ISOZYMES OF THE NA, K-ATPASE--MONOCLONAL ANTIBODY PROBES

心脏糖苷类药物引起的变力作用被认为是由于 抑制Na，KATPase。在大鼠、豚鼠和人身上 心，高亲和力和低亲和力的占有率 已经发现哇巴因结合位点与intoropy相关， 高亲和力大小的比例已经被发现 发育和病理条件的变化。我 先前发现的Na，KATPase的两种不同的同工酶 在大脑中，并证明它们有明显的不同 哇巴因的亲缘关系。这份修订后的提案提供了证据 心脏组织含有相似但不完全相同的同工酶 Na，KATPase。目标是识别、描述和 定位高亲和力和低亲和力的Na，KATPase 细胞或自主神经末梢。 心脏的结构特征一直很少。 Na、KATPase及其高亲和力的分子同一性 哇巴因遗址直到现在才为人所知。相比之下，我们有 纯化大鼠脑轴膜Na，KATPase(高亲和力) 和肾脏(低亲和力)，并确定它们具有结构性 和抗原性差异。我们现在有证据表明 来自心脏的高亲和力形式是抗原性的 与大脑的不同。因为这些细胞的同工酶 NA、KATPase是密切相关的，选择性很强的探针 需要区分它们。我们现在想要开发出具体的 抗轴膜、肾脏、心脏和心脏的单抗 交感神经元同工酶作为研究交感神经同工酶的分子工具 它们的结构和分布。 识别不同抗原的单抗将是 用一种抑制免疫学的策略获得 对共同抗原的反应。Na，KATPase的同一性 心脏将通过检测特定的 它们的蛋白水解肽图谱中的决定因素，以及它们的 结构将与轴膜的结构和 肾钠，KATPase。同工酶的分布 将确定心脏组织及其交感神经支配 通过免疫细胞化学。我们将评估哇巴因亲和力 不同Na、KATPase的差异，为预测 它们不同的生理作用。

Cardiac glycosideelicited inotropy is thought to be due to inhibition of the Na, KATPase. In the rat, guinea pig, and human hearts, the occupancy of highaffinity as well as lowaffinity ouabain binding sites has been found to correlate with intoropy, and the propportion of high affinity sizes has been found to change in development and in pathological conditions. I previously discovered two distinct isozymes of the Na,KATPase in the brain, and demonstrated that they have markedly different affinites for ouabain. This revised proposal presents evidence that cardiac tissue contains similar, but not identical isozymes of the Na,KATPase. The objective is to identify, characterize, and localize high- and lowaffinity Na,KATPases, either in cardiac cells or in autonomic nerve endings. There has been little structural characterization of the cardiac Na,KATPase, and the molecular identity of its high affinity ouabain site has not been known until now. In contrast, we have purified the Na,KATPases of rat brain axolemma (high affinity) and kidney (low affinity) and established that they have structural and antigenic differences. We now have evidence that suggests that the high affinity form from the heart is antigenically different from that of the brain. Because the isozymes of the Na,KATPase are closely related, very selective probes are needed to distinguish them. We now want to develop specific monoclonal antibodies to the axolemma, kidney, cardiac, and sympathetic neuron isozymes, as molecular tools for the study of their structure and distribution. Monoclonal antibodies that recognize distinct antigens will be obtained with a strategy for supressing the immunological response to shared antigens. The identity of the Na,KATPases in the heart will be determined by detection of specific determinants in their proteolytic peptide maps, and their structures will be compared with those of the axolemma and kidney Na,KATPases. The distribution of the isozymes in cardiac tissue and its sympathetic innervation will be determined by immunocytochemistry. We will evaluate the ouabain affinities of the different Na,KATPases, to provide a basis for predicting their different physiological roles.