Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors.
We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.
Messner
D J
DJ
Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Griffiths
G
G
Kornfeld
S
S
D000818:Animals; D002417:Cattle; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D006600:Hexosephosphates / Q000378:metabolism*; D007158:Immunologic Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D008099:Liver / Q000032:analysis* / Q000648:ultrastructure; D008360:Mannosephosphates / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis; D008854:Microscopy, Electron; D008970:Molecular Weight; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D006600:Hexosephosphates; D008360:Mannosephosphates; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; C027693:mannose-6-phosphate