Pig kidney angiotensin converting enzyme. Purification and characterization of amphipathic and hydrophilic forms of the enzyme establishes C-terminal anchorage to the plasma membrane

Abstract

Angiotensin converting enzyme from pig kidney was isolated by affinity chromatography after solubilization from the membrane by one of four different procedures. Solubilization with Triton X-100, trypsin or by an endogenous activity in microvillar membranes all generated hydrophilic forms of the enzyme as assessed by phase separation in Triton X-114 and failure to incorporate into liposomes. Only when solubilization and purification was effected by Triton X-100 in the presence of EDTA (10 mM) could an amphipathic form of the enzyme (membrane- or m-form) be generated. The m-form of angiotensin converting enzyme (ACE) appeared slightly larger (Mr approx. 180,000) than the hydrophilic forms (Mr approx. 175,000) after SDS/polyacrylamide-gel electrophoresis, and the m-form incorporated into liposomes, consistent with retention of the membrane anchor. The m-form of ACE showed an N-terminal sequence identical with that of preparations of enzyme isolated after solubilization with detergent alone (d-form), with trypsin (t-form) or by the endogenous mechanism (e-form). These data imply that ACE is anchored to the plasma membrane via its C-terminus, in contrast with the N-terminal anchorage of endopeptidase-24.11. No release of ACE from the membrane could be detected with a variety of phospholipases, including bacterial phosphatidylinositol-specific phospholipases C, although an endogenous EDTA-sensitive membrane-associated hydrolase was capable of releasing a soluble, hydrophilic, form of the enzyme.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

“…hydrophilic forms of ACE partitioned predominantly ( 96 %) into the detergent-poor phase and failed to reconstitute into liposomes, indicating that they had been cleaved in the juxtamembrane stalk region, as reported previously [29].…”

“…This was based on C-terminal microsequencing, amino acid analysis of Cterminal peptides, and immunocharacterizations with C-terminal antibodies [22]. eACE and tACE clearly lack the hydrophobic anchoring domain, as shown by their partitioning into the aqueous phase of Triton X-114, their failure to reconstitute into liposomes [29] and their lack of recognition by an antiserum raised against the hydrophobic anchoring domain (Figure 3). These properties were distinctly different from those of the amphipathic mACE, which retains the hydrophobic anchoring domain.…”

“…mACE partitioned predominantly ( 60 %) into the detergent-rich phase on phase separation in Triton X-114 and reconstituted into liposomes (results not shown), indicating that it retained the hydrophobic membrane-anchoring domain [29]. Hydrophilic forms of ACE lacking the membrane-anchoring domain were released from the membrane either by treatment with trypsin or by the action of the endogenous secretase.…”

“…Porcine somatic ACE was purified from plasma (pACE) or kidney cortex by affinity chromatography on lisinoprilSepharose as described previously [10,29]. ACE was solubilized from kidney membranes by one of three methods : (a) with 20 % (v\v) Triton X-100 (7 : 1 detergent :protein ratio) in the presence of 10 mM EDTA for 1 h at 4 mC, for isolation of the amphipathic form of ACE (mACE) ; (b) with trypsin [1 : 10 (w\w) trypsin :protein ratio] for 1 h at 37 mC, for isolation of the trypsincleaved hydrophilic form of ACE (tACE) ; or (c) by incubation of the membranes on their own at 37 mC for 1 h, which generates the endogenous secretase-cleaved hydrophilic form of ACE (eACE).…”

“…Proteins were resolved by SDS\PAGE on 7-17 % or 5-20 % polyacrylamide gradient gels and analysed by Western-blotting, as described in [29], using either a polyclonal antiserum (RP183) raised against porcine kidney ACE ectodomain [31], at a dilution of 1 : 4000, or the anti-peptide antibodies at a dilution of 1 : 1000.…”