Identification and Characterisation of a Novel Human Zinc Metalloprotease with Homology to Angiotensin Converting Enzyme

2000 ◽  
Vol 28 (3) ◽  
pp. A81-A81
Author(s):  
Sarah R. Tipnis ◽  
Nigel M. Hooper ◽  
Anthony J. Turner
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
Zinc Metalloprotease

scholarly journals Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer

1997 ◽  
Vol 327 (1) ◽  
pp. 37-43 ◽  
Author(s):  
S. PARVATHY ◽  
Sylvester Y. OPPONG ◽  
Eric H. KARRAN ◽  
Derek R. BUCKLE ◽  
Anthony J. TURNER ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Angiotensin Converting Enzyme ◽  
Lipid Bilayer ◽  
Proteolytic Processing ◽  
Full Length ◽  
Converting Enzyme ◽  
Pig Kidney ◽  
Zinc Metalloprotease ◽  
Metalloprotease Inhibitors ◽  
Triton X

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 μM, whereas TAPI-2 was slightly less potent (IC50 18 μM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2′ substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is related to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. 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 secretases. ACE secretase could be solubilized from the membrane with Triton X-100 and CHAPS, but not with n-octylβ-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.

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