STRUCTURE OF A PROTEIN CATALYZING ACTIVE TRANSPORT

钠钾离子依赖性腺苷三磷酸酶 ((Na+ + K+）-ATP酶）是在质膜中的酶， 所有负责主要活性的动物细胞 将钠从细胞质中运输出来，将钾运输到细胞质中。 是 强心类固醇如洋地黄的有效靶点 和地高辛。 本发明的催化性α-多肽的序列 (Na+ + K+）-ATP酶最近已成为可用的。 十 该序列中的疏水片段已被命名为 作为跨越质膜双层的候选物 当酶处于其天然结构时。 我们的目标是 确定这10个序列中的哪一个完成了这个角色， 确定在质膜的哪一侧， 在这些疏水链段之间存在亲水区域。 一 这些氨基酸中每一种中特定赖氨酸、谷氨酸或酪氨酸 亲水性区域被选择为合适的目标， 通过不渗透试剂改性。 血浆囊泡 高浓度（Na+ + K+）-ATP酶膜 并密封在一个右侧出方向将是标本 用于这些修改中的每一个。 给定的产品 在靶氨基酸之一处的修饰将被分离 通过消化蛋白质并通过免疫吸附分离， 修饰的氨基酸位于其中的肽。 的 用于分离的免疫吸附剂将由 针对含有以下的合成肽的抗体： 修饰的氨基末端或羧基末端序列 肽。 通过确定每种靶向氨基酸 酸位于细胞质或细胞质外 在膜的表面上，α-多肽的拓扑结构 将建立天然的（Na+ + K+）-ATP酶。 这将确认 那些跨越双层并形成中心的序列 钾和钠进出的隔室 的细胞。 这些信息将增加我们对 （Na+ + K+）-ATP酶的作用机制 重要的是那些生理过程的节奏心跳， 动力液体在肾脏和肠道中流动， 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.