Reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. Mode of membrane insertion and morphology.
Pig intestinal microvillus aminopeptidase (EC 3.4.11.2) was reincorporated into lipid membranes by using either beta-octyl glucoside or sodium deoxycholate. The results showed that for this enzyme the deoxycholate-dialysis method was the preferable one. By using this method, microvillus aminopeptidase was inserted almost quantitatively into either phosphatidylcholine vesicles or microvillus-lipid vesicles. By proteolytic treatment of the vesicles, by probing the aminopeptidase with an inhibitory antibody and by monitoring the positioning of the anchor with the aid of [125I]iodonaphthyl azide, it was demonstrated that the catalytically active part was located outside the liposomes and the anchoring peptide(2) was associated with the membrane. Electron-microscopic observation on this model system demonstrated a dimeric symmetrical structure of aminopeptidase (dimensions about 13.5 nm X 5.5 nm) separated by a 5 nm gap from the membrane. This distance corresponds to a molecular weight of 2000-5000 for this junctional segment of the anchor connecting the intramembrane part of the anchor with the catalytically active main part of the aminopeptidase.
D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018826:CD13 Antigens; D002499:Centrifugation, Density Gradient; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism*; D008722:Methods; D008871:Microvilli / Q000201:enzymology; D008954:Models, Biological; D013552:Swine
D003902:Detergents; D005960:Glucosides; D008081:Liposomes; D003840:Deoxycholic Acid; C018619:octyl-beta-D-glucoside; D000626:Aminopeptidases; D018826:CD13 Antigens