title: Incorporation of mannose 6-phosphate receptors into liposomes. Receptor topography and binding of alpha-mannosidase.
abstract: A receptor that binds the lysosomal enzyme alpha-mannosidase via mannose 6-phosphate moieties (mannose 6-phosphate receptor) was purified from Swarm-rat chondrosarcoma and bovine liver microsomal membranes. Receptor-reconstituted liposomes were prepared by dialysis of taurodeoxycholate-dispersed lipids with purified mannose 6-phosphate receptor. Liposomes appeared by electron microscopy as 60-120 nm unilamellar vesicles. Receptor-reconstituted liposomes retained the ability to bind alpha-mannosidase specifically. Binding was saturable with an apparent Kd of 1 nM and was competitively inhibited by mannose 6-phosphate (Ki 2mM). Liposomes containing entrapped 125I-bovine serum albumin were used to demonstrate that treatment with 0.045% taurodeoxycholate rendered liposomes permeable to macromolecules without solubilizing the membrane. Receptor orientation in the liposome membrane was established by measuring binding of ligand to intact and detergent-treated liposomes. Unlike coated vesicles, which contain cryptic mannose 6-phosphate receptors [Campbell, Fine, Squicciarini & Rome (1983) J. Biol. Chem. 258, 2526-2533], treatment of liposomes with detergent revealed no additional cryptic binding sites. In addition, treatment of liposomes with 0.75% trypsin abolished total receptor binding activity. The results suggest that the receptor is inserted with its binding site facing the outside of the liposome.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002813:Chondrosarcoma / Q000032:analysis; D008081:Liposomes / Q000378:metabolism*; D008099:Liver / Q000032:analysis; D008360:Mannosephosphates / Q000494:pharmacology; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D051381:Rats; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D012513:Sarcoma, Experimental / Q000032:analysis; D013657:Taurodeoxycholic Acid / Q000494:pharmacology; D043323:alpha-Mannosidase
chemical_list: D008081:Liposomes; D008360:Mannosephosphates; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; C027693:mannose-6-phosphate; D013657:Taurodeoxycholic Acid; D008361:Mannosidases; D043323:alpha-Mannosidase