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Comparative kinetics of phosphomannosyl receptor‐mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them
Author(s) -
Fischer H. David,
Creek Kim E.,
Strisciuglio Pietro,
Sly William S.
Publication year - 1983
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240220202
Subject(s) - glycopeptide , pinocytosis , concanavalin a , biochemistry , pronase , chemistry , mannose , lectin , receptor , enzyme , endocytosis , trypsin , in vitro , antibiotics
In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH 4 Cl‐induced fibroblast secretions with those of concanavalin A‐binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125 I‐labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6‐phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125 I‐labeled glycopeptides (K i of 1.5 × 10 −6 M) and for the uptake of secretion acid hydrolases (K i of 2.2 × 10 −6 M) were remarkably similar. Furthermore, the K i for mannose 6‐phosphate inhibition of pinocytosis of glycopeptide uptake (3 × 10 −5 M) compares closely to that previously determined for the pinocytosis of intact “high‐uptake” acid hydrolases (3–6 × 10 −5 M). “High‐uptake” fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A‐binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6‐phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand. The pinocytosis rates (% of added ligand internalized/mg protein/hr) of the “high‐uptake” fraction of both intact acid hydrolase (12%/mg/hr) and glycopeptide (6%/mg/hr) differed by only twofold. The apparent Kuprake for both ligands was of the same order of magnitude. The similarity in the kinetics of pinocytosis of the secreted acid hydrolases and of the phosphomannose‐bearing glycopeptides prepared from them suggests that the structural information which confers high‐affinity binding to the phosphomannosyl receptor is contained in the glycopeptide units themselves. No additional information from the intact protein backbone appears essential for phosphomannosyl receptor‐mediated pinocytosis.