title: 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.
authors.affiliation: MRC Membrane Peptidase Research Group, University of Leeds, U.K.
authors.forename: N M
authors.initials: NM
authors.lastname: Hooper
authors.forename: J
authors.initials: J
authors.lastname: Keen
authors.forename: D J
authors.initials: DJ
authors.lastname: Pappin
authors.forename: A J
authors.initials: AJ
authors.lastname: Turner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D008081:Liposomes / Q000378:metabolism; D017830:Octoxynol; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification / Q000378:metabolism*; D010740:Phospholipases / Q000494:pharmacology; D011092:Polyethylene Glycols; D013552:Swine
chemical_list: D003902:Detergents; D008081:Liposomes; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010740:Phospholipases; D007703:Peptidyl-Dipeptidase A