PURIFICATION AND SIGNIFICANCE OF RENIN INHIBITORS

肾素抑制剂的纯化及其意义

• 批准号: 3343378
• 负责人: Theodore Kotchen
• 金额: $ 11.75万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3343378
• 关键词: affinity chromatography; angiotensin /renin /aldosterone hypertension; angiotensins; antihypertensive agents; blood proteins; gas chromatography; hemodialysis; human subject; mass spectrometry; radioimmunoassay; renal failure; renin; uremias

The enzymatic activity of renin is increased in plasma of patients with hypertension and patients with chronic renal failure, possibly due to the deficiency of a circulating renin inhibitor. An acetone soluble lipid extract of plasma and two protein containing plasma fractions (a high protein and a low protein fraction) obtained by Sephadex chromatography inhibit renin. The overall objective of this project is to purify these renin inhibitors and to evaluate their functional significance. Protein purification of the renin inhibiting Sephadex fractions will be carried out to determine if: a) a renin inhibiting lipid is associated with a specific protein, and b) a renin inhibiting peptide can be identified. Steps in protein purification will include Sephadex chromatography, chromatofocusing, chromatography with DEAE cellulose and carboxymethyl cellulose, and HPLC. Renin inhibiting lipid extracts and lipids associated with renin inhibiting protein fractions will alsp be purified; the dependence of inhibition on lipid and/or protein will be determined. Neutral and phospholipids will be separated, and lipids will be further purified with GLC. Additionally, affinity chromatography using a Sepharose afffinity column with purified mouse submaxillary renin as ligand will be used to extract and purify renin inhibitors. In all experiments, renin inhibition will be determined in vitro by evaluating the effects of putative inhibitors on the rate of angiotensin I production after addition to renin-renin substrate and in vivo by evaluating their effects on the pressor response to renin in the anephric rat. The hypothesis that increased enzymatic activity of renin in plasma of hypertensive patients and patients with renal failure is related to the deficiency if a renin inhibitor will be evaluated. We will also evaluate the hypothesis that "activation" of renin by acidification of plasma if related to denaturation of a renin inhibitor. We will determine if increased concentrations of inactive renin in plasma of patients with diabetes mellitus is related to increased concentrations of an acid labile renin inhibitor. To further evaluate a physiologic role of renin inhibitors, we will determine if acute and chronic administration of renin inhibitors extracted from plasma lowers blood pressure in a renin dependent model of experimental hypertension. Identification of naturally occurring renin inhibitors will add a new dimension to current understanding of the renin-aldosterone system.

在患者的血浆中，肾素的酶活性增加 高血压和慢性肾衰竭的患者，可能是由于 循环肾素抑制剂的缺乏。 丙酮可溶性脂质 血浆和两个含有血浆级分的蛋白的提取物（高度 通过Sephadex色谱获得的蛋白质和低蛋白质分数） 抑制肾素。 该项目的总体目的是净化这些 肾素抑制剂并评估其功能意义。 蛋白质 将进行抑制Sephadex级分的肾素纯化 确定是否：a）肾素抑制脂质与特定 蛋白质和b）可以鉴定肾素抑制肽。 介入 蛋白质纯化将包括Sephadex色谱， 染色体，色谱与DEAE纤维素和羧甲基 纤维素和HPLC。 肾素抑制脂质提取物和相关的脂质 使用肾素抑制蛋白质分数，ALSP将被纯化；这 将确定抑制对脂质和/或蛋白质的依赖性。 中性和磷脂将分开，脂质将进一步 用GLC纯化。 另外，使用Sepharose的亲和力色谱法 具有纯化的鼠标超颌骨肾素为配体的亲属柱将是 用于提取和纯化肾素抑制剂。 在所有实验中，肾素 抑制作用将在体外通过评估 添加后血管紧张素I I的推定抑制剂 通过评估其对肾素 - 肾素底物和体内的影响 呼吸响应对肾脏大鼠的肾素。 假设是 高血压患者血浆中肾素的酶活性增加 肾衰竭的患者与肾素有关 将评估抑制剂。 我们还将评估以下假设 如果与变性有关 肾素抑制剂。 我们将确定浓度是否增加 糖尿病患者血浆中的非活性肾素与 酸不稳定肾素抑制剂的浓度增加。 进一步 评估肾素抑制剂的生理作用，我们将确定是否急性 从血浆降低提取的肾素抑制剂的长期给药 在肾素依赖性高血压模型中的血压。 识别自然存在的肾素抑制剂将增加一个新的 当前对肾素 - 醛固酮系统的理解的维度。