Panté N
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
J Struct Biol. 1994 Sep-Oct;113(2):148-63. doi: 10.1006/jsbi.1994.1047.
Paramyosin is the main structural component of the thick filament of molluscan smooth muscles. These filaments consist of a large paracrystalline core of paramyosin with myosin arranged on its surface. The detailed molecular packing of paramyosin in the core and the array of myosin on the surface of the paramyosin core remain unknown. An unsolved problem is the polarity of the paramyosin molecules within these thick filaments (i.e., it is not known whether the paramyosin molecules assemble with their NH2-terminal ends pointing toward the center or toward the end of the thick filament). Here a method to distinguish between the NH2- and the COOH-terminal ends of the paramyosin molecule by electron microscopy is described and used to determine their polarity in synthetic paracrystalline arrays. This method consists of labeling the cysteine residues of paramyosin molecules with the avidin-biotin system developed by Sutoh et al. (1984). Accordingly, the sulfhydryl groups of paramyosin--isolated from the anterior byssus retractor muscle (ABRM) of Mytilus edulis--were modified with maleimide-biotin, and the biotinylated thiols were visualized in the electron microscope after glycerol spraying/rotary metal shadowing by attaching monomeric avidin to them. Avidin-biotin labeling of the native molecule and its carboxypeptidase fragments revealed that ABRM paramyosin contains one pair of cysteine at its NH2-terminal end and one pair at approximately 30 nm from its COOH-terminal end. Synthetic paracrystalline arrays of paramyosin with known axial arrangement were also labeled with the avidin-biotin system. The location of the bound avidin in these paracrystals indicated the polarity of paramyosin in these arrays. The polarity was also determined by comparison of the transverse band-like staining pattern of paracrystals of alpha-paramyosin (intact protein) and beta-paramyosin (a proteolytically cleaved alpha-paramyosin that has lost a small segment at its COOH-terminal end). Both methods revealed that paramyosin assembles with its NH2-terminal end pointing toward the center of the paracrystals. The implications of this result for the polarity of paramyosin in the native filament core, and for the arrangement of myosin on the surface of molluscan thick filaments, are discussed.
副肌球蛋白是软体动物平滑肌粗肌丝的主要结构成分。这些肌丝由一个大的副肌球蛋白副晶状核心组成，肌球蛋白排列在其表面。副肌球蛋白在核心中的详细分子堆积以及副肌球蛋白核心表面的肌球蛋白排列仍然未知。一个尚未解决的问题是这些粗肌丝中副肌球蛋白分子的极性（即，不知道副肌球蛋白分子组装时其NH2末端是指向中心还是指向粗肌丝的末端）。本文描述了一种通过电子显微镜区分副肌球蛋白分子的NH2末端和COOH末端的方法，并用于确定其在合成副晶状阵列中的极性。该方法包括用Sutoh等人（1984年）开发的抗生物素蛋白-生物素系统标记副肌球蛋白分子的半胱氨酸残基。因此，从紫贻贝的前足丝收缩肌（ABRM）中分离出的副肌球蛋白的巯基用马来酰亚胺-生物素进行修饰，通过将单体抗生物素蛋白附着到生物素化的硫醇上，在甘油喷雾/旋转金属阴影处理后在电子显微镜下观察生物素化的硫醇。天然分子及其羧肽酶片段经抗生物素蛋白-生物素标记显示，ABRM副肌球蛋白在其NH2末端含有一对半胱氨酸，在距其COOH末端约30 nm处含有一对半胱氨酸。具有已知轴向排列的副肌球蛋白合成副晶状阵列也用抗生物素蛋白-生物素系统进行标记。这些副晶体中结合的抗生物素蛋白的位置表明了这些阵列中副肌球蛋白的极性。通过比较α-副肌球蛋白（完整蛋白）和β-副肌球蛋白（一种在COOH末端失去一小段的蛋白水解切割的α-副肌球蛋白）副晶体的横向带状染色模式也确定了极性。两种方法都表明，副肌球蛋白组装时其NH2末端指向副晶体的中心。讨论了该结果对天然肌丝核心中副肌球蛋白极性以及软体动物粗肌丝表面肌球蛋白排列的影响。
