title: Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.
abstract: The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D004705:Endocytosis; D000795:Fabry Disease / Q000201:enzymology* / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism; D005696:Galactosidases / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D000519:alpha-Galactosidase / Q000378:metabolism*
chemical_list: D005696:Galactosidases; D000519:alpha-Galactosidase