Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.
A receptor that binds the phosphomannosyl recognition marker of bovine testicular beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was isolated from bovine liver membranes. The receptor was extracted from crude plasma membrane preparations with Triton X-100 and immunoprecipitated as a receptor--beta-galactosidase complex with anti-beta-galactosidase. The receptor was dissociated from the precipitate with mannose 6-phosphate, labeled with 125I, and purified on a beta-galactosidase-Sepharose 4B affinity matrix. A quantitative binding assay employing anti-beta-galactosidase and IgGsorb (formalin-fixed Staphylococcus aureus) was devised to study the binding of 125I-labeled receptor to beta-galactosidase. Maximal binding of receptor to enzyme occurred at pH values between 5.7 and 6.5. Divalent cations were not required for binding. The values of the dissociation constant obtained for beta-galactosidase varied between 200 nM observed with "lower uptake" forms and 20 nM for "higher uptake" forms of the enzyme. A number of phosphorylated monosaccharides were tested as inhibitors of binding of enzyme to receptor; mannose 6-phosphate and fructose 1-phosphate served as inhibitors and exhibited Ki values of 0.064 mM and 0.24 mM, respectively. The receptor has a subunit molecular weight of 215,000. Similar receptors were also demonstrated in Triton X-100 extracts of human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. These cell types are known to assimilate lysosomal enzymes containing covalently bound mannose 6-phosphate residues.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D005696:Galactosidases / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013737:Testis / Q000201:enzymology; D001616:beta-Galactosidase / Q000378:metabolism*
D006023:Glycoproteins; D007527:Isoenzymes; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D005696:Galactosidases; D001616:beta-Galactosidase