STRUCTURE OF A PROTEIN CATALYZING ACTIVE TRANSPORT

钠钾离子依赖性三磷酸腺苷酶[(Na++ K+)-ATPase]是所有动物细胞质膜中的酶， 负责钠的主要主动运输出和 钾进入细胞质。它是美国政府的有效目标 强心类固醇，如洋地黄和地高辛。11个疏水症 催化亚单位序列中的片段已被指定 作为跨越质膜双层的候选者。它是一个 我们的目标是确定这11个序列中的哪一个能够实现 这种作用是通过确定质膜哪一侧的亲水性来实现的 这些疏水片段之间的区域。一种特定的赖氨酸， 这些亲水性区域中的每一个都选择了半胱氨酸或酪氨酸 作为一个合适的靶子，可以用非故意的试剂进行修饰。囊泡 质膜含有高浓度的(Na++K+)-ATPase和 以右侧向外的方向密封的是用于每个 在这些修改中。某一给定修改的乘积 目标氨基酸将通过消化蛋白质和 免疫吸附分离修饰氨基酸的多肽 已经找到了。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 修饰的多肽的氨基末端或羧基末端序列。通过 确定每个目标氨基酸是否位于 膜的胞质或胞外表面，拓扑学 将建立天然(Na++K+)-ATPase中的α-多肽。 这将识别那些跨越双分子层并形成 钾和钠进出的中央隔室 牢房。我们的另一个目标是研究 当阳离子的这个隔间打开和关闭时出现的蛋白质。 赖氨酸、酪氨酸和半胱氨酸的可及性，位于 膜跨段的边缘，到 水相将根据构型的不同而变化 通过使用相同的免疫化学策略来分离经修饰的 含有靶标的多肽。这一信息将增加我们的 洞察(Na++K+)-ATPase的作用机制 对那些控制心跳速度的生理过程至关重要， 动力流体在肾脏和肠道中流动，并产生作用 神经系统的潜能。

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. Eleven hydrophobic segments within the sequence of the catalytic subunit have been designated as candidates for spanning the bilayer of the plasma membrane. It is one of our objectives to determine which of these eleven sequences do fulfill this role by determining on which side of the plasma membrane hydrophilic regions between these hydrophobic segments lie. A specific lysine, cysteine 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 monitored 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 carboxy-terminal sequence of the modified peptide. By determining whether each of the targeted amino acids is located on the cytoplasmic or the extracytoplasmic 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. Our other objective is to examine changes in structure of the protein that occur as this compartment for the cations opens and closes. The accessibility of lysines, tyrosines, and cysteines, located at the edges of membrane-spanning segments, to electrophilic reagents in the aqueous phase will be followed as a function of the configuration of the compartment by using the same immunochemical strategy to isolate modified peptides containing the targets. 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.