Uptake of alpha-D-mannosidase and beta-D-glucosidase from Dictyostelium discoideum via the phosphohexosyl receptor on normal human fibroblasts.
alpha-D-Mannosidase and beta-D-glucosidase from Dictyostelium discoideum are efficiently endocytosed into mutant human fibroblasts through a saturable, mannose 6-phosphate (Man-6-P)-inhibitable uptake system (Freeze, H. H., Kaplan, A., and Miller, A. L. (1980) J. Biol. Chem. 255, 11081-11084). We have extended this study using both of these active, purified enzymes and 125I-labeled beta-glucosidase for uptake into normal human fibroblasts. The pH optimum of uptake is 6.0 for both enzymes and greater than 95% is inhibited by 2 mM Man-6-P (Ki = 5 X 10(-5) M). A variety of mono-and diesterified mannans or mannan derivatives also inhibited uptake of the enzymes. Both enzymes compete with each other for uptake (Ki, 2.0 X 10(-9) M) and have Kuptake of 1.0-2.2 X 10(-9) M and a Vmax of 0.35-0.48 pmol/mg of cell protein/h. The specific binding of 125I-beta-glucosidase to fibroblasts was measured at 0-4 degrees C and found to have a Kd of 1.0 X 10(-9) M with approximately 15,900 +/- 900 receptors/cell. The receptors could be internalized every 5-7 min at saturating concentrations of enzyme at 37 degrees C. The 125I-beta-glucosidase previously bound to the cells at 4 degrees C could be released by continued incubation at 4 degrees C in the presence of Man-6-P, however, after brief warming to 37 degrees C followed by reincubation at 4 degrees C, Man-6-P could no longer release the ligand. Chloroquine inhibited 95% of the uptake of 125I-beta-glucosidase at 50 microM. Following internalization of the enzyme, it is degraded to trichloroacetic acid-soluble fragments with a half-life of approximately 6.5 h. These data suggest that the slime mold enzymes are bound to the same receptors which function in the uptake of mammalian lysosomal enzymes and make the slime mold lysosomal enzymes useful models to study uptake involving this receptor in normal human fibroblasts.
D001692:Biological Transport; D004023:Dictyostelium / Q000201:enzymology*; D005260:Female; D005333:Fetus; D005347:Fibroblasts / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008361:Mannosidases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011247:Pregnancy; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043323:alpha-Mannosidase; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*
D007457:Iodine Radioisotopes; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D005959:Glucosidases; D008361:Mannosidases; D001617:beta-Glucosidase; D043323:alpha-Mannosidase