Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them.
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 NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-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 125I-labeled glycopeptides (Ki of 1.5 X 10(-6) M) and for the uptake of secretion acid hydrolases (Ki of 2.2 X 10(-6) M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 X 10(-5) M) compares closely to that previously determined for the pinocytosis of intact "high-uptake" acid hydrolases (3-6 X 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.
D000643:Ammonium Chloride / Q000494:pharmacology; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006020:Glycopeptides / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008360:Mannosephosphates / Q000494:pharmacology; D010873:Pinocytosis; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear; D013661:Tay-Sachs Disease / Q000378:metabolism / Q000473:pathology; D001619:beta-N-Acetylhexosaminidases
D006020:Glycopeptides; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C034304:cation-dependent mannose-6-phosphate receptor; D000643:Ammonium Chloride; D003208:Concanavalin A; C027693:mannose-6-phosphate; D006867:Hydrolases; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases