Effect of muscle and non-muscle tropomyosins in reconstituted skeletal muscle actomyosin

Smooth and non-muscle tropomyosins were found to produce a 2-3-fold Ca-insensitive stimulation of the ATPase activity of reconstituted skeletal muscles actomyosin at normal MgATP concentrations and physiological ratios of myosin to actin. Under the same conditions skeletal muscles tropomyosin had no effect. Similar effects of these three tropomyosins were observed for the low myosin/F-actin ratios necessary for kinetic measurements. Since it could be established that this actomyosin system, with or without tropomyosin, obeyed Michaelian kinetics, the tropomyosin effects could be interpreted in terms of their influence on maximal turnover (V) or on the affinity of myosin for actin (Kapp). Accordingly, gizzard tropomyosin had practically no effect on the affinity and reduced only slightly the value of V, compared to pure actin. In contrast to gizzard tropomyosin, brain tropomyosin produced an approximately twofold increase in both Kapp and V; i.e. it increased the turnover rate but decreased the affinity. It is apparent from the data that brain tropomyosin acts as an uncompetitive activator with respect to pure actin, while having the same V as the actin plus gizzard tropomyosin complex. Further studies on these tropomyosins show that only skeletal and smooth muscle tropomyosin have similar functional properties with respect to troponin inhibition and the activation of the ATPase at low ATP concentrations. It is suggested that the noted increases in V by tropomyosin are caused by the acceleration of the dissociation of the myosin head from actin at the end point of the cross bridge movement.     

Tropomyosin from smooth and skeletal muscles initiates various conformational changes in skeletal F-actin

Changes in F-actin conformation in myosin-free single ghost fibers of rabbit skeletal muscle induced by the binding of skeletal and gizzard tropomyosin to F-actin were studied by measuring intrinsic tryptophan-polarized fluorescence of F-actin. It was found that skeletal and gizzard tropomyosin binding to F-actin initiate different conformational changes in actin filaments. Skeletal tropomyosin inhibits, while gizzard tropomyosin activates the Mg2+-ATPase activity of skeletal actomyosin. It is supposed that in muscle fibers tropomyosin modulates the ATPase activity of actomyosin via conformational changes in F-actin.     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.     

Calcium binding of arterial tropomyosin: involvement in the thin filament regulation of smooth muscle

Bovine aortic tropomyosin has been isolated by DEAE-Sepharose chromatography following isoelectric precipitation and ammonium sulfate fractionation. A single polypeptide [Mr 36 000 on a sodium dodecyl sulfate (SDS)-polyacrylamide gel] was obtained under different electrophoretic conditions. The amino acid composition of bovine tropomyosin was very similar to that of rabbit skeletal muscle; the amino-terminal residue is blocked. The molecular weight of the native tropomyosin (76 000), which is twice that calculated from the SDS-polyacrylamide gel, suggests that the molecule is a dimer. The diffusion coefficient of 3.4 X 10(-7) cm2 s-1 and the frictional coefficient of 1.7 indicate that the molecule is asymmetric. Comparative high-pressure liquid chromatography peptide mapping of rabbit skeletal and bovine aortic tropomyosins shows primary structure variation. Bovine aortic tropomyosin binds calcium under physiological conditions of pH and ionic strength (22 mol of Ca2+/mol of tropomyosin with a Kd of 1.4 mM). Such a property is not shared by skeletal tropomyosin. In low Mg2+ concentration, both skeletal and aortic actin activations of the skeletal myosin ATPase activity are calcium independent. Addition of aortic tropomyosin to a hybrid actomyosin (aortic actin, skeletal myosin) yields an enhancement of the actin activation of the myosin ATPase activity, but the addition of skeletal tropomyosin yields a decrease of this activity. However, both the enhancement and decrease are calcium dependent. Addition of skeletal or aortic tropomyosin to an actomyosin system, where both actin and myosin come from skeletal muscle, yields only an enhancement of the actin activation of the myosin ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin

The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin-label rigidly bound at Cys-374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin. Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F-actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F-actin, suggesting that the slow formation of interfilamental cross-links in concentrated F-actin solutions contributes to this process. We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F-actin in its rotational mobility but differs from the complex of F-actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub-millisecond internal motion in actin polymer and for a non-cooperative change in the intramonomer conformation around Cys-374 on the binding of HMM.     

Nonlinear dependence of actin-activated Mg2+-ATPase activity on the extent of phosphorylation of gizzard myosin and H-meromyosin

1. The actin-activated Mg2+-ATPase activity of gizzard HMM increased in proportion to the square of the extent of LC phosphorylation. This result indicates that the LCs of HMM are randomly phosphorylated, and the phosphorylation of both heads of HMM is required for the activation of HMM Mg2+-ATPase by F-actin. 2. In 75 mM KCl, the Mg2+-ATPase activity of gizzard myosin was activated by F-actin only slightly when a half of the total LC was phosphorylated. From 1 to 2 mol LC phosphorylation, the activity was enhanced by F-actin almost linearly. In 30 mM KCl, the activity of acto-gizzard myosin increased sigmoidally with increase in the extent of LC phosphorylation. On electron microscopy, side-by-side aggregates of myosin filaments were observed in 30 mM KCl, but not in 75 mM KCl. It was suggested that the activation of the Mg2+-ATPase activity of acto-gizzard myosin LC phosphorylation is modified by formation of myosin filaments and their aggregates. 3. The relationship between the actin-activated Mg2+-ATPase activity of HMM or myosin and the extent of LC phosphorylation was unaffected by tropomyosin.     

Comparison of Ca2+-dependent effects of caldesmon-tropomyosin-calmodulin and troponin-tropomyosin complexes on the structure of F-actin in ghost fibers and its interaction with myosin heads

Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.     

Myosin-linked calcium regulation in vertebrate smooth muscle.

By the use of a new procedure, actomyosin may be extracted in high yield and purity from fowl gizzard which exhibits a calcium-dependent actin-activated ATPase activity comparable to that of the parent myofibril-like preparation. Studies of this vertebrate smooth muscle actomyosin show that the regulation of the actin-myosin interaction is effected, as in molluscan muscles, by the myosin molecule itself and not by an actin-linked regulatory system, as found in vertebrate skeletal muscle.Thus, calcium-sensitive smooth muscle actomyosin is composed of only myosin, actin and tropomyosin, any troponin-like components being absent. Myosin is the only component that binds significant amounts of calcium and shows a calcium-dependent actin-activated ATPase activity in the presence of F-actin from either gizzard or rabbit skeletal muscle.The cross-reaction of gizzard thin filaments with skeletal muscle myosin produces an actomyosin whose actin-activated ATPase is calcium-insensitive, showing that smooth muscle thin filaments do not serve a regulatory function.The effect of Mg²⁺ and pH, and evidence for the involvement of one of the myosin light chains in calcium regulation are described and discussed.     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.     

A 72,000-mol-wt protein from tomato inhibits rabbit acto-S-1 ATPase activity

Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.     

Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.     

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.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

Potentiation of actomyosin ATPase activity by filamin

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

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