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

被引:99
作者
OPPONG, SY [1 ]
HOOPER, NM [1 ]
机构
[1] UNIV LEEDS, DEPT BIOCHEM & MOLEC BIOL, LEEDS LS2 9JT, W YORKSHIRE, ENGLAND
关键词
D O I
10.1042/bj2920597
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant 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 affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA. an inhibitor concentration of 2.5 mM causing 50 % inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15 % at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83 %) by Mg2+ ions, and partially (34 % and 29 %) by Zn2+ and Mn2+ 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 human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.
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页码:597 / 603
页数:7
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