Superprecipitation Of Myosin Research Articles

P2-155 - Relationship between the phosphorylation level of myosin light chain (MLC) and superprecipitation of myosin B from bovine stomach smooth muscle

P2-155 - Relationship between the phosphorylation level of myosin light chain (MLC) and superprecipitation of myosin B from bovine stomach smooth muscle

6-Tridecylresorcylic acid, a novel ATPase inhibitor that blocks the contractile apparatus of skeletal muscle proteins.

6-Tridecylresorcylic acid (TRA) isolated from a primula Lysimachia japonica Thunb. inhibited contraction of myofibrils, superprecipitation of myosin B, and ATPase activities of myosin and actomyosin prepared from rabbit skeletal muscle in a dose-dependent manner. The IC50 values in molarity of TRA were as follows: myosin (K+,EDTA)-ATPase, 3.5 X 10(-6); myosin Ca2+-ATPase, 3.5 X 10(-5); and actomyosin Mg2+-ATPase, 1.6 X 10(-5). The inhibition of ATPase activity of myofibrils by TRA was virtually reversed by washing with the fresh saline solution. Kinetic analysis of inhibitory effects of TRA suggests that the inhibition of (K+,EDTA)-ATPase activity of myosin or subfragment-1 is parabolic noncompetitive. TRA had no effect on alkaline phosphatase and choline acetyltransferase activities. TRA may provide a useful chemical tool for the study of the molecular mechanisms of actin-myosin contractile systems.

Effects of methylxanthines on superprecipitation of myosin B extracted from rabbit fast skeletal muscle.

Myosin B was extracted from rabbit fast skeletal muscle. Caffeine (2-70 mM), theophylline (2-30 mM), and theobromine (2 mM) were examined for their effects on the rate and extent of myosin B superprecipitation at a low ionic strength (50 mM KCl) and a low concentration of ATP (40 microM) by the turbidimetric method. The rate of the superprecipitation was significantly (p less than 0.05 and p less than 0.01) reduced by 30-70 mM caffeine and 2-30 mM theophylline, while the extent was significantly (p less than 0.01) increased by 10-30 mM theophylline. The onset of the superprecipitation was also delayed and a clearing phase was even induced by 50-70 mM caffeine. Theobromine had no significant effect on the rate or extent of the superprecipitation at the concentration used. These results indicate that caffeine and theophylline are retardants of the myosin B superprecipitation reaction in the concentration ranges investigated.

Effects of Ca ion and ADP on superprecipitation of myosin B from slime mold, Physarum polycephalum.

Effects of Ca ion and ADP on superprecipitation of myosin B from slime mold, Physarum polycephalum.

分光光度計のセル自動試料添加攪拌装置: Myosin Bの超沈澱への応用

従来の自記分光光度計のセル試料添加攪拌装置の欠点を克服する目的で，新しくインペラーと試料添加装置を一体化した，往復型インペラーによるセル自動試料添加攪拌装置を工夫した．ブレード型インペラーの往復運動回数21 strokes/sec， 往復運動距離8mm，ブレード面積0.5cm2の実験条件において， メチレンブルー10μl添加時のmixing timeは0.6secであった．ウサギ白筋より抽出したmyosin Bの超沈殿の実験に本装置を応用した．ATPの添加と攪拌を，a)手による振り混ぜ，b)manual mixing plunger， およびc)本装置によりそれぞれ行ない比較した．a，bいずれも初期反応の記録が中断されたが，cの場合は連続記録された．測定値の再現性の点でも手動式に比較してよい結果が得られた．本装置は材料が簡単に入手でき， 自作も容易でしかも費用も低廉である．インペラー駆動部を試料室外部に設置すれば， ほとんどの既製の自記分光光度計に応用が可能である．

Local Migration of Myosin in F-Actin plus ATP Solution on the Boundary of a Diffusion Cell1

The diffusion phenomena of myosin (myosin A, H-meromyosin or subfragment-1) in F-actin plus ATP solutions were investigated. The upper part of the diffusion cell was filled with F-actin plus ATP, and the lower part was filled with F-actin, ATP, and myosin, then both parts were brought into contact so that a boundary of the two solutions was formed and the diffusion of myosin in F-actin plus ATP solutions started. The diffusion pattern was observed with a schlieren lens system. When almost all the ATP in the lower part of the cell had been consumed by actomyosin, a hyper-sharp schlieren pattern appeared near the boundary. On analyzing this pattern, it was found that a local fast migration of proteins was occurring. Simple Brownian motion of myosin molecules could not explain the hyper-sharp phenomenon. This phenomenon occurred in ther pesence of Mg2+ or Ca2+, but very little in the presence of EDTA. Although it is well known that the superprecipitation of myosin B suspension occurs only at physiological ionic strength, this phenomenon occurred over a relatively wide range of ionic strengths. The molecular mechanism of this phenomenon is discussed in relation to the basic mechanism of the interaction between myosin and F-actin.

Involvement of Calcium in Cyclic Nucleotide Metabolism in Human Vascular Smooth Muscle

When cyclic nucleotide phosphodiesterase was purified from isolated smooth muscle layer of human aorta by DEAE-cellulose column chromatography, separated cyclic GMP phosphodiesterase activity was markedly stimulated in the presence of 10-20 micrometer of Ca2+ by a protein modulator which has similar physicochemical properties to troponin C. Synthetic compound, N-(6-aminohexl)-5-chloro-1-naphthalensulfonamide, which produced relaxations of arteries contracted by prostaglandin F2alpha or KCl was found to inhibit selectively this Ca2+-dependent cyclic GMP phosphodiesterase. This compound produced inhibition of superprecipitation of myosin B system obtained from mouse skeletal muscle and also inhibited adenosine triphosphatase activity of myosin B. Our data suggest that calcium is involved through a protein modulator in cyclic nucleotide metabolism of vascular smooth muscle and that the calcium-dependent protein modulator probably participates in the regulation of contractile response of vascular smooth muscle by affecting actomyosin ATPase activity.

Action of peptide-B from bovine fibrinogen on ATPase activity and superprecipitation of myosin B.

Bovine peptide-B from fibrinogen was capable of accelerating the structural and enzymatic effects associated with superprecipitation of myosin B. The rate of superprecipitation coupled with the hydrolysis of ATP are increased during the structural transformation. In the concentration range from 10-8 to 10-4 M peptide-B, the rate of superprecipitation is increased 12-fold while the hydrolysis of ATP doubles and the time to reach the final extent of superprecipitation is decreased 68%. Under these same conditions, the hydrolysis of ATP by myosin A was unaffected. The concentrations of magnesium and calcium were between 10 and 20 muM, and no additional divalent metal ions were added to the system. Superprecipitation was treated as a model for muscle contraction to explain the in vivo studies of bovine peptide-B in which the peptide behaves as a vasopressor substance producing vascular vasoconstriction. A possible mechanism for the participation of bovine peptide-B in the model for muscle contraction, based on the polarizing interaction of the highly charged density of negativity of the peptide with the actomyosin complex, is presented. Furthermore, bovine peptide-B is speculated as participating in vasoconstriction via attachment to some smooth muscle receptor.

A third effect of calcium on superprecipitation of myosin B: activation of superprecipitation by high concentrations of calcium or manganese at alkali pH.

A third effect of calcium on superprecipitation of myosin B: activation of superprecipitation by high concentrations of calcium or manganese at alkali pH.

Effect of catecholamines on energy metabolism of myocardium.

The effects of catecholamines on the oxidative phosphorylation of mitochondria and superprecipitation of myosin B was studied. Mitochondrial oxidative phosphorylations was inhibited by catecholamines. But super-precipitation of myosin B was activated by isoproterenol when the superprecipitation was inhibited by the microsomal fraction.

Relation between the inhibitory action of native tropomyosin and the dual effect of magnesium on the ATPase activity of actomyosin

The effects of various concentrations of Mg 2+ on the ATPase activities of native tropomyosin-free synthetic actomyosin, myosin B, and synthetic actomyosin to which native tropomyosin was added, and on the superprecipitation of myosin B, were investigated in the presence of 6.2 × 10 −8 m Ca 2+ and 1.7 × 10 −5 m Ca 2+. At 6.2 × 10 −8 m Ca 2+, ATPase activity of synthetic actomyosin was enhanced by 5 × 10 −6 to 1 × 10 −4 m Mg 2+. Even with supramaximal amounts of Mg 2+, the ATPase activity was not inhibited below the original level attained without added Mg 2+. At 1.7 × 10 −5 m Ca 2+, the ATPase was markedly activated by Mg 2+ in concentrations lower than 10 −5 m. Further addition of Mg 2+ decreased the ATPase activity toward, but not below, the original level. Unlike synthetic actomyosin, Mg 2+-activated ATPase activity of myosin B and of synthetic actomyosin to which native tropomyosin was added falls below the original level at high Mg 2+ concentrations if Ca 2+ level is very low. This inhibitory effect of Mg 2+ is abolished by high concentrations of Ca 2+. On the basis of these and related findings, we suggest mechanisms for the inhibitory action of native tropomyosin and for the dual effect of Mg 2+.

Structural changes in myosin B during the process of superprecipitation

Superprecipitation of myosin B was studied, using the techniques of light scattering (turbidity and polarization ratio) and electron microscopy. With a dilute myosin B suspension (<0.05 mg/ml) in 5 mM KCl containing 20 mM Tris-HCl (pH 7.4) and 5 mM MgCl 2, the rate of turbidity change caused by addition of ATP was found to follow first order kinetics with respect to the myosin B concentration. The measurement of the polarization ratio, ϱ, as a function of scattering angle θ, showed that a myosin B suspension, with or without ATP, gives a ϱ-θ relationship, indicating a minimum value at 90°, but that the ϱ value in the presence of ATP is substantially higher than that in the absence of ATP. The electron micrographs revealed that myosin B in 5 mM KCl assumes the “arrow head” structures. Immediately after addition of ATP the “arrow heads” tended to disappear and the dense clusters became attached to the thin filaments. From these results, three possible steps involved in superprecipitation are discussed.

Simultaneous measurement of ATPase and superprecipitation of myosin B

A combination is described of two laboratory instruments which simultaneously measures the extent of increasing turbidity and an accompanying enzymic reaction. The instrument consists of a Beckman DU spectrophotometer (serving as a densitometer) and Radiometer Titrator (serving as a pH-stat) which are made to record the progress of the two phenomena on a Texas Instrument dual-channel recorder. The two concomitant phenomena, in this instance, are the superprecipitation of myosin B and the accompanying enzymic dephosphorylation of ATP. Experimental results are presented to demonstrate the efficiency of the instrumentation for quantitating these phenomena.

Adenosine triphosphatase activated by magnesium and superprecipitation of myosin B.

THE importance of the divalent cations calcium and magnesium in muscle contraction is widely acknowledged1,2. An ordinary preparation of myosin B has been found to contain the “extra proteins” which are responsible for the effects of calcium on the magnesium-activated super-precipitation and adenosine triphosphatase (ATPase)3,4. As to the effects of magnesium, however, it is not clear whether this ion acts directly on the protein or indirectly by forming a complex with ATP5. How the activity of ATPase is related to the superprecipitation is still not known. We have attempted to ascertain the relationship of the two functional properties of myosin B by observing the effects of magnesium ions on the behaviour of the protein which shows no sensitivity to calcium.

A study on the mechanism of superprecipitation of myosin B.

A study on the mechanism of superprecipitation of myosin B.

Superprecipitation of Myosin B Caused by ATP as a Nucleated Growth Process*

Superprecipitation of Myosin B Caused by ATP as a Nucleated Growth Process*

The removal of the bound ADP of F-actin

F-actin essentially free of bound nucleotide has been prepared by replacing bound ADP with AMP under sonic vibration and subsequently removing bound AMP by charcoal treatment. A comparison of nucleotide-deficient and normal F-actin revealed no differences between the two types in reversible depoly-merization, viscosity, combination with myosin at high ionic strength, stimulation of Mg2+ -ATPase activity of myosin at low ionic strength or superprecipitation with myosin. Nucleotide-deficient F-actin bound ATP stoichiometrically. ATP, on combining with this F-actin, was hydrolyzed into inorganic phosphate and F-actin-bound ADP. ATP was bound to nucleotide-deficient F-actin under various conditions, both in the presence and absence of myosin, and whether or not the actomyosin was superprecipitated, e.g. in the presence of Mg2+ , EDTA or Ca2+. Nucleotide-deficient F-actin did not appreciably bind deoxyATP or CTP either in the presence or absence of myosin, but stimulated the Mg2+ -deoxy-ATPase and Mg2+ -CTPase activity of myosin several-fold as normal F-actin does. Mg2+-deoxyATP and Mg2+ -CTP caused superprecipitation of myosin with nucleotide-deficient F-actin to the same extent that it did with normal F-actin. It is suggested that the bound ADP of F-actin is not involved in the physiologically important properties of F-actin, namely, the activation of the ATPase activity of myosin and superprecipitation with myosin in the presence of ATP.

PREPARATION OF VESICULAR RELAXING FACTOR FROM BOVINE HEART TISSUE.

A particulated fraction, consisting mainly of protein and phospholipid, was prepared from bovine heart muscle by differential centrifugation. This microsomal suspension was found to inhibit superprecipitation of myosin B and to have a strong calcium-binding capacity. Although the preparation appeared to be very labile, a constant degree of activity was obtained by adding α-tocopherol before the homogenization of cardiac tissue. Evidence was gained for an interaction of calcium ions with synthetic lecithin in water suspensions. However, this interaction was competitively inhibited by the presence of magnesium and ATP, which are required cofactors for calcium binding by microsomes. It is proposed that in cardiac muscle, sarcoplasmic reticulum plays essentially the same role as in skeletal muscle.

The Role of Magnesium in the Superprecipitation of Myosin B

In 1951 and 1952, Spicer (2-5) made a series of further studies on this phenomenon and described the “clearing” response, a phenomenon associated with the superprecipitation. The use of this model has been revived in recent studies (6-11) on the physiological function of “relaxing factors,” including ethylenediaminetetraacetate. In these studies, however, atten- tion has been focused on the role of Ca2+ in the superprecipita- tion in the presence of physiological (relatively ‘higher) concen- trations of Mg*, and the primary requirement of Mg*+ for superprecipitation has been somewhat overlooked. In the earlier studies, Szent-Gyorgyi (1) reported that addi- tion of Mg* in very low concentrations accelerated the process of superprecipitation. IMarugama and Tonomura (12), using crayfish myosin B, reported the inhibition of superprecipitation by ethylenediaminetetraacetate. However, no positive evidence has been reported that Mg*, but not Ca’+, is the primary re- quirement for the superprecipitation. In this paper, such posi- tive evidence for the Mg* requirement for superprecipitation will be presented.