Direct Demonstration of Binding of a Lysosomal Enzyme, α -L-Iduronidase, to Receptors on Cultured Fibroblasts

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 α -L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to recover, and used in suspension. They were incubated with urinary α -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 α -L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kdof 1 × 10-9M for high-uptake α -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 × 10-4M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of α -L-iduronidase at 35 degrees C, the receptors were regenerated every few minutes, even in the absence of protein synthesis.