Reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. Mode of membrane insertion and morphology

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Abstract
Pig intestinal microvillus aminopeptidase (EC 3.4.11.2) was reincorporated into lipid membranes using .beta.-octyl glucoside or sodium deoxycholate. For this enzyme the deoxycholate-dialysis method was the preferable one. By using this method, microvillus aminopeptidase was inserted almost quantitatively into 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(s) was associated with the membrane. EM observation on this model system demonstrated a dimeric symmetrical structure of aminopeptidase (dimensions about 13.5 nm .times. 5.5 nm) separated by a 5 nm gap from the membrane. This distance corresponds to a MW 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.

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