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. 1983 Aug 15;214(2):413–419. doi: 10.1042/bj2140413

Incorporation of mannose 6-phosphate receptors into liposomes. Receptor topography and binding of alpha-mannosidase.

C H Campbell, A L Miller, L H Rome
PMCID: PMC1152262  PMID: 6311183

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.

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Selected References

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