Biochemical analysis of the movement of a major lysosomal membrane glycoprotein in the endocytic membrane system.
HRP-anti LGP107Fab' and 125I-anti LGP107IgG were used as probes to study the movement of LGP107 in the endocytic membrane transport system in primary cultured hepatocytes of rats. Following the addition of HRP-anti LGP107Fab' to the culture medium, the transfer of the antibody conjugate from the cell surface of lysosomes was examined by cell fractionation on Percoll density gradients. The HRP tracer showed a bimodal subcellular distribution, in plasma membrane and lysosomal fractions. The amount of HRP found in the lysosomal fractions became larger as the period of cell incubation was increased. The rate of HRP accumulation in lysosomes was 0.13% of the administered load per hour per 10(6) cells. When cells were given 125I-anti LGP107 IgG, the antibody was not stored but was rapidly degraded in the lysosomes. The uptake of 125I-IgG by the cells, which was assessed by measuring the TCA-soluble radiolabeled degradation products released into the medium, increased proportionally to the administered concentration of the antibody and to the incubation time. The rate of uptake of the polyvalent 125I-IgG was comparable to that for the uptake of the monovalent HRP-Fab', and remained unchanged even after long exposure of the cells to a saturating concentration of the polyvalent IgG. This uptake process continued for many hours in the cells exposed to the protein synthesis inhibitor, cycloheximide. These results suggest that there is a continuous circulation of LGP107 between the cell surface and lysosomes in hepatocytes.
Furuno
K
K
Faculty of Pharmaceutical Sciences, Kyshu University, Fukuoka.
Yano
S
S
Akasaki
K
K
Tanaka
Y
Y
Yamaguchi
Y
Y
Tsuji
H
H
Himeno
M
M
Kato
K
K
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D006735:Horseradish Peroxidase; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D012795:Sialoglycoproteins; D006735:Horseradish Peroxidase