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. 1985 Jul 1;229(1):213–219. doi: 10.1042/bj2290213

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

M F Dean, S Diment, C Ostlünd, B M Jenne, S Contractor
PMCID: PMC1145169  PMID: 4038256

Abstract

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Chern C. J. Detection of active heteropolymeric beta-glucuronidase in hybrids between mouse cells and human fibroblasts with beta-glucuronidase deficiency. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2948–2952. doi: 10.1073/pnas.74.7.2948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Contractor S. F., Oakey M. Use of borate buffers for purification of placental beta-glucuronidase on DEAE-cellulose. Anal Biochem. 1977 Mar;78(1):279–282. doi: 10.1016/0003-2697(77)90033-1. [DOI] [PubMed] [Google Scholar]
  4. Dean M. F., Diment S. Exchange of lysosomal enzymes between cells. Biochem Soc Trans. 1984 Jun;12(3):524–526. doi: 10.1042/bst0120524. [DOI] [PubMed] [Google Scholar]
  5. Dean M. F., Olsen I., Muir H. Identification of rabbit and mouse beta-glucuronidases in human fibroblasts following direct interaction with lymphocytes. Biochim Biophys Acta. 1982 Dec 30;721(4):441–448. doi: 10.1016/0167-4889(82)90100-8. [DOI] [PubMed] [Google Scholar]
  6. Diment S., Dean M. F. Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages. Biochim Biophys Acta. 1983 Apr 5;762(2):165–174. doi: 10.1016/0167-4889(83)90068-x. [DOI] [PubMed] [Google Scholar]
  7. Freeze H. H., Miller A. L., Kaplan A. Acid hydrolases from Dictyostelium discoideum contain phosphomannosyl recognition markers. J Biol Chem. 1980 Dec 10;255(23):11081–11084. [PubMed] [Google Scholar]
  8. Freeze H. H., Yeh R. Y., Miller A. L. Uptake of alpha-D-mannosidase and beta-D-glucosidase from Dictyostelium discoideum via the phosphohexosyl receptor on normal human fibroblasts. J Biol Chem. 1983 Jul 25;258(14):8928–8933. [PubMed] [Google Scholar]
  9. Homandberg G. A. Chemical modification of mouse beta-glucuronidase implicates lysyl, carboxyl and tyrosyl residues as catalytically essential and causes a reversible dissociation of the subunits. Biochem Biophys Res Commun. 1982 Apr 14;105(3):1109–1114. doi: 10.1016/0006-291x(82)91084-1. [DOI] [PubMed] [Google Scholar]
  10. Kaplan A., Achord D. T., Sly W. S. Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts. Proc Natl Acad Sci U S A. 1977 May;74(5):2026–2030. doi: 10.1073/pnas.74.5.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Owens J. W., Gammon K. L., Stahl P. D. Multiple forms of beta-glucuronidase in rat liver lysosomes and microsomes. Arch Biochem Biophys. 1975 Jan;166(1):258–272. doi: 10.1016/0003-9861(75)90387-2. [DOI] [PubMed] [Google Scholar]
  13. Rome L. H., Weissmann B., Neufeld E. F. Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts. Proc Natl Acad Sci U S A. 1979 May;76(5):2331–2334. doi: 10.1073/pnas.76.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stahl P. D., Rodman J. S., Miller M. J., Schlesinger P. H. Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1399–1403. doi: 10.1073/pnas.75.3.1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Stahl P., Gordon S. Expression of a mannosyl-fucosyl receptor for endocytosis on cultured primary macrophages and their hybrids. J Cell Biol. 1982 Apr;93(1):49–56. doi: 10.1083/jcb.93.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Stahl P., Schlesinger P. H., Sigardson E., Rodman J. S., Lee Y. C. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell. 1980 Jan;19(1):207–215. doi: 10.1016/0092-8674(80)90402-x. [DOI] [PubMed] [Google Scholar]
  17. Ullrich K., Mersmann G., Weber E., Von Figura K. Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety. Biochem J. 1978 Mar 15;170(3):643–650. doi: 10.1042/bj1700643. [DOI] [PMC free article] [PubMed] [Google Scholar]

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