STRUCTURE OF A PROTEIN CATALYZING ACTIVE TRANSPORT

(NA+ + K+）腺苷三磷酸酶是一种酶，存在于细胞质膜上， 所有负责主动运输钠的动物细胞 和钾进入细胞质。 它也是主要的目标， 强心类固醇，如洋地黄和地高辛。 我们的目标是 获得有关其分子结构的广泛信息，并使用此 了解主动转运机制的知识。 它有 已经证明，功能亚基，α，是非常大的（1100 残留物）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+）-ATP酶，并且全部以右侧向外的方向密封， 羟乙硫酰基共价修饰 3-（N-2，4-二硝基苯基）-3-氨基丙酰亚胺酸盐，一种不渗透试剂， 与蛋白质中暴露的赖氨酸残基反应。 上的赖氨酸 与试剂反应的酶的细胞质外表面将被 通过分离和测序它们所处的肽来鉴定 所在 强心类固醇的结合位点也位于 该细胞质外表面，以及衍生自这些表面的亲和试剂， 药物和具体的修改本网站是可用的。 他们 将用于鉴定该蛋白的氨基酸序列的部分， 形成这个位点的蛋白质。 最后，通过利用 α链中特定巯基残基的反应性 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 蛋白质整个序列中的这些巯基将 测定 所有这些实验将增加我们对 (Na+ + K+）腺苷三磷酸酶，其功能至关重要 这些生理机制使心跳加快， 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.