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) 184000 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 184000-M(r) protein is generated from a single-polypeptide, mannose-rich precursor of ACE (M(r) 175000) 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 I-125-labelled 3-(trifluoromethyl)-3-(m{formylamino}phenyl)diazirine (I-125-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 S-35-labelled inside-out membrane vesicles with trypsin revealed that ACE possesses a cytoplasmic tail, and therefore has a transmembraneous orientation.