Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates.
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.
Distler
J J
JJ
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
Patel
R
R
Jourdian
G W
GW
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008722:Methods; D008970:Molecular Weight; D011137:Polystyrenes; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000032:analysis*; D018160:Receptors, Cytoplasmic and Nuclear; D013737:Testis / Q000201:enzymology; D001616:beta-Galactosidase / Q000032:analysis
D011137:Polystyrenes; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C034304:cation-dependent mannose-6-phosphate receptor; D001616:beta-Galactosidase