STRUCTURE OF A PROTEIN CATALYZING ACTIVE TRANSPORT

(Na++K+)腺苷三磷酸酶是植物细胞质膜上的一种酶 负责主动转运钠的所有动物细胞 和钾进入细胞质。它也是 强心类固醇，如洋地黄和地高辛。我们的目标是 获取有关其分子结构的广泛信息，并使用此 了解主动运输机制的知识。它有 已经证明，功能亚基Alpha非常大(1100 残基)，并跨越质膜，但更详细的信息是 缺乏。质膜小泡，含有高浓度的 (Na++K+)-ATPase和所有密封在右侧向外的，将是 异硫基共价修饰 3-(N-2，4-二硝基苯基)-3-氨基丙酰亚胺，一种不受欢迎的试剂 与蛋白质中暴露的赖氨酸残基发生反应。歌词中的赖氨酸 与试剂反应的酶的胞外表面将是 通过分离和测序它们所在的多肽来鉴定 找到了。强心类固醇的结合部位也位于 这种胞质外表面，以及从这些表面衍生的亲和试剂 本网站有用于修改的药物和特效药。他们 将被用来鉴定这一氨基酸序列的部分 形成这个部位的蛋白质。最后，通过利用 α链上特定巯基残基在反应过程中的反应性 构象的变化，我们将能够识别这些区域 参与这些构象变化的蛋白质。的位置 这些巯基在蛋白质的总序列中将是 下定决心。所有这些实验都将增加我们对 (Na++K+)三磷酸腺苷酶，其功能至关重要 到那些调整心跳速度并产生 神经系统的动作电位。

(NA+ + K+) Adenosine triphosphatase is the enzyme in the plasma membrane of all animal cells that is responsible for the active transport of sodium out of and potassium into the cytoplasm. It is also the primary target of the cardiotonic steroids such as digitalis and digoxin. It is our objective to obtain extensive information about its molecular structure and use this knowledge to gain insight into the mechanism of active transport. It has been demonstrated that the functional subunit, Alpha, is very large (1100 residues) and spans the plasma membrane, but more detailed information is lacking. Vesicles of plasma membrane, containing high concentrations of (Na+ + K+)-ATPase and all sealed in a right-side-out orientation, will be modified covalently with isethionyl 3-(N-2,4-dinitrophenyl)-3-aminopropionimdate, an impermeant reagent that reacts with the exposed lysine residues of the protein. The lysines on the extracytoplasmic surface of the enzyme that react with the reagent will be identified by isolating and sequencing the peptides in which they are located. The binding site for the cardiotonic steroids is also located on this extracytoplasmic surface, and affinity reagents derived from these drugs and specific for the modification of this site are available. They will be used to identify the portions of the amino acid sequence of this protein that form this site. Finally, by exploiting changes that occur in the reactivity of specific sulfhydryl residues in the Alpha chain during the changes in conformation, we will be able to identify those regions in the protein involved in these conformational changes. The location of these sulfhydryls in the overall sequence of the protein will be determined. All of these experiments will increase our understanding of (Na+ + K+) adenosine triphosphatase whose function is of central importance to those physiological mechanisms which pace heartbeat and create the action potentials of the nervous system.