title: Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.
abstract: Receptor-binding of "high-uptake" forms of lysosomal enzymes to human diploid skin fibroblasts had been predicted from the Michaelis--Menten kinetics of uptake of these enzymes [e.g., Sando, G.N. & Neufeld, E.F. (1977) Cell 12, 619--627]. We have now demonstrated such binding directly by using a sensitive assay for the bound enzyme. Cells deficient in alpha-L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to recover, and used in suspension. They were incubated with urinary alpha-L-iduronidase at 0 degrees C for 90 minutes and then washed by centrifugation through concentrated bovine serum albumin; the activity of the cell-associated enzyme was measured with 4-methylumbelliferyl alpha-L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kd of 1 x 10(-9) M for high-uptake alpha-L-iduronidase; binding of the low-uptake form was barely detectable. Mannose 6-phosphate, a known competitive inhibitor of uptake, inhibited the binding competitively, with Ki = 1 x 10(-4) M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of alpha-L-iduronidase at 35 degrees C, the receptors were regenerated every few minutes, even in the absence of protein synthesis.
mesh_terms: D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006801:Humans; D007068:Iduronidase / Q000378:metabolism*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D010873:Pinocytosis; D011485:Protein Binding; D011955:Receptors, Drug / Q000378:metabolism; D012867:Skin
chemical_list: D011955:Receptors, Drug; D006026:Glycoside Hydrolases; D007068:Iduronidase; D002118:Calcium