CHARACTERIZATION OF A SECRETASE ACTIVITY WHICH RELEASES ANGIOTENSIN-CONVERTING ENZYME FROM THE MEMBRANE

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both memb rane-bound and soluble forms. Phase separation in Triton X-114 and a c ompetitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound fo rm of ACE in pig kidney microvilli into a hydrophilic, soluble form. T his secretase activity was enriched to a similar extent as other micro villar membrane proteins, was tightly membrane-associated, being resis tant to extensive washing of the microvillar membranes with 0.5 M NaCl , and displayed a pH optimum of 8.4. The ACE secretase was not affecte d by inhibitors of serine-, thiol- or aspartic-proteases, nor by reduc ing agents or alpha2-macroglobulin. The metal chelators, EDTA and 1,10 -phenanthroline, inhibited the secretase activity, with, in the case o f EDTA. an inhibitor concentration of 2.5 mM causing 50 % inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15 % at a co ncentration of 10 mM. The inhibition of EDTA was reactivated substanti ally (83 %) by Mg2+ ions, and partially (34 % and 29 %) by Zn2+ and Mn 2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and hu man and pig intestinal brush-border membranes. The form of ACE release d from the microvillar membrane by the secretase co-migrated on SDS/PA GE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in th e post-translational proteolytic cleavage of membrane-bound ACE to gen erate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.