CHARACTERIZATION OF A NOVEL, HIGH-MOLECULAR-WEIGHT, ACIDIC, ENDOTHELIN-1 INACTIVATING METALLOENDOPEPTIDASE FROM THE RAT-KIDNEY

Objective: To characterize endothelin-1 inactivating peptidase (ET-1 p eptidase) recently isolated from rat kidney. Methods: ET-1 peptidase w as purified from the membranes of whole Wistar-Kyoto (WKY) rat kidneys using differential centrifugation, detergent solubilization, ion-exch ange chromatography, ultrafiltration and preparative electrophoresis. The enzyme activity in the presence of increasing concentrations of un labelled peptides, inhibitors and other substances was determined at p H 5.5 and 37 degrees C using fixed amounts of [(125)l]-ET-1 as the sub strate. Results: On non-denaturing gels, the purified enzyme migrated in the form of a compact, low-mobility (R(f) 0.07), high relative mole cular mass (approximately 250000) protein band. During denaturing poly acrylamide gel electrophoresis this protein separated into three fract ions with apparent relative molecular masses 158000, 110000 and 61000. Using different buffers, the optimum pH for this enzyme was found to be 5.5. Zinc (3.7mmol/l), nickel (4.0mmol/l), citrate (0.6mmol/l), pho sphate (1.3 mmol/l) and barbital ions (2.5 mmol/l) inhibited ET-1 pept idase activity by 50%, whereas magnesium, calcium, cobalt, manganous, sodium and berate ions were without effect. The most powerful inhibito rs of the enzyme included: phenanthroline [median inhibitory concentra tion (IC50) 28 mu mol/l], phosphoramidon (IC50 8.0nmol/l), thiorphan ( IC50 32nmol/l) and N-carboxymethyl-Phe-teu (IC50 12 mu mol/l). Also, b acitracin (25 mu mol/l), cyclosporine A (20 mu mol/l) and sodium dodec yl sulphate (0.5%) inhibited enzyme activity by 50%, whereas bestatin, puromycin, aprotinin, phenylmethylsulphonyl fluoride, amanitin (50-10 0 mu mol/l) and cardiotoxin (25 mu g/assay) had no effect. The Michael is constant (K-m) values of 70 and 66 nmol/l were found towards ET-1 a nd the ET(16-21) fragment, respectively, whereas the K-m values in res pect to big-ET-1, sarafotoxin S6b, sulphated cholecystokin octapeptide , gastrin, glucagon, insulin, gastric inhibitory peptide and growth ho rmone ranged from 1.5 to approximately 50 mu mol/l. The enzyme showed no apparent affinity for enkephalins, bradykinin, angiotensins, cholec ystokinin tetrapeptides and kyotorphin. Conclusions: The present data suggest that the ET-1 peptidase that we isolated from rat kidney displ ays inhibitory characteristics similar to that of other known metalloe ndopeptidases. 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 an d the ET(16-21) fragment compared with other peptides either related o r unrelated to endothelin.