Janas J, Sitkiewicz D, Warnawin K, Janas R M
Department of Clinical Biochemistry, National Institute of Cardiology, Warsaw, Poland.
J Hypertens. 1994 Oct;12(10):1155-62.
To characterize endothelin-1 inactivating peptidase (ET-1 peptidase) recently isolated from rat kidney.
ET-1 peptidase was purified from the membranes of whole Wistar-Kyoto (WKY) rat kidneys using differential centrifugation, detergent solubilization, ion-exchange chromatography, ultrafiltration and preparative electrophoresis. The enzyme activity in the presence of increasing concentrations of unlabelled peptides, inhibitors and other substances was determined at pH 5.5 and 37 degrees C using fixed amounts of [125I]-ET-1 as the substrate.
On non-denaturing gels, the purified enzyme migrated in the form of a compact, low-mobility (Rf 0.07), high relative molecular mass (approximately 250,000) protein band. During denaturing polyacrylamide gel electrophoresis this protein separated into three fractions with apparent relative molecular masses 158,000, 110,000 and 61,000. Using different buffers, the optimum pH for this enzyme was found to be 5.5. Zinc (3.7 mmol/l), nickel (4.0 mmol/l), citrate (0.6 mmol/l), phosphate (1.3 mmol/l) and barbital ions (2.5 mmol/l) inhibited ET-1 peptidase activity by 50%, whereas magnesium, calcium, cobalt, manganous, sodium and borate ions were without effect. The most powerful inhibitors of the enzyme included: phenanthroline [median inhibitory concentration (IC50) 28 mumol/l], phosphoramidon (IC50 8.0 nmol/l), thiorphan (IC50 32 nmol/l) and N-carboxymethyl-Phe-Leu (IC50 12 mumol/l). Also, bacitracin (25 mumol/l), cyclosporine A (20 mumol/l) and sodium dodecyl sulphate (0.5%) inhibited enzyme activity by 50%, whereas bestatin, puromycin, aprotinin, phenylmethylsulphonyl fluoride, amanitin (50-100 mumol/l) and cardiotoxin (25 micrograms/assay) had no effect. The Michaelis constant (Km) values of 70 and 66 nmol/l were found towards ET-1 and the ET(16-21) fragment, respectively, whereas the Km values in respect to big-ET-1, sarafotoxin S6b, sulphated cholecystokin octapeptide, gastrin, glucagon, insulin, gastric inhibitory peptide and growth hormone ranged from 1.5 to approximately 50 mumol/l. The enzyme showed no apparent affinity for enkephalins, bradykinin, angiotensins, cholecystokinin tetrapeptides and kyotorphin.
The present data suggest that the ET-1 peptidase that we isolated from rat kidney displays inhibitory characteristics similar to that of other known metalloendopeptidases. However, this enzyme exhibits several unique properties such as high molecular mass, an apparent complex subunits structure, pH optimum at 5.5, and very high substrate specificity towards ET-1 and the ET(16-21) fragment compared with other peptides either related or unrelated to endothelin.
