Muscle contraction and free energy transduction in biological systems

Muscle contraction occurs when the actin and myosin filaments in muscle are driven past each other by a cyclic interaction of adenosine triphosphate (ATP) and actin with cross-bridges that extend from myosin. Current biochemical studies suggest that, during each adenosine triphosphatase cycle, the myosin cross-bridge alternates between two main conformations, which differ markedly in their strength of binding to actin and in their overall structure. Binding of ATP to the cross-bridge induces the weak-binding conformation, whereas inorganic phosphate release returns the cross-bridge to the strong-binding conformation. This cross-bridge cycle is similar to the kinetic cycle that drives active transport and illustrates the general principles of free energy transduction by adenosine triphosphatase systems.     

Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum

The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.     

Tritiated digoxin binding to (Na+ + K+)-activated adenosine triphosphatase: possible allosteric site

Tritiated H(3)-digoxin specifically binds to a cardiac (Na(+) + K(+))-activated adenosine triphosphatase. In the presence of adenosine triphosphate and other nucleoside di- and triphosphates, binding is stimulated by sodium ion, the apparent rate constant being similar to that reported for phosphorus-32 incorporation from adenosine triphosphate and for the adenosine triphosphatase activity. In the presence of magnesium, manganese, inorganic phosphate, or other ions, sodium ion inhibits binding. The data support an allosteric type of sodium-potassium ion pump.     

Actomyosin-like protein isolated from mammalian brain

A protein with characteristics similar to actomyosin has been isolated from whole brain of rat and cat. It is soluble in 0.6 molar potassium chloride and insoluble in 0.1 molar potassium chloride. It superprecipitates with magnesium ions and adenosine triphosphate. It has adenosine triphosphatase activity stimulated by either magnesium or calcium ions. Both superprecipitation and adenosine triphosphatase activity are inhibited by p-chloromercuribenzoate and Mersalyl but not by ouabain.     

Catecholamine and dibutyryl cyclic AMP effects on myosin adenosine triphosphatase in cultured rat heart cells

Catecholamines and dibutyryl adenosine 3', 5'-monophosphate (dibutyryl cyclic AMP) increase the activity of myosin adenosine triphosphatase in cultured rat heart cells. Dichloroisoproterenol, an inhibitor of the beta receptor of the catecholamines, inhibits the action of the catecholamines but not of cyclic AMP.     

Imaging elemental distribution and ion transport in cultured cells with ion microscopy

Both elemental distribution and ion transport in cultured cells have been imaged by ion microscopy. Morphological and chemical information was obtained with a spatial resolution of approximately 0.5 micron for sodium, potassium, calcium, and magnesium in freeze-fixed, cryofractured, and freeze-dried normal rat kidney cells and Chinese hamster ovary cells. Ion transport was successfully demonstrated by imaging Na+-K+ fluxes after the inhibition of Na+- and K+ -dependent adenosine triphosphatase with ouabain. This method allows measurements of elemental (isotopic) distribution to be related to cell morphology, thereby providing the means for studying ion distribution and ion transport under different physiological, pathological, and toxicological conditions in cell culture systems.     

Sodium and potassium effects on skeletal muscle microsomal adenosine triphosphatase and calcium uptake

The relationship between the (Na(+) and K(+))-activated adenosine triphosphatase enzyme system implicated in sodium-transport by cell membranes and the calcium-activated adenosine triphosphatase, which is generally associated with calcium uptake, was examined in microsomes from skeletal muscle. Whereas sodium and potassium did not modify the relatively low adenosine triphosphatase activity seen in the absence of calcium, a pattern similar to that of the sodium-transport enzyme system was seen afer the addition of CaCl(2). The calcium-activated adenosine triphosphatase was stimulated equally by sodium or potassium alone, but both the rate and extent of calcium uptake were enhanced more by potassium than by sodium at concentrations below 0.12 mole per liter. In the absence of either of these ions addition of calcium failed to activate adenosine triphosphatase although significant amounts of calcium were taken up by the microsomes.     

Mercurial-induced transformation of myosin prevented by adenosine triphosphate and pyrophosphate

Adenosine triphosphate and pyrophosphate prevent the loss of Ca(//)-activated adenosine triphosphatase activity caused by high concentrations of mercurial sulfhydryl reagent. They concomitantly prevent the transformation of myosin into faster-sedimenting products. This is adduced as support for the hypothesis that the strategic sulfhydryl group is not binding adenosine triphosphate at the active site, but is initiating a conformational change upon its reaction with the mercurial reagent.     

The crystal structure of uncomplexed actin in the ADP state

The dynamics and polarity of actin filaments are controlled by a conformational change coupled to the hydrolysis of adenosine 5'-triphosphate (ATP) by a mechanism that remains to be elucidated. Actin modified to block polymerization was crystallized in the adenosine 5'-diphosphate (ADP) state, and the structure was solved to 1.54 angstrom resolution. Compared with previous ATP-actin structures from complexes with deoxyribonuclease I, profilin, and gelsolin, monomeric ADP-actin is characterized by a marked conformational change in subdomain 2. The successful crystallization of monomeric actin opens the way to future structure determinations of actin complexes with actin-binding proteins such as myosin.     

日粮中锰对公鸡胆固醇含量及ATP酶活性的影响

摘要经添加锰0(Ⅰ组)、50(Ⅱ组)、100(Ⅲ组)和300(Ⅳ组)PPM日粮处理后的种用公鸡,检测其胆固醇含量和三磷酸腺苷酶(ATP酶)活性。结果表明,缺锰或高锰日粮使肝脏、睾丸匀浆中胆固醇含量下降,血清、肝脏和睾丸匀浆中ATP酶活性降低。结果提示,日粮中锰含量的高低对胆固醇的合成有影响作用,而这种影响作用同锰引起的ATP酶活性的改变以及ATP供能速率的变化有关。     

Control of myocardial contraction: the sensitivity of cardiac actomyosin to calcium ion

The control by calcium ion of the adenosine triphosphatase activity of cardiac actomyosin is similar to that of white skeletal actomyosin. This finding indicates that the slower contraction and relaxation of heart muscle do not reflect different levels to which free calcium ion concentration around the myofibrils must be adjusted during contraction and relaxation and suggests a mechanism whereby myocardial contractility may be regulated.     

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.     

Phosphoryl transfer and calcium ion occlusion in the calcium pump

A tight coupling between adenosine triphosphate (ATP) hydrolysis and vectorial ion transport has to be maintained by ATP-consuming ion pumps. We report two crystal structures of Ca2+-bound sarco(endo)plasmic reticulum Ca2+-adenosine triphosphatase (SERCA) at 2.6 and 2.9 angstrom resolution in complex with (i) a nonhydrolyzable ATP analog [adenosine (beta-gamma methylene)-triphosphate] and (ii) adenosine diphosphate plus aluminum fluoride. SERCA reacts with ATP by an associative mechanism mediated by two Mg2+ ions to form an aspartyl-phosphorylated intermediate state (Ca2-E1 approximately P). The conformational changes that accompany the reaction with ATP pull the transmembrane helices 1 and 2 and close a cytosolic entrance for Ca2+, thereby preventing backflow before Ca2+ is released on the other side of the membrane.     

Erythrocytes: 5'' -adenylic acid deaminase requirement for ammonia or monovalent metal ion

5'-Adenylic acid deaminase free from sodium and potassium ion is prepared from erythrocytes by a convenient method. Like adenosine triphosphatase and adenylic kinase from erythrocytes, the deaminase is activated by some monovalent cations, but unlike these enzymes it requires the presence of a monovalent cation. In all three instances the pattern of activation by ions is similar and suggests a common mechanism.     

Renal concentrating mechanism: possible role for sodium-potassium activated adenosine triphosphatase

Sodium-potassium activated adenosine triphosphatase activity was found to be almost twice as high in renal medulla as in cortex. Infusion of digoxin, a specific inhibitor of the enzyme, into one renal artery of the dog resulted in unilateral natriuresis, impaired concentrating capacity, and reduction of the enzyme activity in both cortex and medulla. It is suggested that the sodium-potassium adenosine triphosphatase plays an important role in urine concentration mechanisms.     

Nonequilibrium mechanics of active cytoskeletal networks

Cells both actively generate and sensitively react to forces through their mechanical framework, the cytoskeleton, which is a nonequilibrium composite material including polymers and motor proteins. We measured the dynamics and mechanical properties of a simple three-component model system consisting of myosin II, actin filaments, and cross-linkers. In this system, stresses arising from motor activity controlled the cytoskeletal network mechanics, increasing stiffness by a factor of nearly 100 and qualitatively changing the viscoelastic response of the network in an adenosine triphosphate-dependent manner. We present a quantitative theoretical model connecting the large-scale properties of this active gel to molecular force generation.     

Adenosine triphosphate usage by flagella

Comparison of beat frequencies with rates of dephosphorylation of adenosine triphosphate by glycerinated sea urchin spermatozoa as functions of adenosine triphosphate concentration suggests that each molecule of the flagellar adenosine triphosphatase, dynein, dephosphorylates one adenosine triphosphate molecule during each beat cycle.     

宰后肌肉中肌球蛋白磷酸化调控肌动球蛋白解离作用机制

【目的】研究宰后肌肉中肌球蛋白磷酸化与肌动球蛋白解离之间的关系,分析其磷酸化水平的变化对肌动球蛋白解离的影响,探究肌球蛋白磷酸化对宰后肌肉肌节长度与嫩度的作用。【方法】取宰后30 min内的羊背最长肌,在4℃条件下分别成熟6、24、48和72 h,通过SDS-PAGE电泳、Pro-Q染色和蛋白质免疫印迹测定肌球蛋白的磷酸化水平和肌动球蛋白解离程度随宰后时间的变化;测定肌动球蛋白ATP酶的活性,分析宰后不同时间点肌球蛋白与肌动蛋白结合作用力的强弱;采用透射电镜分析宰后肌节长度随时间的变化。【结果】研究发现宰后肌肉中肌球蛋白轻链2的磷酸化水平在0.5—48 h快速降低(P0.05),并在48 h达到最低点,在48—72 h有所升高(P0.05),但其最终磷酸化水平明显低于初始值。肌动球蛋白的解离程度在宰后初期(0.5—6 h)显著降低(P0.05),在6—48 h显著升高(P0.05),并于48—72 h维持稳定,其最终解离程度显著高于宰后0.5 h的初始值。肌动球蛋白ATPase活性在宰后初期(0.5—6 h)略有升高,6—24 h快速上升(P0.05),并在24 h达到最高点,24—72 h逐渐降低;而肌节长度的变化则与之相反,呈先下降后上升的趋势,并在24 h达到肌节最短点。【结论】羊宰后肌肉中的肌球蛋白轻链2磷酸化水平的变化对肌球蛋白与肌动蛋白的相互作用有较大的影响,且肌节收缩(肌球蛋白与肌动蛋白的相互作用力)与肌动球蛋白的解离(肌球蛋白与肌动蛋白的相互作用量)并不是一个同步的进程。肌球蛋白轻链2的磷酸化修饰负向调控肌动球蛋白解离和肌动球蛋白ATPase活性,导致肌节的收缩与舒张,进而调控肉品最终的嫩度。     

Cochlear function and sodium and potassium activated adenosine triphosphatase

The maintenance of the cation gradients between endolymph and perilymph in the cochlea requires the operation of a cation pump. An adenosine triphosphatase system activated by sodium and potassium is present in high activity in the cochlear membranes (tegmentum vasculosum and stria vascularis). The cochlear microphonic potential is inhibited by perilymphatic perfusion of ouabain and erythrophleine. Since the microphonic potential depends on the high concentration of potassium ions in the endolymph, our findings strongly suggest the operation of an adenosine triphosphatase cation pump system activated by sodium and potassium, in the generation of cochlear cation gradients.     

蛋白质磷酸化对肌动球蛋白解离及其乙酰化水平的影响

[目的]研究肌球蛋白重链和肌动蛋白磷酸化对其乙酰化水平、肌动球蛋白解离及ATP酶活性的影响,为通过调控磷酸化水平改善肉品嫩度提供理论依据.[方法]以羊背最长肌为材料制备肌肉匀浆液,采用碱性磷酸酶抑制剂(抑制去磷酸化)和蛋白激酶抑制剂(抑制磷酸化)调控其磷酸化水平,在4℃分别孵育0、0.5、4、12、24、48和72 h...