PURIFICATION OF ENDOTHELIN-1-INACTIVATING PEPTIDASE FROM THE RAT-KIDNEY

Objective: To identify and purify endothelin-1-inactivating peptidase from rat tissues. Methods: Subcellular fractions of rat kidney, aorta, heart, lung, liver and blood cells were prepared by differential cent rifugation. Kidney membrane-bound peptidase was solubilized with Trito n X-100, chromatographed on the diethylaminoethyl-cellu lose, ultrafil tered through a membrane of relative molecular mass 100000 cutoff and subjected to electrophoresis on a non-denaturing polyacrylamide gel. T he enzyme activity assay was performed at pH 5.5 using [I-125]-endothe lin-1 as the substrate. The trichloroacetic acid precipitation test, a n endothelin-1 immunoreactivity assay, reverse-phase high-performance liquid chromatography and a receptor-binding assay were applied for th e detection of degradation products. Results: High-activity endothelin -1-degrading peptidase coincided with the fraction from the kidney mem branes of both Wistar-Kyoto and spontaneously hypertensive rats, but n ot with any other of the tissues that were studied. The membrane (0.5 mu g protein/assay) degraded [I-125]-endothelin-1 (5-100 pmol/l) withi n a half-time of about 10 min at 37 degrees C. The enzyme was purified to an apparent homogeneity with non-denaturing gel electrophoresis, b y which it was identified as a low-mobility (R(f) 0.07) protein fracti on of high relative molecular mass (>250000). The optimum pH was 5.5, with a little activity found outside the range 5.0-7.0. The activity o f 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, i mplicating a metalloenzyme. Bestatin, puromycin, phenylmethylsulphonyl fluoride and thiorphan were without effect. Unlabelled endothelin-1 i nhibited the degradation of [I-125]-endothelin-1 (half-maximal inhibit ory concentration 100 nmol/l), whereas 100 mu mol/l methionine enkepha lin or angiotensin I did not. High-performance liquid chromatography a nalyses of the [I-125]-endothelin-1 incubated with purified peptidase revealed a time-dependent accumulation of one major radioactive fracti on that was soluble in trichloroacetic acid. This product (or products ) was not further hydrolysed. It did not react with the endothelin ant ibodies or with the specific, myocardial membrane receptors. Conclusio n: Our data suggest that the rat kidney contains an acidic metalloprot einase of high relative molecular mass that is able to hydrolyse endot helin-l rapidly and efficiently in vitro. The enzyme may participate i n the inactivation of circulating or tissue endothelins, or both.