Janas J, Sitkiewicz D, Pulawska M F, Warnawin K, Janas R M
Department of Clinical Biochemistry, National Institute of Cardiology, Warsaw, Poland.
J Hypertens. 1994 Apr;12(4):375-82.
To identify and purify endothelin-1-inactivating peptidase from rat tissues.
Subcellular fractions of rat kidney, aorta, heart, lung, liver and blood cells were prepared by differential centrifugation. Kidney membrane-bound peptidase was solubilized with Triton X-100, chromatographed on the diethylaminoethyl-cellulose, ultrafiltered through a membrane of relative molecular mass 100,000 cutoff and subjected to electrophoresis on a non-denaturing polyacrylamide gel. The enzyme activity assay was performed at pH 5.5 using [125I]-endothelin-1 as the substrate. The trichloroacetic acid precipitation test, an endothelin-1 immunoreactivity assay, reverse-phase high-performance liquid chromatography and a receptor-binding assay were applied for the detection of degradation products.
High-activity endothelin-1-degrading peptidase coincided with the fraction from the kidney membranes of both Wistar-Kyoto and spontaneously hypertensive rats, but not with any other of the tissues that were studied. The membrane (0.5 microgram protein/assay) degraded [125I]-endothelin-1 (5-100 pmol/l) within a half-time of about 10 min at 37 degrees C. The enzyme was purified to an apparent homogeneity with non-denaturing gel electrophoresis, by which it was identified as a low-mobility (Rf 0.07) protein fraction of high relative molecular mass (> 250,000). The optimum pH was 5.5, with a little activity found outside the range 5.0-7.0. The activity of the peptidase was inhibited by 0.5 mmol/l 1,10 phenanthroline (half-maximal inhibitory concentration 0.03 mmol/l), and by 1 mmol/l EDTA, implicating a metalloenzyme. Bestatin, puromycin, phenylmethylsulphonyl fluoride and thiorphan were without effect. Unlabelled endothelin-1 inhibited the degradation of [125I]-endothelin-1 (half-maximal inhibitory concentration 100 nmol/l), whereas 100 mumol/l methionine enkephalin or angiotensin I did not. High-performance liquid chromatography analyses of the [125I]-endothelin-1 incubated with purified peptidase revealed a time-dependent accumulation of one major radioactive fraction that was soluble in trichloroacetic acid. This product (or products) was not further hydrolysed. It did not react with the endothelin antibodies or with the specific, myocardial membrane receptors.
Our data suggest that the rat kidney contains an acidic metalloproteinase of high relative molecular mass that is able to hydrolyse endothelin-1 rapidly and efficiently in vitro. The enzyme may participate in the inactivation of circulating or tissue endothelins, or both.
从大鼠组织中鉴定并纯化内皮素 -1 失活肽酶。
通过差速离心制备大鼠肾脏、主动脉、心脏、肺、肝脏和血细胞的亚细胞组分。用 Triton X -100 溶解肾脏膜结合肽酶，经二乙氨基乙基纤维素柱层析，通过相对分子质量截留值为 100,000 的膜超滤，并在非变性聚丙烯酰胺凝胶上进行电泳。以 [125I] -内皮素 -1 为底物，在 pH 5.5 条件下进行酶活性测定。采用三氯乙酸沉淀试验、内皮素 -1 免疫反应性测定、反相高效液相色谱法和受体结合试验检测降解产物。
高活性内皮素 -1 降解肽酶与 Wistar - Kyoto 大鼠和自发性高血压大鼠肾脏膜的组分一致，但与所研究的其他任何组织均不一致。该膜（0.5 微克蛋白/测定）在 37℃下约 10 分钟的半衰期内降解 [125I] -内皮素 -1（5 - 100 皮摩尔/升）。通过非变性凝胶电泳将该酶纯化至表观均一，鉴定其为相对分子质量高（> 250,000）、迁移率低（Rf 0.07）的蛋白质组分。最适 pH 为 5.5，在 5.0 - 7.0 范围之外活性较低。该肽酶的活性受到 0.5 毫摩尔/升 1,10 -菲咯啉（半数最大抑制浓度 0.03 毫摩尔/升）和 1 毫摩尔/升 EDTA 的抑制，提示其为金属酶。贝抑素、嘌呤霉素、苯甲基磺酰氟和硫磷酰胺无作用。未标记的内皮素 -1 抑制 [125I] -内皮素 -1 的降解（半数最大抑制浓度 100 纳摩尔/升），而 100 微摩尔/升甲硫氨酸脑啡肽或血管紧张素 I 则无此作用。对与纯化肽酶孵育的 [125I] -内皮素 -1 进行高效液相色谱分析，显示出一种主要放射性组分随时间的积累，该组分可溶于三氯乙酸。该产物（或这些产物）不再进一步水解。它不与内皮素抗体或特异性心肌膜受体反应。
我们的数据表明，大鼠肾脏含有一种相对分子质量高的酸性金属蛋白酶，该酶在体外能够快速、有效地水解内皮素 -1。该酶可能参与循环或组织内皮素的失活，或两者皆参与。
