Purification and characterization of myosin from wheat mitochondria

Myosin was purified from wheat mitochondria using DE-52 anion exchange chromatography and Sephacryl S-300 gel filtration. The molecular weight of its heavy chain is about 210 ku, similar to that of muscle myosin Ⅱ(205 ku), and it could be recognized by the polyclonal antibodies against human skeletal muscle myosin Ⅱ. The ATPase activity of the mitochondrial myosin stimulated by F-actin from chicken muscle is 202.5 nmoles Pi/min·mg. The mitochondrial myosin could be activated by Ca²⁺ and was not inhibited by Ca²⁺ at high concentration. The results demonstrate that the myosin of wheat mitochondria shares some similarities with the skeletal muscle myosin Ⅱ.     

Modulation of ATP-sensitive K+ channels in skeletal muscle by intracellular protons

Since their discovery in cardiac muscle, ATP-sensitive K+(KATP) channels have been identified in pancreatic beta-cells, skeletal muscle, smooth muscle and central neurons. The activity of KATP channels is inhibited by the presence of cytosolic ATP. Their wide distribution indicates that they could have important physiological roles that may vary between tissues. In muscle cells the role of K+ channels is to control membrane excitability and the duration of the action potential. In anoxic cardiac ventricular muscle KATP channels are believed to be responsible for shortening the action potential, and it has been proposed that a fall in ATP concentration during metabolic exhaustion increases the activity of KATP channels in skeletal muscle, which may reduce excitability. But the intracellular concentration of ATP in muscle is buffered by creatine phosphate to 5-10 mM, and changes little, even during sustained activity. This concentration is much higher than the intracellular ATP concentration required to half block the KATP-channel current in either cardiac muscle (0.1 mM) or skeletal muscle (0.14 mM), indicating that the open-state probability of KATP channels is normally very low in intact muscle. So it is likely that some additional means of regulating the activity of KATP channels exists, such as the binding of nucleotides other than ATP. Here I present evidence that a decrease in intracellular pH (pHi) markedly reduces the inhibitory effect of ATP on these channels in excised patches from frog skeletal muscle. Because sustained muscular activity can decrease pHi by almost 1 unit in the range at which KATP channels are most sensitive to pHi, it is likely that the activity of these channels in skeletal muscle is regulated by intracellular protons under physiological conditions.     

Calcium regulation of molluscan myosin ATPase in the absence of actin

In the myosin-linked regulatory mechanism typified by the molluscan scallop adductor muscle, contraction is controlled by Ca2+ binding to sites on the thick filament protein, myosin. The regulatory light chains of myosin heads are involved directly in this mechanism and early studies suggested that, in the absence of Ca2+, these subunits prevent the interaction of a myosin-adenosine nucleotide complex with the actin-containing thin filament. Subsequently, Ashiba et al. reported that the steady-state ATPase of molluscan myosin exhibits a limited degree of Ca2+ activation in the absence of actin. Recently, however, we have shown that steady-state ATPase activity in relaxing conditions is dominated by the unregulated molecules in the myosin preparation. Single-turnover kinetic methods are required to monitor the highly suppressed ATPase activity of the regulated population. Using the latter approach, we report here that scallop myosin ATPase is reduced about 100-fold on removal of Ca2+. The regulatory light chains maintain the relaxed state via conformational changes which suppress the product release steps, irrespective of the presence of actin.     

Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle

Muscle contraction results from a sliding movement of actin filaments induced by myosin crossbridges on hydrolysis of ATP, and many non-muscle cells are thought to move using a similar mechanism. The molecular mechanism of muscle contraction, however, is not completely understood. One of the major problems is the mechanochemical coupling at high velocity under near-zero load. Here, we report measurements of the sliding distance of an actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle in an unloaded condition. We used single sarcomeres from which the Z-lines, structures which anchor the thin filaments in the sarcomere, had been completely removed by calcium-activated neutral protease (CANP) and trypsin, and measured both the sliding velocity of single actin filaments along myosin filaments and the ATPase activity during sliding. Our results show that the average sliding distance of the actin filament is less than or equal to 600 A during one ATP cycle, much longer than the length of power stroke of myosin crossbridges deduced from mechanical studies of muscle, which is of the order of 80 A (for example, ref. 15).     

去神经前后肌组织ATP酶活性改变

对中华大蟾蜍快肌和慢肌组织的ＡＴＰ酶活性进行比较研究发现,快肌缝匠肌肌组织比慢肌腹直肌肌组织的ＡＴＰ酶活性要高,去神经后缝匠肌ＡＴＰ酶的活性有显著下降,这些差异和变化具有一定的生理意义．     

Rapid regeneration of the actin-myosin power stroke in contracting muscle.

At the molecular level, muscle contraction is the result of cyclic interaction between myosin crossbridges, which extend from the thick filament, and the thin filament, which consists mainly of actin. The energy for work done by a single crossbridge during a cycle of attachment, generation of force, shortening and detachment is believed to be coupled to the hydrolysis of one molecule of ATP. The distance the actin filament slides relative to the myosin filament in one crossbridge cycle has been estimated as 12 nm by step-length perturbation studies on single fibres from frog muscle. The 'mechanical' power stroke of the attached crossbridge can therefore be defined as 12-nm shortening with a force profile like that shown by the quick recovery of force following a length perturbation. According to this definition, power strokes cannot be repeated faster than the overall ATPase rate. Here, however, we show that the power stroke can be regenerated much faster than expected from the ATPase rate. This contradiction can be resolved if, in the shortening muscle, the free energy of ATP hydrolysis is used in several actin-myosin interactions consisting of elementary power strokes each of 5-10 nm.     

Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum of skeletal muscle is a specialized form of endoplasmic reticulum that controls myoplasmic calcium concentration and, therefore, the contraction-relaxation cycle. Ultrastructural studies have shown that the sarcoplasmic reticulum is a continuous but heterogeneous membranous network composed of longitudinal tubules that surround myofibrils and terminal cisternae. These cisternae are junctionally associated, via bridging structures called 'feet', with sarcolemmal invaginations (the transverse tubules) to form the triadic junction. Following transverse tubule depolarization, a signal, transmitted along the triadic junction, triggers Ca2+ release from terminal cisternae, but the mechanism of this coupling is still unknown. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has recently been shown to mobilize Ca2+ from intracellular stores, referable to endoplasmic reticulum, in a variety of cell types (see ref. 8 for review), including smooth muscle cells of the porcine coronary artery and canine cardiac muscle cells. Here we show that Ins(1,4,5)P3 releases Ca2+ from isolated, purified sarcoplasmic reticulum fractions of rabbit fast-twitch skeletal muscle, the effect being more pronounced on a fraction of terminal cisternae that contains morphologically intact feet structures; and elicits isometric force development in chemically skinned muscle fibres.     

Stimulation of Acanthamoeba actomyosin ATPase activity by myosin-II polymerization

Phosphorylation of the regulatory light chains of vertebrate smooth muscle or cytoplasmic myosins alters the structure of myosin monomers, favours myosin filament formation and stimulates the actin-activated Mg2+-ATPase of myosin. Similarly, in Dictyostelium and Acanthamoeba phosphorylation of the myosin heavy chains exhibits both polymerization and actin-activated Mg2+ATPase. Unfortunately, the relationships between phosphorylation, myosin assembly and activation of ATP hydrolysis are not fully understood in any of these systems, as there has been no way of varying the extent of polymerization of intact myosin without changing solution conditions or the level of myosin phosphorylation, parameters that may have independent effects on ATPase activity. Taking an entirely new approach, we have used monoclonal antibodies against the tail of Acanthamoeba myosin-II that cause filament disassembly to show that myosin polymerization itself stimulates actomyosin ATPase activity. With a fixed level of myosin-II phosphorylation and constant solution conditions, depolymerization of myosin-II filaments by antibodies causes a concomitant loss of actin-activated ATPase activity.     

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil alpha-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP). Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.     

藏系绵羊心肌、骨骼肌MDH和LDH活力及LDH同工酶谱

为了探索草地藏系绵羊高原适应性的分子机制,对草地藏系绵羊骨骼肌、心肌中乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)活力进行测定,并采用聚丙烯酰胺凝胶电泳分析了其心肌、骨骼肌、肝脏中LDH同工酶谱.结果显示:藏系绵羊心肌MDH/LDH活力比值极显著高于骨骼肌(P<0.01,LDH同工酶的表达在心肌、骨骼肌、肝脏中表现出组织特异性,LDH在肌肉中总活力最高,在电泳凝胶上表现为5条酶带,其中LDH5相对活力最强.     

Synthesis of fast myosin induced by fast ectopic innervation of rat soleus muscle is restricted to the ectopic endplate region

Skeletal muscle fibres, long multinucleated cells, arise by fusion of mononucleated myoblasts to form a myotube that matures into the adult fibre. The two major types of mature fibre, fast and slow fibres, differ physiologically in their rate of isotonic shortening. At the molecular level these type-specific physiological properties are ascribed to different isoforms of myosin, a major protein involved in shortening. Differentiation of fast and slow fibres seems to be under the control of motoneurones, and mature fibres are innervated by only one motoneurone. When rat soleus muscle (SOL, a slow muscle) is dually innervated with a fast nerve, it acquires some properties of a fast muscle, that is, low sensitivity to caffeine and high glycogen content. We report here that in dually innervated soleus muscle the foreign fast nerve induces synthesis of fast isoforms of myosin, but only in the segment of the muscle fibre that is close to the foreign endplate. The localized influence of the nerve endplates suggest that factors controlling the phenotypic expression of the muscle fibre have a short range of activity.     

The motor protein myosin-I produces its working stroke in two steps

Many types of cellular motility, including muscle contraction, are driven by the cyclical interaction of the motor protein myosin with actin filaments, coupled to the breakdown of ATP. It is thought that myosin binds to actin and then produces force and movement as it 'tilts' or 'rocks' into one or more subsequent, stable conformations. Here we use an optical-tweezers transducer to measure the mechanical transitions made by a single myosin head while it is attached to actin. We find that two members of the myosin-I family, rat liver myosin-I of relative molecular mass 130,000 (M(r) 130K) and chick intestinal brush-border myosin-I, produce movement in two distinct steps. The initial movement (of roughly 6 nanometres) is produced within 10 milliseconds of actomyosin binding, and the second step (of roughly 5.5 nanometres) occurs after a variable time delay. The duration of the period following the second step is also variable and depends on the concentration of ATP. At the highest time resolution possible (about 1 millisecond), we cannot detect this second step when studying the single-headed subfragment-1 of fast skeletal muscle myosin II. The slower kinetics of myosin-I have allowed us to observe the separate mechanical states that contribute to its working stroke.     

Movement of myosin-coated beads on oriented filaments reconstituted from purified actin

Although the biochemical properties of the actin/myosin interaction have been studied extensively using actin activation of myosin ATPase as an assay, until recently no well-defined assay has been available to measure the mechanical properties of ATP-dependent movement of myosin along actin filaments. The first direct measurements of the rate of myosin movement in vitro used a naturally occurring, biochemically ill-defined array of actin filaments from the alga Nitella. We report here the construction of an oriented array of filaments reconstituted from purified muscle actin and the use of this array in a biochemically defined quantitative assay for the directed movement of myosin-coated polystyrene beads. We demonstrate for the first time that actin alone, linked to a substratum by a protein anchor, is sufficient to support movement of myosin at rates consistent with the speeds of muscle contraction and other forms of cell motility.     

Orientation of spin labels attached to cross-bridges in contracting muscle fibres

Electron micrographs showing different cross-bridge orientations in different states of muscle fibres, and X-ray diffraction patterns indicating axial cross-bridge disorder in contracting muscle first suggested that force generation in the contracting muscle involved a change in orientation of the myosin heads that form cross-bridges between thick and thin filaments. This has been supported by subsequent work; the myosin molecule has the required flexibility for changes in orientation. The orientation of muscle tryptophans and of probes attached to the myosin heads of permeable muscle fibres depends on the state of the muscle. Recently, fluorescence polarization fluctuations and time-resolved X-ray diffraction patterns have suggested that cross-bridges of a contracting muscle can rotate. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor the orientation of spin labels attached specifically to a reactive sulphydryl on the myosin heads in glycerinated rabbit psoas skeletal muscle. Previously, it has been shown that the paramagnetic probes are highly ordered in rigor muscle, with a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, approximately 80% of the probes display a random angular distribution as in relaxed muscle while the remaining 20% are highly oriented at the same angle as found in rigor muscle. These findings indicate that a domain of the myosin head does not change orientation during the power stroke of the contractile interaction.     

Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy

FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation-contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation-contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.     

沙苑子对运动训练大鼠骨骼肌自由基代谢的影响

通过建立力竭跑台训练模型,测试大鼠骨骼肌组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量和ATP酶活性,探讨沙苑子对力竭跑台训练大鼠骨骼肌组织自由基代谢、ATP酶活性和运动能力的影响.结果显示,与安静组比较,运动力竭组SOD、CAT和GSH-Px活性降低,MDA含量升高;沙苑子组大鼠骨骼肌组织活性显著高于对照组,MDA含量明显低于相应对照组(P<0.01);运动力竭组Na 、K -ATPase和Ca2 、Mg2 -ATPase活性与安静组比较降低,沙苑子组大鼠骨骼肌组织ATPase活性显著高于对照组.沙苑子可显著提高运动大鼠抗氧化的功能,提高运动大鼠骨骼肌组织ATPase活性,具有较强抗氧化能力,使大鼠运动能力有了明显提高.     

The myosin motor in muscle generates a smaller and slower working stroke at higher load

Muscle contraction is driven by the motor protein myosin II, which binds transiently to an actin filament, generates a unitary filament displacement or 'working stroke', then detaches and repeats the cycle. The stroke size has been measured previously using isolated myosin II molecules at low load, with rather variable results, but not at the higher loads that the motor works against during muscle contraction. Here we used a novel X-ray-interference technique to measure the working stroke of myosin II at constant load in an intact muscle cell, preserving the native structure and function of the motor. We show that the stroke is smaller and slower at higher load. The stroke size at low load is likely to be set by a structural limit; at higher loads, the motor detaches from actin before reaching this limit. The load dependence of the myosin II stroke is the primary molecular determinant of the mechanical performance and efficiency of skeletal muscle.     

Isolation and characterization of the G-actin-myosin head complex

The two main proteins involved in muscular contraction and cell motility, myosin and actin, possess the intrinsic property of being able to form filamentous structures. This property poses a serious impediment to the study of their structures and interactions, and a considerable effort has thus been made to isolate their functional domains. The globular part of myosin, subfragment-1 (S1), which possesses ATPase and actin-binding sites as well as supporting the movement of actin filaments during in vitro assays, has been isolated. But because S1 is efficient in inducing actin polymerization, as is myosin, it has not been possible to prepare and characterize a complex of S1 with monomeric actin (G-actin). We have now used chromatographically purified proteins to show that only the S1 isoenzyme carrying the A1 light-chain subunit promotes actin polymerization. The other isoenzyme, S1 (A2), carrying the A2 light-chain subunit, binds to actin, forming a tight complex of G-actin and S1 in a 1:1 ratio. This new functional difference between myosin isoforms directly implicates the A1 light-chain in myosin-induced actin polymerization. Additionally, this finding should lead to the purification of the stable G-actin-S1 complex needed to resolve the structure and to understand the molecular dynamics of the actin-myosin system.     

热敏灸对运动大鼠运动能力和心肌、骨骼肌细胞线粒体过氧化损伤的影响

目的研究热敏灸对运动大鼠运动能力和心肌、骨骼肌细胞线粒体过氧化损伤的影响。方法将40只大鼠随机分为正常对照组、单纯运动组、运动+普通悬灸组和运动+热敏灸组,采用14 d递增大强度跑台运动训练和生物化学的方法,观察热敏灸对运动大鼠运动能力和心肌、骨骼肌细胞线粒体过氧化损伤的影响。结果运动+热敏灸组大鼠疲劳相关症状显著改善,跑台力竭时间显著高于运动+普通悬灸组和单纯运动组。运动+热敏灸组大鼠心肌、骨骼肌细胞线粒体SOD酶活性高于单纯运动组、运动+普通悬灸组,心肌细胞线粒体SOD酶活性低于正常对照组,但骨骼肌细胞线粒体SOD酶活性与正常对照组比较,差异无显著性;MDA含量低于单纯运动组和运动+普通悬灸组,但高于正常对照组。结论热敏灸足三里穴具有明显的抗运动性疲劳作用,机理与热敏灸降低过度运动大鼠心肌和骨骼肌细胞线粒体过氧化损伤有关。