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

NA、K-ATP酶同工酶--单克隆抗体探针

• 批准号: 3351126
• 负责人: Kathleen J Sweadner
• 金额: $ 13.47万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3351126
• 关键词: adenosinetriphosphatase; affinity chromatography; antigens; axon; enzyme structure; gel filtration chromatography; genetic manipulation; heart cell; hybridomas; immunochemistry; isozymes; kidney; monoclonal antibody; nerve endings; neurons; ouabain

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.

心脏糖苷引起的变力作用被认为是由于 抑制Na，KATP酶。 在大鼠、豚鼠和人类中， 心，高亲和低亲和的占有 已经发现哇巴因结合位点与内复制相关， 并且已经发现高亲和力尺寸的比例 在发展和病理条件的变化。 我 先前发现了Na，KATP酶的两种不同的同工酶 在大脑中，并证明他们有显着不同的 哇巴因的近亲 这一修订提案提出了证据， 心脏组织含有相似但不完全相同的 Na，KATP酶。 目的是识别、表征和 定位高和低亲和力Na，KATP酶，无论是在心脏 细胞或自主神经末梢。 几乎没有心脏的结构特征 Na，KATP酶的分子特性及其高亲和力 哇巴因的地点至今尚未查明。 相比之下， 纯化大鼠脑轴膜Na、KATP酶（高亲和力） 和肾脏（低亲和力），并确定它们具有结构性 和抗原差异。 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，KATP酶密切相关，非常有选择性的探针， 我们需要区分它们。 我们现在要制定具体的 针对轴膜、肾脏、心脏和 交感神经元同工酶，作为研究的分子工具 其结构和分布。 识别不同抗原的单克隆抗体将被 通过抑制免疫反应的策略获得 对共同抗原的反应。 Na，KATP酶的鉴定 心脏将通过检测特定的 蛋白水解肽图谱中的决定簇， 结构将与轴膜的结构进行比较， 肾Na+、KATP酶。 同工酶的分布 将确定心脏组织及其交感神经支配 免疫细胞化学 我们会评估哇巴因的亲和力 不同的Na，KATP酶，以提供预测的基础 不同的生理作用。