蜜蜂间接飞翔肌粗肌丝的微丝结构

用能溶解肌球蛋白但不溶解副肌球蛋白的溶液(300 mM KCI,pH6.0)处理分离的蜜蜂间接飞翔肌粗肌丝,经数分钟后可以看到粗肌丝端头散开成为多根微丝,微丝数最多为7根。延长处理时间,可以见到粗肌丝中央部分只剩下直径约为5 nm的徽丝。实验结果支持我们以前提出的蜜蜂间接飞翔肌粗肌丝的结构模式,并指示贯穿肌小节、两端都和Z线相连的内芯至少部分由副肌球蛋白组成。只存在于A带的,由6根微丝形成的外套是由肌球蛋白分子组成。     

螯虾腹屈肌肌原纤维副肌球蛋白和原肌球蛋白的分离和结晶

螯虾Procambarus clarki腹屈肌肌原纤维副肌球蛋白和原肌球蛋白已经分离和结晶。在离子强度近于0.3和pH 6.0的溶液中透析,螯虾副肌球蛋白类晶体析出,与原肌球蛋白可以彼此完全分离。经含200mmol KCl,10mmol磷酸缓冲液,pH 6.0溶液或者50mmol BaCl_2 50mmol Tris.HCl_1 pH7.8溶液透析,可以得到副肌球蛋白针状类晶体,周期为14.5或72.5nm。原肌球蛋白溶液经含2％饱和硫酸铵,10mmol醋酸缓冲液,pH 5.1之溶液透析,呈现针状或梭形结晶,具有39nm周期。     

螯虾腹屈肌的超微结构

用电子显微镜观察,发现螯虾(Procambarus clarkii)腹屈肌浅层(慢肌,tonic fiber)肌纤维和深层(快肌,twitch fiber)肌纤维的超微结构存在显著差异。浅层腹屈肌肌原纤维有相对长的肌节(5—10μm),肌原纤维直径较大,每根粗肌丝周围有9—12根细肌丝环绕,细肌丝与粗肌丝数量比约为6∶1;深层腹屈肌有相对短的肌节(3—4.5μm),肌原纤维直径较小,每根粗肌丝周围有6根细肌丝环绕,细肌丝与粗肌丝数量比为3∶1。以上结果提示,在决定螯虾腹屈肌收缩速度方面,可能肌原纤维直径大小比肌节长度更为重要。细、粗肌丝排列方式也可能与收缩速度有关。这与脊椎动物骨骼肌的情况是不同的。     

副肌球蛋白在意大利蜜蜂间接飞翔肌中的定位

肌肉收缩是由于肌原纤维中粗、细肌丝相互滑行的结果(Huxley,1988;Huxley,1983;Squire,1986)。许多无脊椎动物肌肉粗肌丝中除含有肌球蛋白外,还存在着含量不同的副肌球蛋白(陈明等,1984、1985)。我们曾经进行过一系列关于意大利蜜蜂(Apis mellifera ligustica Spin)间接飞翔肌原纤维排列及其粗肌丝亚丝结构的研究。间接飞翔肌的粗肌丝从Z-线延伸至另一Z-线(范世藩等,1966),分离的天然粗肌丝经变性剂(脲、胍)处理,可以散开成直径约为5nm的数根亚丝,在一些亚丝上     

穴居狼蛛腿肌的显微和亚显微结构

用光学显微镜、电子显微镜和小角 X 射线衍射技术研究了穴居狼蛛(Lycosa singriensis)腿肌的构造。腿肌的肌原纤维显示 I 带和 A 带构成的典型的横纹,肌节长度比脊椎动物骨胳肌长得多,约为5.3μm。A 带中间存在 H 带和 M 线区。在肌原纤维中,长4.3μm,直径200(?)的粗肌丝排成中心间距为约600(?)的六角阵列,每根粗肌丝周围环绕着8—12根直径60(?)的纽肌丝。粗肌丝中心的显著浅染提示粗肌丝可能由肌球蛋白围绕着副肌球蛋白核心组成。X射线衍射实验结果指示,静息状态活肌肉中,肌球蛋白横桥形成435(?)重复螺旋,横桥水平的轴向间距为145(?)。静息状态在位固定的腿肌的电镜照片还显示与肌动蛋白细肌丝接触的肌球蛋白横桥,文中讨论了这一颇有兴趣的结果。     

甲壳动物横纹肌的收缩蛋白质与调节机制

甲壳动物横纹肌肌原纤维的肌丝陈列,收缩蛋白质和收缩的Ca²⁺依赖性调节机制与脊椎动物横纹肌有不少差异.脊椎动物横纹肌、甲壳动物快肌与慢肌的粗丝与细丝的数量比依次为1:2,1:3和1:6,肌丝阵列各异.甲壳动物粗肌丝由肌球蛋白和副肌球蛋白组成,其分子装配与脊椎动物不同.细肌丝含有肌动蛋白、原肌球蛋白和肌钙蛋白,肌钙蛋白-T分子量较高,肌钙蛋白-C仅1个Ca²⁺结合位点.甲壳动物横纹肌兼有细肌丝调节与粗肌丝调节.     

肌原纤维上蛋白质定位问题的研究 Ⅰ.标记抗体的电子显微镜观察在副肌球蛋白定位问题上的初步应用

(一)应用铁蛋白标记抗体的技术对尖蚌闭壳肌白色部分的粗丝作了副肌球蛋白的定位研究。结果支持了许多作者根据小角度X-光衍射和电子显微镜分别从副肌球蛋白肌丝和晶体反映出来的共同特征周期所作的推测,即副肌球蛋白是粗丝的组成部分。从免疫化学反应的结果来看,副肌球蛋白分布于粗丝的表面。(二)粗丝与标记的副肌球蛋白抗体反应后,再经反染,周期为360A的横纹结构更为清晰。此数值恰为一般在副肌球蛋白天然及人工纤维和晶体中所见到的720A周期的一半。这个观察和平行的物化研究以及对副肌球蛋白分子的电子显微镜观察,都证明副肌球蛋白可能具有亚基结构,亚基的长度为360A。(三)观察到粗丝在与标记抗体反应后,有侧向偶联现象。     

栉江珧平滑闭壳肌收缩装置的超微结构

利用电子显微镜观察了栉江珧平滑闭壳肌收缩装置的精细结构,它含有粗肌丝、细肌丝和致密体。分离的天然粗肌丝含肌球蛋白和副肌球蛋白,呈现带状和Bear-Selby网格状图象,其周期为14.5nm和7.2nm。以不含ATP的低离子强度溶液处理粗肌丝,则其近侧集聚大量细肌丝;而以微酸性、中等离子强度溶液处理粗肌丝,可以溶去肌球蛋白,但不破坏其周期性结构。以2M脲处理粗肌丝,它纵向分散成直径约有10nm的长带。     

肌球蛋白磷酸化的研究进展

肌球蛋白是肌原纤维粗丝的组成单位,由多条重链与多条轻链组成,被视为一种分子马达。在肌肉收缩、趋化性胞质分裂、胞引作用、膜泡运输以及信号传导等生理过程中起重要作用。目前肌球蛋白磷酸化是研究的一个热点,它对细胞的迁移、收缩、胞质分裂以及其他未知功能都有着至关重要的作用。肌球蛋白磷酸化分为重链的磷酸化与轻链的磷酸化。根据国内外的最新相关研究报道,分别从肌球蛋白的结构与功能、磷酸化的作用机制、磷酸化的生物学功能以及最新研究成果等方面,对肌球蛋白的磷酸化研究进展进行阐述。     

非肌肉Ⅱ型肌球蛋白在细胞骨架网络中动力学行为的图像分析(已撤稿2016-06-15)

贴壁细胞的形状和弹性(硬度)与细胞骨架网络的形态以及纤维的交联方式密切相关．而细胞骨架网络的形态和组成与细胞中的二型肌球蛋白的活动,尤其是肌球蛋白组装成的肌球蛋白粗丝(minifilament)的活动有关．细胞通过与其外部环境的机械传感(mechanosensing)来调节二型肌球蛋白的活动和肌球蛋白粗丝的相互作用,从而实现对细胞骨架网络重组(remodeling)的控制．当前对活体细胞内二型肌球蛋白的研究从实验测量到理论模型的建立之间还有不小距离,主要是因为直接测量会对细胞结构和生理活动产生影响,而间接测量不能得到肌球蛋白在细胞内活动的准确数据．因此本文提出利用新的免疫荧光显微图像分析技术,例如免疫荧光蛋白图像追踪和局部图像相关函数分析技术,分析HeLa细胞体内肌球蛋白在细胞骨架网络中的动态分布,总结出肌球蛋白主导的细胞骨架和张力纤维组装与分解过程中的基本动力学规律．图像分析结果说明：肌球蛋白纤维在细胞骨架网络构建过程中依次动态处于组装与分解状态,通过其粗丝相对旋转对齐与收缩产生张力以维持纤维束稳定,并形成有不同肌球蛋白和粘着斑数量与分布形态的三类稳定性肌动球蛋白网络,其稳定性和收缩力大小呈正相关、与所结合肌球蛋白数量密度成正比．     

Neuromuscular relationships in the abdomen of the Californian shore crab Pachygrapsus crassipes

There are two pairs of muscles in each abdominal segment of the crab; one pair of flexors and one pair of extensors. In the early larval stages the muscles have short sarcomeres--a property of fast fibers--and high thin to thick filament ratios--a property of slow fibers. In the adult the abdominal muscles are intermediate and slow, since they have fibers with intermediate and long sarcomeres, high thin to thick filament ratios, low myofibrillar ATPase activity, and high NADH diaphorase activity. The different fiber types are regionally distributed within the flexor muscle. Microelectrode recordings from single flexor muscle fibers in the adult showed that most fibers are supplied by three excitatory motor axons, although some are supplied by as many as five efferents. One axon supplies all of the flexor muscle fibers in its own hemisegment, and the evoked junctional potentials exhibit depression. This feature together with the innervation patterns of the fibers are similar to those reported for the deep flexor muscles of crayfish and lobsters. Therefore, in the adult crab, the abdominal flexor muscles have some features in common with the slow superficial flexors of crayfish and other features in common with the fast deep flexor muscles.     

Myosin from abdominal flexor muscle in a crayfish, Procambarus clarki Girard.

1. Crayfish (Procambarus clarki) myosin was obtained from abdominal flexor muscle. The Ca2+-ATPase activity of crayfish myosin was much lower than that of rabbit skeletal myosin. However, F-actin-activated Mg2+-ATPase of crayfish and its superprecipitation closely resembled those of rabbit skeletal myosin. This fact suggests that the ability of crayfish myosin to combine with F-actin is essentially the same as that of skeletal myosin, although the chemical structures of both the myosin molecules when involved in their Ca2+-ATPast activity must be different from each other. 2. Crayfish and rabbit skeletal myosins were subjected to SDS-polyacrylamide gel electrophoresis. Crayfish myosin was found to have one heavy chain and two distinct light chain components (CF-gl and CF-g2), which have molecular weights of 18,000 and 16,000, respectively. These light chains correspond in molecular weight to the light chains (SK-g2 and SK-g3) in rabbit skeletal myosin. 3. CF-g1 could be liberated from the crayfish myosin molecule reacting with 5,5'-dithio-bis (2-nitrobenzoic acid), (Nbs2), without recovery of ATPase activity by the addition of DTT. These properties are equivalent to those of SK-g2 in rabbit skeletal myosin, although Nbs2-treated crayfish myosin did not recover its ATPase activity at all.     

THE MYOFILAMENT LATTICE: STUDIES ON ISOLATED FIBERS : I. The Constancy of the Unit-Cell Volume with Variation in Sarcomere Length in a Lattice in which the Thin-to-Thick Myofilament Ratio is 6:1

The spacing between the thick myofilaments of muscle fibers from the walking legs of crayfish (Orconectes) was determined by optical transform analysis of electron micrograph plates of fixed single fibers and by X-ray diffraction of living single fibers. Sarcomere lengths were determined by light diffraction prior to fixation and prior to the in vivo experiments. From these combined measurements, it is demonstrated that the unit-cell volume of the myofilament lattice is constant during muscle shortening, indicating that the myofilament lattice works in a constant-volume manner. It is further demonstrated with X-ray diffraction measurements of living single fibers that the myofilament lattice continues to work at constant volume after the sarcolemma is removed from the fiber. This indicates that the constant-volume behavior of muscle is inherent to the myofilament lattice.     

横纹肌肌原纤维的第三肌丝──肌联蛋白

实验研究证明,在动物横纹肌肌原纤维中,除包含有粗肌丝、细肌丝外,还有纤肌丝的存在,肌联蛋白（肌巨蛋白）是具有挠性的线状蛋白质,分子量为3000 000,长度约为0.9μm,跨越肌原纤维的M－线和Z－线,形成纤肌丝.其生理功能是在粗肌丝装配中具有分子模板作用,并将粗肌丝稳定于肌原纤维肌小节中央以及可参与肌球蛋白活性的调节.     

螯虾横纹肌粗肌丝超微结构的计算机图象分析

本文在微机ＡＳＴ／３８６和真彩色图形采集卡ＣＡ－５４０构成的趁科象处理系统上开发了一个软件,包括用于对动物横纹肌粗肌丝的超微结构进行分析的专用模块,一般图象处理系统的通用模块和文件管理模块三大部分。本系统对动物横纹肌粗肌丝的亚结构从旋转功率谱的角度分析了其对称性的存在,并运用图象的迭加,旋转平衡和旋转滤波等手段来处理,显示其对称性;同时,对存在的不对称结构也作了相应的分析处理。利用本系统,对螯虾的     

Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions.     

SOME ASPECTS OF THE STRUCTURAL ORGANIZATION OF THE MYOFIBRIL AS REVEALED BY ANTIBODY-STAINING METHODS

From observations of fluorescent antibody staining and antibody staining in electron microscopy, evidence is presented for the following: (a) Direct contact of the actin and myosin filaments occurs at all stages of contraction. This results in inhibition of antibody staining of the H-meromyosin portion of the myosin molecule in the region of overlap of the thin and thick filaments. (b) Small structural changes occur in the thick filaments during contraction. This leads to exposure of antigenic sites of the L-meromyosin portion of the myosin molecule. The accessibility of these antigenic sites is dependent upon the sarcomere length. (c) The M line is composed of a protein which is weakly bound to the center of the thick filament and is not actin, myosin, or tropomyosin. (d) Tropomyosin as well as actin is present in the I band. (e) If actin or tropomyosin is present in the Z line, it is masked and unavailable for staining with antibody.