STRUCTURE OF A PROTEIN CATALYZING ACTIVE TRANSPORT

钠钾离子依赖型三磷酸腺苷酶 ((Na++K+)-ATPase)是细胞质膜上的一种酶 所有负责初级活动的动物细胞 把钠运出细胞质，把钾运入细胞质。它是 洋地黄等强心类固醇的有效靶点 和地高辛。催化α-多肽的序列分析 (Na++K+)-ATPase最近出现了。十 该序列中的疏水片段已被指定 作为跨越质膜双层的候选者 当酶处于其天然结构中时。我们的目标是 通过以下方式确定这十个序列中的哪一个确实履行了这一角色 确定在质膜的哪一侧 这些疏水片段之间的亲水性区域。一个 每一种都含有特定的赖氨酸、谷氨酸或酪氨酸 亲水性区域已被选为合适的目标 被非本意的试剂修改的。血浆小泡 含高浓度(Na++K+)-ATPase的膜 并将标本以右侧向外的方向密封 用于这些修改的每一个。给定值的乘积 目标氨基酸之一的修饰将被分离出来 通过消化蛋白质并通过免疫吸附分离 修饰后的氨基酸所在的肽。这个 用于分离的免疫吸附剂将由 针对合成肽的抗体，合成肽包含 修饰后的氨基末端或羧基末端序列 多肽。通过确定每个靶向氨基是否 酸位于细胞质或胞外。 膜表面，α-多肽的拓扑结构 将建立天然的(Na++K+)-ATPase。这将确定 那些跨越双层并形成中心的序列 钾和钠进出的隔间 在牢房里。这些信息将增加我们对 具有中枢功能的(Na++K+)-ATPase机制 对那些控制心跳的生理过程的重要性， 动力流体在肾脏和肠道中流动，并创造 神经系统的动作电位。

Sodium and potassium ion-dependent adenosine triphosphatase ((Na+ + K+)-ATPase) is the enzyme in the plasma membranes of all animal cells that is responsible for the primary active transport of sodium out of and potassium into the cytoplasm. It is the effective target of the cardiotonic steroids such as digitalis and digoxin. The sequence of the catalytic alpha-polypeptide of (Na+ + K+)-ATPase has recently become available. Ten hydrophobic segments within this sequence have been designated as candidates for spanning the bilayer of the plasma membrane when the enzyme is in its native structure. It is our objective to determine which of these ten sequences do fulfill this role by determining on which side of the plasma membrane each of the hydrophilic regions between these hydrophobic segments lies. A specific lysine, glutamic acid, or tyrosine in each of these hydrophilic regions has been chosen as a suitable target to be modified by an impermeant reagent. Vesicles of plasma membrane containing high concentrations of (Na+ + K+)-ATPase and sealed in a right-side-out orientation will be the specimens used for each of these modifications. The product of a given modification at one of the targeted amino acids will be isolated by digesting the protein and isolating by immunoadsorption the peptide in which the modified amino acid is located. The immunoadsorbent used for the isolation will be made from antibodies directed against a synthetic peptide containing the amino terminal or carboxyl terminal sequence of the modified peptide. By determining whether each of the targeted amino acids is located on the cytoplasmic or the extra-cytoplasmic surface of the membrane, the topology of the alpha-polypeptide in native (Na+ + K+)-ATPase will be established. This will identify those sequences that span the bilayer and form the central compartment through which potassium and sodium pass in and out of the cell. This information will increase our insight into the mechanism of (Na+ + K+)-ATPase whose function is of central importance to those physiological processes that pace heartbeat, power fluid flows in the kidney and intestine, and create the action potentials of the nervous system.