NDLI: Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.

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Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.

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Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Weissmann, B. Rome, L. H. Neufeld, E. F.
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.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	5
Volume Number	76
Language	English
Publisher	National Academy of Sciences
Publisher Date	1979-08-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Fibroblasts Metabolism Glycoside Hydrolases Iduronidase Calcium Pharmacology Cells, Cultured Kinetics Lysosomes Enzymology Pinocytosis Protein Binding Receptors, Drug Skin Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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