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.     

The contractile proteins of smooth muscle. Properties and components of a Ca2+-sensitive actomyosin from chicken gizzard.

The preparation and characterization of a Ca²⁺-sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg²⁺ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myosin ATPase by actin, and the Ca²⁺ sensitivity were confined to the neutral pH region. The subunit composition of the Ca²⁺-sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of ~130,000.     

Myosin and actin from ascidian smooth muscle and their interaction

Myosin and actin were purified from ascidian smooth muscle. Ascidian myosin contained two classes of light chains and the pH dependence of Ca2+-activated ATPase and the KCl dependence of actin-activated ATPase of ascidian myosin differed from those of vertebrate skeletal myosin. Troponin-tropomyosin complex from ascidian increased the ATPase activity of ascidian reconstituted actomyosin in a Ca2+-dependent manner. Ascidian myosin provided the reconstituted actomyosin with the responsiveness to calcium ions. Two actin isoforms were present in ascidian, which were distinguished by isoelectric points.     

Superprecipitation of an actomyosin-like complex isolated from Naegleria gruberi amoebae

A protein complex similar to muscle actomyosin and plasmodial myosin B has been isolated from Naegleria gruberi amoebae. This extract, which comprises approximately 0.7% of the total cell protein, has the solubility properties of actomyosin, displays Ca²⁺-activated, Mg²⁺-inhibited ATPase activity, forms microfilaments, and undergoes a strong superprecipitation reaction. Superprecipitation is initiated by ATP and is preceded by a very brief clearing phase. Although added Mg²⁺ is not essential for superprecipitation of the extract, the reaction proceeds maximally when 7 mM Mg²⁺ is provided. This extract does not appear to have a Ca²⁺ requirement, and superprecipitation is in fact inhibited by added Ca²⁺ ion at all concentrations greater than 0.1 mM. Both ATPase activity and superprecipitation of the actomyosin-like complex are inhibited by the sulfhydryl inhibitor salyrgan.     

Effect of phosphorylation on the actin-activated atpase activity of myosin

The purpose of this study was to test the hypothesis that the phosphorylation of myosin is solely responsible for the activation of the Mg²⁺-ATPase activity of gizzard actomyosin. Using a washed natural actomyosin and a reconstituted actomyosin it was shown that phosphorylation alone caused only a slight activation of ATPase activity. Full activity was obtained only when proteins in addition to the myosin light chain kinase were added. It is evident from these results that: 1) there is no simple relationship between the extent of myosin phosphorylation and the specific Mg²⁺-ATPase activity of actomyosin and 2) in order for full activation by actin of the Mg²⁺-ATPase activity of phosphorylated myosin additional factors are required.     

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.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Extraction of an actomyosin-like pootein from amoebae of Dictyostelium discoideum

An actomyosin-like protein has been extracted from amoebae of Dictyostelium discoideum, V-12. The purified protein exhibited a reversible change in viscosity upon addition of ATP, indicating an ATP sensitivity of 75–85% and a specific viscosity of 0.1. At low ionic strength in the presence of Mg⁺⁺ and ATP the amoeba protein displayed the phenomenon of superprecipitation. The protein extract was found to be an adenosinetriphosphatase (ATP'ase) hydrolyzing ATP to ADP and inorganic phosphate. Both Mg⁺⁺ and Ca⁺⁺ at low ionic strength accelerated the ATP ase activity whereas at high ionic strength only Ca⁺⁺ stimulated ATP hydrolysis. The ATP'ase activity was inhibited by ethylene-diamine-tetraacetic-acid, Mersayl and p-chloromercuribenzoate. The physico-chemical and enzymatic properties of the extracted amoeba protein are qualitatively comparable to those of muscle actomyosin, and very similar in quantitative properties to smooth muscle actomyosin and the actomyosin-like proteins of blood platelets, leucocytes and slime mold plasmodia. The significance of the presence of this actomyosin-like protein in Dictyostelium amoebae is discussed in relation to amoeboid form and movement.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.     

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.     

Novel Mode of Cooperative Binding between Myosin and Mg-actin Filaments in the Presence of Low Concentrations of ATP

Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca²⁺-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg²⁺-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg²⁺-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.     

Inhibition of Mg ++ ATPase activity of actin-activated Acanthamoeba myosin by muscle troponin-tropomyosin: implications for the mechanism of control of amoeba motility and muscle contraction

Troponin-tropomyosin is known to inhibit the Mg⁺⁺ATPase activity of muscle actomyosin in the absence, but not in the presence, of Ca⁺⁺. In contrast, we have now found that muscle troponin-tropomyosin inhibits the Mg⁺⁺ATPase activity of muscle actin-activated $\text{Acanthamoeba}$ myosin both in the presence and the absence of Ca⁺⁺. Addition of purified tropomyosin and troponins-I, C and T demonstrated that it is troponin-T that acts differently in the two systems which differ only in the source of the myosin. These data suggest that myosin, as well as actin, plays a role in the troponin-tropomyosin control of muscle contraction and make it unlikely that control proteins identical to troponin-tropomyosin function in this amoeba.     

Generation of force by single-headed myosin.

Myosin has two heads, each of which can interact with actin and ATP. We have investigated the possibility that co-operative interactions occur between the heads by measuring the force generated by single-headed myosin in reconstituted actomyosin threads. Myofibrils were digested with papain, actomyosin was extracted from the myofibrils, and one-headed myosin was purified by cycles of sedimentation with actin. The one-headed myosin was approximately 90 to 95% pure as determined by densitometer scans of polyacrylamide gels run in 20 mm-PP₁ (impurities consisted of 1 to 5% of myosin and 1 to 5% myosin rod). The ATPase activity per mole of single-headed myosin was one half that of myosin under conditions where the activity was activated by Ca²⁺, K⁺ or actin. One-headed myosin could also participate in superprecipitation, although with a rate that was at least one order of magnitude slower than that for myosin. Myosin or one-headed myosin was mixed with actin, threads were formed via extrusion into low ionic strength, and the isometric forces and isotonic velocities generated by the threads were measured. The ratio of the isometric tension produced per head by the one-headed myosin to the isometric tension produced per head by myosin was 1·0 ± 0·1. The maximum velocity of thread contraction for the one-headed myosin was also not different from the control myosin. Thus, the absence of one head does not appear to impair the generation of force or motion by the remaining head.     

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

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

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

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

Calcium-Dependent Myosin from Insect Flight Muscles

Calcium regulation of the insect actomyosin ATPase is associated with the thin filaments as in vertebrate muscles, and also with the myosin molecule as in mollusks. This dual regulation is demonstrated using combinations of locust thin filaments with rabbit myosin and locust myosin with rabbit actin; in each case the ATPase of the hybrid actomyosin is calcium dependent. The two regulatory systems are synergistic, the calcium dependency of the locust actomyosin ATPase being at least 10 times that of the hybrid actomyosins described above. Likewise Lethocerus myosin also contains regulatory proteins. The ATPase activity of Lethocerus myosin is labile and is stabilized by the presence of rabbit actin. Tropomyosin activates the ATPase of insect actomyosin and the activation occurs irrespective of whether the myosin is calcium dependent or rendered independent of calcium.     

Activation by actin of ATPase activity of chemically modified gizzard myosin without phosphorylation.

The ATPase activity of myosin from chicken gizzard measured in the presence of either Mg²⁺ or Ca²⁺ is increased in the absence of dithiothreitol or upon reaction with Cu²⁺, o-iodosobenzoate, or N-ethylmaleimide. Iodosobenzoate or Cu²⁺ produce no change in K⁺(EDTA)-ATPase while N-ethylmaleimide produces a decrease. These treatments also make the actin-activated ATPase insensitive to Ca²⁺ when assayed in the presence of tropomyosin and a partially purified myosin light chain kinase. Phosphorylation of N-ethylmaleimide modified myosin remains dependent on Ca²⁺ and therefore appears not to be required for activation by actin of the ATPase activity of modified myosin.     

The effect of phosphorylation of gizzard myosin on actin activation

Gizzard myosin is phosphorylated by a kinase found in chicken gizzards. The 20,000 dalton light chains are the only subunits to show an appreciable extent of ³²P incorporation. Phosphorylation requires trace amounts of Ca²⁺. The Mg²⁺-ATPase activity of gizzard myosin in the phosphorylated form is activated to an appreciable extent by skeletal actin, whereas the activation of the non-phosphorylated myosin is verylow. These results suggest that the Ca²⁺-sensitive regulatory mechanism of gizzard actomyosin is mediated via a kinase. In the presence of Ca²⁺ the onset of contraction and the resultant increase of the Mg²⁺-ATPase activity we suggest is due, at least partly, to the phosphorylation of the 20,000 dalton light chains. Whether or not Ca²⁺ binding by myosin is also essential remains to be established.     

The reaction of myosin with N-ethylmaleimide in the presence of ADP

Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg²⁺-ATPase activity. At the point at which actin activation was lost the K⁺-EDTA-ATPase activity was also lost, but the Ca²⁺-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect K_app (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg²⁺-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin.