MYOSIN, ACCESSORY PROTEIN: ARRANGEMENT IN THICK FILAMENT

我们希望研究A波段和粗丝的两个特殊方面 结构。其中之一涉及粗丝辅助蛋白，如 作为M-、C-和H-蛋白和肌动蛋白。我们计划确定这一关系 这些蛋白在A带(M蛋白)和粗丝中的存在 (C-蛋白，H-蛋白，肌动蛋白)完整性，两者都在脊椎动物中 (鸡胸)肌肉，这些蛋白质已知存在的地方，以及在 无脊椎动物的长肌节横纹肌(蝌蚪、狼蛛、蝎子、 龙虾、藤壶)，它们尚未被鉴定为成分 A-乐队的成员。在这方面的研究中，我们还将调查 C-蛋白在维持合成酶完整性中的作用 无脊椎动物和非肌肉肌球蛋白的细丝。我们还将使用光和 分离的A段肌原纤维的电子显微镜和图像分析 和粗丝，以及合成肌球蛋白聚集体，与 利用单抗进行免疫定位技术的研究 这些辅助蛋白的存在、定位和交叉反应 在不同的粗丝中。 这项建议的第二个方面是调查 环境调整，如pH、离子强度(40-200 mm)。二价 阳离子浓度(Mg++-1到8 mM，Ca++0到1 mM)和缺勤与 在交叉桥阵列和核心的数量级上存在ATP 分枝状肌球蛋白结构的单丝粗丝。在这里我们 假设从原生的、放松的条件变化将是容易的 可识别的，并且可以模拟跨桥位置在 收缩周期，原位。我们将用电子来检验孤立的细丝。 显微光学衍射和获得确定的结构信息 采用计算机图像分析方法。这些实验将被比较 在皮肤完整的收缩装置上得到的结果 美国马克斯·普朗克医学研究所约翰·S·雷博士所著 研究，海德堡，西德。 这些研究的结果将为深入了解在 收缩装置的两根粗丝的组织 进入有序的A带和肌球蛋白交叉桥的位置与 在肌肉活动过程中的细丝主干。

We wish to study two particular aspects of A-band and thick filament structure. One of these deals with thick filament accessory proteins, such as M-, C- and H-proteins and titin. We plan to determine the relationship of the presence of these proteins to A-band (M-protein) adn thick filament (C-protein, H-protein, titin) integrity, both in vertebrate striated (chicken breast) muscle, where these proteins are known to exist, and in invertebrate long sarcomere striated muscles (Limulus, tarantula, scorpion, lobster, barnacle), where they have not yet been identified as components of the A-band. In this aspect of the study we will also investigate the effect of C-protein on the maintenance of the integrity of the synthetic filaments of invertebrate and nonmuscle myosin. We will also use light and electron microscopy and image analysis of myofibrils, isolated A-segments and thick filaments, and synthetic myosin aggregates, in combination with immunological localization techniques using monoclonal antibodies to study the presence, location and crossreactivity among these accessory proteins in different thick filaments. The second aspect of this proposal is to investigate the effects of environmental modifications such as pH, ionic strength (40-200mM). divalent cation concentration (Mg++ -1 to 8mM, Ca++ 0 to 1mM) and absence vs. presence of ATP on the order of the crossbridge array and the core paramyosin structure of isolated Limulus thick filaments. Here we hypothesize that changes from the native, relaxed condition will be readily identifiable and may mimic crossbridge positional changes during a contractile cycle,in situ. We will examine isolated filaments by electron microscopy optical diffraction and obtain definitive structural information by computer image analytic methods. These exeperiments will be compared with results obtained on the intact contractile apparatus in skinned Limulus fiber by Dr. John S. Wray, Max-Planck Institute for Medical Research, Heidelberg, West Germany. The results of these studies will provide insight into factors effective in the organization of both the thick filaments of the contractile apparatus into ordered A-bands and the position of myosin crossbriges in relation to the filament backbone, during muscle activity.