Hooper, N. M., Keen, J., Pappin, D. J., Turner, A. J. (October 1987) Pig kidney angiotensin converting enzyme. Purification and characterization of amphipathic and hydrophilic forms of the enzyme establishes C-terminal anchorage to the plasma membrane. Biochem J, 247 (1). pp. 85-93. ISSN 0264-6021 (Print)0264-6021 (Linking)
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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Amino Acid Sequence Animals Cell Membrane/enzymology Detergents Electrophoresis, Polyacrylamide Gel Kidney/ enzymology Liposomes/metabolism Peptidyl-Dipeptidase A/isolation & purification/ metabolism Phospholipases/pharmacology Polyethylene Glycols Swine |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > angiotensin converting enzyme biotechnology > chromatography > protein purification |
| CSHL Authors: | |
| Communities: | CSHL labs > Pappin lab |
| Depositing User: | Matt Covey |
| Date: | 1 October 1987 |
| Date Deposited: | 09 Sep 2014 21:12 |
| Last Modified: | 09 Sep 2014 21:12 |
| PMCID: | PMC1148373 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30770 |
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