STRUCTURE OF A PROTEIN CATALYZING ACTIVE TRANSPORT

钠和钾离子依赖性腺苷三磷酸酶[（Na+ + K+）-ATP酶]是所有动物细胞质膜中的酶， 负责钠的主要主动转运， 钾进入细胞质。 它是有效的目标， 强心类固醇，如洋地黄和地高辛。 十一疏水 催化亚基序列中的片段已被指定为 作为跨越质膜双层的候选物。 是一 我们的目标是确定这11个序列中的哪一个满足 这一作用是通过确定质膜的哪一侧亲水 位于这些疏水链段之间的区域。 一种特殊的赖氨酸， 在这些亲水区域中的每一个中选择半胱氨酸或酪氨酸 作为被不渗透试剂修饰的合适靶。 囊泡 质膜含有高浓度的（Na+ + K+）-ATP酶， 密封在一个右侧出方向将是用于每个样本 这些修改。 给定修改在其中一个 通过消化蛋白质来监测目标氨基酸， 通过免疫吸附分离其中修饰的氨基酸 所在 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： 修饰肽的氨基末端或羧基末端序列。 通过 确定每种靶向氨基酸是否位于所述多肽上， 细胞质或细胞质外表面的膜，拓扑结构 将建立天然（Na+ + K+）-ATP酶中α-多肽的活性。 这将识别跨越双层并形成双层的那些序列。 钾和钠进出的中央室 牢房 我们的另一个目标是研究结构的变化， 蛋白质在阳离子区室打开和关闭时出现。 赖氨酸，酪氨酸和半胱氨酸的可及性，位于 跨膜片段的边缘，亲电试剂在 水相将作为所述组合物的构型的函数而被跟踪。 通过使用相同的免疫化学策略分离修饰的 含有目标的肽。 这些信息将增加我们的 （Na+ + K+）-ATP酶的作用机制 对那些控制心跳的生理过程至关重要， 动力液体在肾脏和肠道中流动，并产生动作 神经系统的潜力。

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.