Incorporation of Pure Hydrolases Isolated from Brush Border Membranes in Single-Bilayer Lecithin Vesicles

Abstract

1. Both hydrolases, sucrase-isomaltase and aminopeptidase, when in their integral form interact with lecithin to form stable lipoprotein complexes. 2. In the reconstituted lipoprotein complex the hydrophobic part of the amphipathic hydrolase is incorporated in the lecithin bilayer. The total protein mass in contact with lecithin is less than 5%. 3. With sucrase-isomaltase the hydrophobic part incorporated in the lecithin bilayer belongs to the isomaltase subunit. 4. The sucrase-isomaltase is incorporated in the lecithin bilayer such that the bulk of the protein is not in contact with the lipid surface but sitting on a stalk of a minimum length of ~ 10 A. 5. The reconstituted lipoprotein/membrane system is a lecithin single-bilayer vesicle (diameter ~ 300 A) with all the enzyme incorporated being oriented towards the external aqueous phase. 6. Lecithin forms a single closed bilayer surrounding a waterfilled cavity. The properties of the lipid bilayer — molecular packing, segmental and cooperative motion, and ion permeability — are unchanged compared to the reference state of a pure lecithin bilayer vesicle. 7. No hydrolysis-related sugar transfer is measured in the recon-stituted membrane system. 8. The following properties of the reconstituted system resemble those observed in “native” brush border membranes: (1) the asymmetric incorporation of the enzyme; (2) the attachment of sucrase-isomaltase to the membrane is accomplished by the hydrophobic “anchor”, which belongs to the isomaltase subunit; (3) the susceptibility of membrane-bound hydrolases to detachment by papain treatment: the proteins released from either system are indistinguishable in their sedimentation behavior and in their electrophoretic mobility in polyacrylamide gels.