Microsequencing of a Putatve Myosin I Isoform

术语“肌球蛋白”描述了一类运动蛋白，其特征是当它们与肌动蛋白结合时能够水解ATP。肌球蛋白II是肌球蛋白家族中最熟悉和最具特征的成员，是一种丝状蛋白，它利用ATP水解时释放的能量使丝状蛋白相互移动，这是肌肉收缩和细胞质分裂的重要步骤。与我们对肌球蛋白II生理特性的理解相反，对这个超家族的其他成员知之甚少，其中许多成员不形成细丝。在这些非丝状肌凝蛋白中，肌凝蛋白I被研究得最为广泛。肌球蛋白I最初在棘阿米巴原虫中被描述，现在已知在哺乳动物细胞中广泛分布。该蛋白已在分子和生化水平上进行了广泛的研究，并且有大量关于肌球蛋白i的细胞分布的信息，但该蛋白的生理作用，特别是在哺乳动物细胞中的作用还不太明确。为了研究肌球蛋白I在哺乳动物细胞中的生理作用，从牛肾上腺中纯化了该蛋白，并制备了针对其的多克隆抗体。在鉴定这些抗体时，观察到对肌球蛋白I的亲和纯化抗体染色了间期细胞的细胞核。根据最近其他人在细胞核中肌动蛋白样蛋白的证明，这一数据提示了肌凝蛋白I参与细胞核功能的假设。然而，在研究这种假定的作用之前，必须明确地确定细胞核中与抗体发生反应的蛋白质。因此，本研究的目标是纯化足够的蛋白质，以便通过微测序对其进行鉴定。* * *

9631833 de Lanerolle The term "myosin" describes a family of motor proteins that are characterized by their ability to hydrolyze ATP when they bind actin. Myosin II, the most familiar and best characterized member of this family, is a filamentous protein that uses the energy released when ATP is hydrolyzed to move filaments past each other, an essential step in muscle contraction and in cytokinesis. In contrast to our understanding of the physiological properties of myosin II, little is known about the other members of this superfamily, many of which do not form filaments. Of these non-filamentous myosins, myosin I has been studied most extensively. Myosin I was initially described in Acanthamoeba and is now known to be widely distributed in mammalian cells. This protein has been studied extensively at the molecular and biochemical levels, and there is an abundance of information about the cellular distribution of myosin I. The physiological role of this protein, particularly in mammalian cells, is less well defined. In an effort the investigate the physiological role of myosin I in mammalian cells, the protein was purified from bovine adrenal glands and a polyclonal antibody was raised against it. While characterizing these antibodies, it was observed that the affinity-purified antibodies to myosin I stained the nucleus in interphase cells. In light of the recent demonstration by others of actin-like proteins in the nucleus, this data prompted the hypothesis that myosin I is involved in nuclear functions. However, before such a putative role can be investigated, the protein in the nucleus which reacts with the antibody must be unambiguously identified. Therefore, the goal of this research is to purify sufficient protein to identify it by microsequencing it. ***