ANGIOTENSIN-CONVERTING ENZYME SECRETASE IS INHIBITED BY ZINC METALLOPROTEASE INHIBITORS AND REQUIRES ITS SUBSTRATE TO BE INSERTED IN A LIPID BILAYER

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of s everal proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For AC E we have previously identified a metalloprotease (secretase) responsi ble for this proteolytic cleavage. The effect of a range of structural ly related zinc metalloprotease inhibitors on the activity of the secr etase has been examined. Batimastat (BB94) was the most potent inhibit or of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 mu M, whereas TAPI-2 was slightly less potent (IC50 18 m u M). Removal of the thienothiomethyl substituent adjacent to the hydr oxamic acid moiety or the substitution of the P2' substituent decrease d the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibi tory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is related to, but distinct from, the matri x metalloproteases. The full-length amphipathic form of ACE was labell ed selectively with 3-trifluoromethyl-3-(m-[I-125]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was abl e to cleave the hydrophobic anchoring domain from the bulk of the prot ein, there was no cleavage of full-length ACE by a Triton X-100-solubi lized pig kidney secretase preparation when the substrate was in deter gent solution. In contrast, the Triton X-100-solubilized secretase pre paration released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipat hic form of ACE when it was incorporated into artificial lipid vesicle s. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein se cretases. ACE secretase could be solubilized from the membrane with Tr iton X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Fur thermore trypsin could release the secretase from the membrane, implyi ng that like its substrate, ACE, it too is a stalked integral membrane protein.