Purification of endothelin-1-inactivating peptidase from the rat kidney

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.