肌球蛋白工作循环的一个新模型

分析总结关于分子马达肌球蛋白的最新研究结果,给出一个新的肌球蛋白工作循环的机械化学偶联模型.从新模型出发,用一组化学动力学方程描述肌肉中大量肌球蛋白的集体工作行为.利用动力学方程的非平衡定态解,并结合Pate和Cooke的实验结果得到了力作为变量的肌肉态方程.理论结果同热力学原理一致,与传统的肌肉收缩理论有一定区别.根据肌肉的特殊结构,对肌肉态方程做了进一步讨论.     

肌肉收缩Hill特性式的理论研究

从肌球蛋白工作循环的机械化学偶联模型出发,利用化学动力学方法和生物化学热力学原理,结合肌球蛋白单分子实验结果,从能量转化的观点给出了肌肉收缩的Hill特性式,加深了对Hill特性式及肌肉收缩过程中能量转化的理解,在整合肌球蛋白单分子性质与肌肉收缩宏观性质的信息方面做了尝试。     

锯缘青蟹主要过敏原的纯化与鉴定

以锯缘青蟹为研究对象,从免疫鉴定、分离纯化、抗体制备和免疫学分析等方面对其主要过敏原进行研究。首先利用过敏者血清的免疫印迹法,确定锯缘青蟹的主要过敏原为分子量约38kD的蛋白。然后通过制备丙酮粉、等电点沉淀、硫酸铵沉淀及加热处理对分子量为38kD的主要过敏蛋白进行了高度纯化。该蛋白的pI约为4.5,与虾的原肌球蛋白Pena1性质相近,证实了锯缘青蟹的主要过敏原为原肌球蛋白。通过免疫新西兰大白兔,制备了原肌球蛋白的抗血清,采用Protein A Sepharose亲和层析柱对动物抗体进行了纯化。该抗血清效价高,经4×105倍稀释后仍能与抗原进行反应。该抗体与甲壳类动物及软体动物的原肌球蛋白具有较强的免疫交叉反应,可用于食品过敏原检测。       

丝瓜肌球蛋白的电子显微学研究

从丝瓜 (Luffacylindrica (L .)Roem .)卷须中纯化得到分子量为 174kD的肌球蛋白 ,并对其进行了酶学与电子显微学的研究。这种肌球蛋白具有肌动蛋白激活的MgATPase活性 ,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别。电子显微学研究表明 :它有两个头部 (大小和形状与动物肌肉的肌球蛋白相似 )和一条相对较短的尾部。还对丝瓜卷须的肌动蛋白进行了观测 ,偶尔发现一些尾部有球状结构的肌球蛋白。该肌球蛋白的免疫特性和超微结构证明了它由 2条重链组成 ,并与传统的肌球蛋白相似。然而 ,这种 174kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究。     

草鱼和鲢鱼肌球蛋白Ca2+-ATPase热力学活化参数的季节变化

肌球蛋白构成了鱼肌肉肌原纤维蛋白的50%以上,它的性质主要决定了肌原纤维蛋白的性质,进而影响了鱼肌肉蛋白质的加工适性.本文对来自春季(4月份)、夏季(8月份)、秋季(11月份)和冬季(1月份)的草鱼(Ctenopharyngodon idellus Valenciennes)和鲢鱼(Hypophthalmichthys molitrix Valenciennes)骨骼肌肌球蛋白进行提取和纯化,通过测定不同季节的两种淡水鱼肌球蛋白Ca2 -ATPase的热力学活化参数,从化学反应的热力学理论上证明了淡水鱼肌球蛋白的热稳定性与季节温度变化之间的关系.研究结果表明,草鱼和鲢鱼肌球蛋白Ca2 -ATPase的热力学活化参数活化能Ea、活化焓ΔH 和活化熵ΔS 伴随栖息环境温度的上升而增大,而活化自由能ΔG 的变化幅度很小,证明了肌球蛋白的热稳定性夏季鱼明显优于冬季鱼,而春季鱼和秋季鱼分别与夏季鱼和冬季鱼的相似.另一方面,ΔH 与ΔS 之间显示了良好的线性相关,证明了淡水鱼能够通过其肌肉蛋白质反应的活化焓与活化熵之间的互补效应,以应对自然界季节环境温度的变化.     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白,并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ,１７．５ｋＤ）组成的大分子,其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性,Ｃａ~（２＋）离子也能提高其活性,Ｍｇ~（２＋）离子无明显影响。钒酸盐,碘乙酸,对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

丝瓜肌球蛋白的电子显微学研究

从丝瓜(Luffa cylindrica (L.) Roem.)卷须中纯化得到分子量为174kD的肌球蛋白,并对其进行了酶学与电子显微学的研究.这种肌球蛋白具有肌动蛋白激活的MgATPase活性,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别.电子显微学研究表明:它有两个头部(大小和形状与动物肌肉的肌球蛋白相似)和一条相对较短的尾部.还对丝瓜卷须的肌动蛋白进行了观测,偶尔发现一些尾部有球状结构的肌球蛋白.该肌球蛋白的免疫特性和超微结构证明了它由2条重链组成,并与传统的肌球蛋白相似.然而,这种174 kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究.     

肌球蛋白动力冲程分布研究

通过建立肌球蛋白工作循环的四态模型,利用化学动力学方法,讨论了动力冲程过程的几率和无机磷酸盐的释放率是影响动力冲程分布的两个重要因素,得到动力冲程的大小近似呈均值为（8-10）nm的高斯分布。     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白，并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ，１７．５ｋＤ）组成的大分子，其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性，Ｃａ＾２＋离子也能提高其活性，Ｍｇ＾２＋离子无明显影响，钒酸盐，碘乙酸，对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

鸭肫平滑肌肌球蛋白的提纯及其ATP酶性质的研究

从鸭肫肌肉中分离纯化了平滑肌肌球蛋白,并对平滑肌肌球蛋白分子的亚单位组成和平滑肌肌球蛋白的ＡＴＰ酶性质进行了分析研究。鸭肫平滑肌肌蛋白的Ｃａ＾２＋－ＡＴＰ酶活力与溶液的离子强度有关。在低于０．２０ｍｏｌ／Ｌ的ＫＣＬ浓度时,酶活力很低;大于０．３０ｍｏｌ／Ｌ的ＫＣＬ浓度时,酶活力较高,Ｃａ＾２＋－ＡＴＰ酶有两个最适ｐＨ值。Ｋ＾＋／ＥＤＴＡ－ＡＴＰ酶活力随ＫＣＬ浓度升高而增加。肌动蛋白激活的磷酸化肌球     

平滑肌肌球蛋白轻链激酶及ATP结合位点突变体对ATP酶活性的调节作用

目的：探寻MLCK的非激酶活性区域对MLCK活性的影响,进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制。方法：利用编码MLCK全长的pColdI表达载体对其ATP结合位点进行定点突变,获得无激酶活性的MLCK突变体;应用Glycerol—PAGE鉴定肌球蛋白磷酸化水平;应用孔雀绿方法检测重组MLCK对肌球蛋白ATP酶活性的影响。结果：MLCK／△ATP（突变型）失去磷酸化肌球蛋白轻链的激酶活性;重组MLCK（野生型）和MLCK／AATP（突变型）均可以在非钙条件下激活非磷酸化肌球蛋白Mg2＋-ATP酶活性,抑制磷酸化肌球蛋白的Mg2＋．ATP酶活性,而且激活与抑制作用均随着MLCK浓度的增加而增大,但二者对肌球蛋白的ATP酶活性的作用没有显著差异（P〉0．05）。结论：平滑肌肌球蛋白轻链激酶及ATP结合位点突变体具有激活非磷酸化肌球蛋白ATP酶活性的作用。     

An electromechanical model of myosin molecular motors

There is a long-running debate on the working mechanism of myosin molecular motors, which, by interacting with actin filaments, convert the chemical energy of ATP into a variety of mechanical work. After the development of technologies for observing and manipulating individual working molecules, experimental results negating the widely accepted 'lever-arm hypothesis' have been reported. In this paper, based on the experimental results so far accumulated, an alternative hypothesis is proposed, in which motor molecules are modelled as electromechanical components that interact with each other through electrostatic force. Electrostatic attractive force between myosin and actin is assumed to cause a conformational change in the myosin head during the attachment process. An elastic energy resulting from the conformational change then produces the power stroke. The energy released at the ATP hydrolysis is mainly used to detach the myosin head from actin filaments. The mechanism presented in this paper is compatible with the experimental results contradictory to the previous theories. It also explains the behavior of myosins V and VI, which are engaged in cellular transport and move processively along actin filaments.     

Muscle myosin filaments: cores, crowns and couplings

Myosin filaments in muscle, carrying the ATPase myosin heads that interact with actin filaments to produce force and movement, come in multiple varieties depending on species and functional need, but most are based on a common structural theme. The now successful journeys to solve the ultrastructures of many of these myosin filaments, at least at modest resolution, have not been without their false starts and erroneous sidetracks, but the picture now emerging is of both diversity in the rotational symmetries of different filaments and a degree of commonality in the way the myosin heads are organised in resting muscle. Some of the remaining differences may be associated with how the muscle is regulated. Several proteins in cardiac muscle myosin filaments can carry mutations associated with heart disease, so the elucidation of myosin filament structure to understand the effects of these mutations has a clear and topical clinical relevance.     

Synthesis and assembly of native myosin on muscle polyribosomes

We have used the overload-induced growth of avian muscles to study the assembly of the newly synthesized myosins which were separated by non-denaturing pyrophosphate-polyacrylamide gel electrophoresis. Using this model, we have observed the appearance of fast-like isomyosins in polyribosomes prepared from slow anterior latissimus dorsi muscle after 72 h of overload. These new isoforms comigrating with native myosin from fast posterior latissimus dorsi muscle were not yet present in cellular extracts from the same muscle. The in vitro translation system utilizing muscle specific polyribosomes directs the synthesis of the corresponding myosin isoforms. Under denaturing conditions, myosin heavy chains and light chains dissociate to the expected subunit composition of each specific isoform. The synthesis and assembly of native myosin on polyribosomes indicate that myosin exists as a single mature protein prior to the incorporation in the thick filament.     

扇贝肌钙蛋白及其对肌球蛋白超沉淀的影响

分离了扇贝闭壳肌肌钙蛋白,其分子量为４６（ＩｎＩ）,４０（ＴｎＴ）,和２２（ＴｎＣ）ｋＤ．肌球蛋白Ｂ含有主要的收缩蛋白质与调节蛋白质,在有Ｃａ２＋和ＡＴＰ存在时,它会发生超沉淀作用．经低离子强度溶液反复沉淀处理,即失去Ｃａ２＋－敏感性,成为去敏肌球蛋白Ｂ．在Ｃａ２＋和ＡＴＰ作用下,它仍可发生超沉淀作用,但仅及最大活性的５０％．若加入肌钙蛋白,则反应活性可完全恢复．兔骨骼肌肌钙蛋白可替代扇贝闭壳肌肌钙蛋白．这表明扇贝闭壳肌兼有肌动蛋白相关调节和肌球蛋白相关调节．     

Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments

Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin''s access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion.     

Solubility-determining domain of smooth muscle myosin rod

Chymotryptic digestion of chicken gizzard light meromyosin (LMM) produced a 72 kDa core fragment, which was fully soluble at 150 mM KCl, pH 6.5–7.5. The fragment showed weak self-association at 50 mM KCl. The homology of the N-terminus amino acid sequence of this fragment with the sequence of the rabbit skeletal myosin rod suggested that the N-terminus of the core fragment originated 5 kDa from the hinge common to both smooth and skeletal myosin rod. Sedimentation experiments indicated that the domain specifying the insolubility of the intact LMM was 13 kDa long. Progressive proteolytic shortening of this region produced LMM fragments of progressively increasing solubility. Electron microscopy of segments formed from full-length LMM and from LMM core suggested that this 13 kDa domain specified the 43 nm parallel and antiparallel molecular overlaps characteristic of self-assembled intact myosin.     

Exploration of the conformational space of myosin recovery stroke via molecular dynamics

Muscle contractions are driven by cyclic conformational changes of myosin, whose molecular mechanisms of operation are being elucidated by recent advances in crystallographic studies and single molecule experiments. To complement such structural studies and consider the energetics of the conformational changes of myosin head, umbrella sampling molecular dynamics (MD) simulations were performed with the all-atom model of the scallop myosin sub-fragment 1 (S1) with a bound ATP in solution in explicit water using the crystallographic near-rigor and transition state conformations as two references. The constraints on RMSD reaction coordinates used for the umbrella sampling were found to steer the conformational changes efficiently, and relatively close correlations have been observed between the set of characteristic structural changes including the lever arm rotation and the closing of the nucleotide binding pocket. The lever arm angle and key residue interaction distances in the nucleotide binding pocket and the relay helix show gradual changes along the recovery stroke reaction coordinate, consistent with previous crystallographic and computational minimum energy studies. Thermal fluctuations, however, appear to make the switch-2 coordination of ATP more flexible than suggested by crystal structures. The local solvation environment of the fluorescence probe, Trp 507 (scallop numbering), also appears highly mobile in the presence of thermal fluctuations.     

Mouse Myosin-19 Is a Plus-end-directed,High-duty Ratio Molecular Motor

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.