Binding of lysosomal enzymes to the mannose 6-phosphate receptor: a novel binding assay that makes use of biotinylated receptor molecules, coupled to avidin-agarose.
Binding assay procedures for receptor-ligand interactions should meet requirements such as ease of operation, reproducibility and low costs. In the case of the mannose 6-phosphate receptor (MPR) for lysosomal enzymes, the earliest assay procedure made use of a crude membrane preparation containing MPR, that was sedimented after incubation with an enzyme solution. The bound enzyme activity was determined thereafter. With purification methods of MPR (CI and CD) available, we found it of interest to compare the binding of different lysosomal enzymes with these molecular MPR preparations. We therefore developed a method in which MPR was biotinylated, followed by coupling to avidin-agarose. Very small quantities of this gel (2 microliters) appear to be needed to bind sufficient amounts of lysosomal enzyme. The bound enzyme activity can be rapidly measured with high reproducibility, by incubating the agarose spheres directly with substrate solutions. We could demonstrate that the binding properties of MPR, although biotinylated and immobilized, were not different from those obtained with crude MPR-preparations from rat liver membranes.
Overdijk
B
B
Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Van Steijn
G J
GJ
D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology*; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012685:Sepharose / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; C066402:avidin-agarose; D001360:Avidin; D001710:Biotin; D012685:Sepharose; D001616:beta-Galactosidase; D001619:beta-N-Acetylhexosaminidases