Angiotensin-converting enzyme of the human small intestine. Subunit and quaternary structure, biosynthesis and membrane association.
Angiotensin-converting enzyme (ACE) was isolated from detergent-derived extracts of human intestinal brush-border membranes (BBMs) by immunoprecipitation using a monoclonal antibody. Analysis of the immunoprecipitates by SDS/PAGE revealed a polypeptide of apparent M(r) 184,000 under reducing and non-reducing conditions, indicating that ACE does not contain intermolecular disulphide bridges. The quaternary structure of ACE was examined using cross-linking experiments with dithiobis[succinimidylpropionate] (DSP) and density gradient centrifugation on sucrose gradients. Both approaches demonstrated that ACE is assembled in the membrane as a monomer. By contrast, the control glycoprotein aminopeptidase N (ApN) exists as a dimer. Biosynthetic labelling experiments in intestinal tissue explants demonstrated that the 184,000-M(r) protein is generated from a single-polypeptide, mannose-rich precursor of ACE (M(r) 175,000) by modification of the carbohydrate side-chains in the Golgi apparatus. The mode of association of the mature form of the enzyme with BBMs was investigated by hydrophobic labelling of right-side-out brush-border vesicles with the photoactivatable carbene-generating reagent 125I-labelled 3-(trifluoromethyl)-3-(m[formylamino]phenyl)diazirine (125I-labelled TID), followed by treatment with trypsin at dilutions that do not cause substantial degradation of ACE. These studies demonstrated that ACE is associated with the membrane via a hydrophobic segment. Furthermore, treatment of 35S-labelled inside-out membrane vesicles with trypsin revealed that ACE possesses a cytoplasmic tail, and therefore has a transmembraneous orientation.
Naim
H Y
HY
Institute of Microbiology, Heinrich-Heine University of Düsseldorf, Germany.
D002462:Cell Membrane / Q000201:enzymology; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology*; D046911:Macromolecular Substances; D008871:Microvilli / Q000201:enzymology; D009924:Organ Culture Techniques; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D013388:Succinimides; D007703:Peptidyl-Dipeptidase A; C011240:dithiobis(succinimidylpropionate)