL) of dephosphorylated HMM responds to changes in ionic strength in essentially the same way as does kH, suggesting that the response of kL to changes in ionic strength can also be attributed to conversion of HMM to the extended form. However, kL for phosphorylated HMM measured in the presence of MgATP exhibits very little dependence on ionic strength.     

Calcium-dependent structural changes in scallop heavy meromyosin

The mechanism of calcium regulation of scallop myosin is not understood, although it is known that both myosin heads are required. We have explored possible interactions between the heads of heavy meromyosin (HMM) in the presence and absence of calcium and nucleotides by sedimentation and electron microscope studies. The ATPase activity of the HMM preparation was activated over tenfold by calcium, indicating that the preparation contained mostly regulated molecules. In the presence of ADP or ATP analogs, calcium increased the asymmetry of the HMM molecule as judged by its slower sedimentation velocity compared with that in EGTA. In the absence of nucleotide the asymmetry was high even in EGTA. The shift in sedimentation occurred with a sharp midpoint at a calcium level of about 0.5 microM. Sedimentation of subfragment 1 was not dependent on calcium or on nucleotides. Modeling accounted for the observed sedimentation behavior by assuming that both HMM heads bent toward the tail in the absence of calcium, while in its presence the heads had random positions. The sedimentation pattern showed a single peak at all calcium concentrations, indicating equilibration between the two forms with a t(1/2) less than 70 seconds. Electron micrographs of crosslinked, rotary shadowed specimens indicated that 81 % of HMM molecules in the presence of nucleotide had both heads pointing back towards the tail in the absence of calcium, as compared with 41 % in its presence. This is consistent with the sedimentation data. We conclude that in the "off" state, scallop myosin heads interact with each other, forming a rigid structure with low ATPase activity. When molecules are switched "on" by binding of calcium, communication between the heads is lost, allowing them to flex randomly about the junction with the tail; this could facilitate their interaction with actin in contracting muscle.     

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.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

The effect of mechanical stretching of the myosin rod component (fragment LMMMM S-2) on the ATPase activity of myosin.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.     

Heavy meromyosin from skipjack tuna, Euthynus pelamis. Preparation and enzymic properties.

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin.     

Two functional heads are required for full activation of smooth muscle myosin

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.     

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.     

Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases

The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.     

The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state

The correlation curve between phosphorylation and MgATPase activity suggests that the 20,000-dalton light chain of both heads of a smooth muscle myosin or heavy meromyosin (HMM) molecule must be phosphorylated before the MgATPase activity of either head can be activated by actin. The two heads of HMM appear to be phosphorylated randomly at equal rates, while those of myosin are phosphorylated in a negatively cooperative manner (Persechini, A., and Hartshorne, D.J. (1981) Science, 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. 91, 1809-1812). We have investigated the cause of this difference between HMM and myosin. We find that if myosin is first phosphorylated at high ionic strength (0.6 M KCl), where it is monomeric, and then assayed for MgATPase activity (in 0.05 M KCl), the data support a model where the two heads are phosphorylated randomly with equal rates (i.e. similarly to HMM). The correlation curves between MgATPase activity and dephosphorylation of fully phosphorylated myosin, both in a filamentous and monomeric state, are also best explained by a model where dephosphorylation of one head is sufficient to deactivate the entire molecule. With monomeric myosin, the dephosphorylation appears to occur randomly with equal rates, whereas with filamentous myosin the dephosphorylation appears to be negatively cooperative. The correlation between dephosphorylation of HMM and its MgATPase activity is more complex and is consistent with a positively cooperative dephosphorylation. Direct analyses of the time courses of phosphorylation of HMM and monomeric myosin show that a single exponential is sufficient to fit the data through greater than 90% of the reaction. However, when phosphorylation is carried out at low ionic strength (0.02 M KCl), where myosin is present as filaments, the time course consists of two exponential functions where the rate constant for the phosphorylation of one myosin head is 6-10 times greater than that for the other head which is located on the same molecule. This suggests that when myosin is polymerized into filaments the two previously indistinguishable heads either become nonequivalent or are subject to head-head interactions leading to a negatively cooperative phosphorylation reaction.     

Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation.

Myosin has been identified in a variety of non-muscle cells, and is believed to play a role in maintenance of cell shape, locomotion, cytokinesis, exocytosis and other cellular functions. In this paper we describe the purification of myosin from a pancreatic acinar-cell carcinoma of the rat which forms solid tumours, but retains many differentiated functions. The purified myosin was composed of a 200,000 Da heavy chain and two or three classes of light chains. Electron-microscopic examination of rotary-shadowed preparations revealed that individual molecules had two globular heads and a long tail measuring approx. 149 nm. The myosin was soluble in high-salt buffers and became sedimentable as the ionic strength was lowered. Examination of negative-stained preparations showed that this sedimentable myosin consisted of short, bipolar, thick filaments which had a strong tendency to aggregate in a head-to-head manner. The ATPase activity of the purified myosin was stimulated by EDTA or Ca2+, but not by Mg2+. In low ionic strength the Mg2+-dependent ATPase activity was activated by muscle f-actin. The pancreatic myosin bound to actin and could be dissociated by the addition of MgATP. Myosin purified from cells cultured in media containing [32P]Pi was phosphorylated on one of the light chains as well as the heavy chain. Thus pancreatic acinar cells contain a typical non-muscle myosin, and the subunits of this molecule are subject to post-translational modification by phosphorylation.     

The tail of myosin reduces actin filament velocity in the in vitro motility assay

It has been observed that heavy meromyosin (HMM) propels actin filaments to higher velocities than native myosin in the in vitro motility assay, yet the reason for this difference has remained unexplained. Since the major difference between these two proteins is the presence of the tail in native myosin, we tested the hypothesis that unknown interactions between actin and the tail (LMM) slow motility in native myosin. Chymotryptic HMM and LMM were mixed in a range of molar ratios (0-5 LMM/HMM) and compared to native rat skeletal myosin in the in vitro motility assay at 30 degrees C. Increasing proportions of LMM to HMM slowed actin filament velocities, becoming equivalent to native myosin at a ratio of 3 LMM/HMM. NH4+ -ATPase assays demonstrated that HMM concentrations on the surface were constant and independent of LMM concentration, arguing against a simple displacement mechanism. Relationships between velocity and the number of available heads suggested that the duty cycle of HMM was not altered by the presence of LMM. HMM prepared with a lower chymotrypsin concentration and with very short digestion times moved actin at the same high velocity. The difference between velocities of actin filament propelled by HMM and HMM/LMM decreased with increasing ionic strength, suggesting that ionic bonds between myosin tail and actin filaments may play a role in slowing filament velocity. These data suggest the high velocities of actin filaments over HMM result from the absence of drag generated by the myosin tail, and not from proteolytic nicking of the motor domain.     

The binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.     

The interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin

Recent findings have suggested that the interaction between the two heads is critical for phosphorylation-dependent regulation of smooth muscle myosin. We hypothesized that the interaction between the two regulatory light chains on two heads of myosin dictates the regulation of myosin motor function. To evaluate this notion, we engineered and characterized smooth muscle heavy meromyosin (HMM), which is composed of one entire HMM heavy chain and one motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM was inactive for both actin-translocating activity and actin-activated ATPase activity in the dephosphorylated state, demonstrating that the interaction between the two RLC domains on the two heads and/or a motor domain and a RLC domain in a distinct head is sufficient for the inhibition of smooth muscle myosin motor activity. When phosphorylated, SMDHMM was activated for both actin-translocating activity and actin-activated ATPase activity; however, these activities were lower than those of double-headed HMM, implying partial release of inhibition by phosphorylation in SMDHMM and/or cooperativity between the two heads of smooth muscle myosin. The present results indicate that the RLC domain is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. On the other hand, similar to double-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, suggesting that the RLC domain is sufficient to regulate the conformational transition in myosin.     

Comparison of effects of smooth and skeletal muscle tropomyosins on interactions of actin and myosin subfragment 1

The ATPase activity of acto-myosin subfragment 1 (S-1) was measured in the presence of smooth and skeletal muscle tropomyosins over a wide range of ionic strengths (20-120 mM). In contrast to the 60% inhibitory effect caused by skeletal muscle tropomyosin at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on ionic strength. At low ionic strength (20 mM), smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM), it potentiates the ATPase activity 3-fold. All of these ATPase activities were measured at very low ratios of S-1 to actin, under conditions at which a 4-fold increase in S-1 concentration did not change the specific activity of the tropomyosin-acto.S-1 ATPase. Therefore, the potentiation of the ATPase activity by smooth muscle tropomyosin at high ionic strength cannot be explained by bound S-1 heads cooperatively turning on the tropomyosin-actin complex. To determine whether the fully potentiated rates are different in the presence of smooth muscle and skeletal muscle tropomyosins, S-1 which was extensively modified by N-ethylmaleimide was added to the ATPase assay to attain high ratios of S-1 to actin. The results showed that, under all conditions, the fully potentiated rates are the same for both tropomyosins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Supramolecular regulation of the actin-activated ATPase activity of filaments of Acanthamoeba Myosin II

Acanthamoeba myosin II has three phosphorylation sites clustered near the end of the tail of each of its two heavy chains (six phosphorylation sites/molecule). Myosin II has little or no actin-activated ATPase activity when four to six of these sites are phosphorylated. Maximal actin-activated ATPase activity is obtained when all six sites are dephosphorylated. Under assay conditions, both phosphorylated and dephosphorylated myosin II form bipolar filaments. Filaments of dephosphorylated myosin II have larger sedimentation coefficients than filaments of phosphorylated myosin II but this difference does not explain the difference in their actin-activated ATPase activities. Heteropolymers, formed by mixing soluble dephosphorylated and phosphorylated myosins and then diluting the mixture into low ionic strength buffer containing MgCl2, have sedimentation coefficients close to those of the homopolymer of phosphorylated myosin. The actin-activated ATPase activities of heteropolymers are, under most conditions, lower than the equivalent mixtures of homopolymers of dephosphorylated and phosphorylated myosins. It is concluded, therefore, that the phosphorylation of myosin tails regulates the actin-activated ATPase activity of Acanthamoeba myosin II by affecting the myosin filament as a whole rather than specifically affecting the heads of the phosphorylated myosin molecules only.     

The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction.

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.     

Visualization of head-head interactions in the inhibited state of smooth muscle myosin

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

New method to prepare single-headed heavy meromyosin with high purity and a high yield

We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.