Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates
- PMID: 2823641
- DOI: 10.1016/0003-2697(87)90546-x
Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates
Abstract
A novel sensitive binding assay for quantitation of a low-molecular-weight phosphomannosyl receptor (41-46 K) was devised. The receptor was immobilized by immunochemical means in the wells of polystyrene multiwell plates. The lysosomal enzyme ligand, testicular beta-galactosidase, was added and receptor-bound beta-galactosidase was measured by conventional colorimetric analysis using p-nitrophenyl beta-galactoside as substrate. Inhibitors of the binding of beta-galactosidase to the receptor were removed prior to addition of beta-galactosidase and did not interfere with the assay. Low-molecular-weight phosphomannosyl receptor was readily quantitated in the range of 4 to 100 ng of receptor protein. Binding of beta-galactosidase to the receptor was specifically inhibited by 5 mM mannose 6-phosphate. The receptor exhibited optimum binding of beta-galactosidase at pH 5.7 and was saturated with beta-galactosidase at 320 munits/ml in the presence of 20 mM MnCl2. The requirement for MnCl2 was greatly diminished at higher concentrations of beta-galactosidase. Application of the assay procedure to the quantitation of the low-molecular-weight phosphomannosyl receptor in mammalian tissues is discussed.
Similar articles
-
Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.Proc Natl Acad Sci U S A. 1981 Jul;78(7):4289-93. doi: 10.1073/pnas.78.7.4289. Proc Natl Acad Sci U S A. 1981. PMID: 6270668 Free PMC article.
-
Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors.Anal Biochem. 1989 Nov 1;182(2):432-7. doi: 10.1016/0003-2697(89)90619-2. Anal Biochem. 1989. PMID: 2558593
-
Characteristics of lysosomal phosphomannosyl-enzyme receptors in the rat heart.Basic Res Cardiol. 1987 May-Jun;82(3):252-60. doi: 10.1007/BF01906857. Basic Res Cardiol. 1987. PMID: 2820372
-
beta-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor.Biochem Biophys Res Commun. 1987 Mar 30;143(3):799-807. doi: 10.1016/0006-291x(87)90319-6. Biochem Biophys Res Commun. 1987. PMID: 3032184
-
Reciprocal modulation of binding of lysosomal enzymes and insulin-like growth factor-II (IGF-II) to the mannose 6-phosphate/IGF-II receptor.Adv Exp Med Biol. 1991;293:311-24. doi: 10.1007/978-1-4684-5949-4_28. Adv Exp Med Biol. 1991. PMID: 1662863 Review. No abstract available.
Cited by
-
Mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the secretory pathway.Biochim Biophys Acta. 2011 Sep;1810(9):815-26. doi: 10.1016/j.bbagen.2011.06.016. Epub 2011 Jun 24. Biochim Biophys Acta. 2011. PMID: 21723917 Free PMC article. Review.
-
Binding receptors for alpha-L-fucosidase in human B-lymphoid cell lines.Glycoconj J. 1992 Feb;9(1):56-62. doi: 10.1007/BF00731178. Glycoconj J. 1992. PMID: 1327338
-
Mannose 6-phosphate receptors and their role in targeting proteins to lysosomes.J Membr Biol. 1988 Jul;103(1):7-16. doi: 10.1007/BF01871928. J Membr Biol. 1988. PMID: 2972840 Review. No abstract available.
-
Glucosidase II and MRH-domain containing proteins in the secretory pathway.Curr Protein Pept Sci. 2015;16(1):31-48. doi: 10.2174/1389203716666150213160438. Curr Protein Pept Sci. 2015. PMID: 25692846 Free PMC article. Review.
-
Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor.J Biol Chem. 2008 Apr 11;283(15):10124-34. doi: 10.1074/jbc.M708994200. Epub 2008 Feb 13. J Biol Chem. 2008. PMID: 18272523 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
