Effects of cross-linked dimers of ribonuclease A or of lysozyme on the processing of endocytosed peroxidase by hepatoma cells.
Cross-linked dimers of ribonuclease, added at a concentration of 0.05 mg/ml to the culture medium of hepatoma (HTC) cells, were previously shown to inhibit intracellular degradation of peroxidase taken up by endocytosis. Intracellular localization showed that endocytosed peroxidase does not reach lysosomes in dimer-treated cells. The present study shows that preloading of lysosomes with fluorescent anti-peroxidase IgG, obtained by exposing HTC cells for 48 h to 0.1 mg of antibody/ml, restores intracellular degradation of endocytosed peroxidase. Moreover, accumulation of peroxidase into lysosomes, which no longer occurs in dimer-treated cells, occurs again under these conditions. We conclude that inhibition of transfer of peroxidase from phagosomes to lysosomes is most likely to be the alteration resulting from the exposure of the cells to ribonuclease dimer, rather than inhibition of fusion between phagosomes and lysosomes. The dimer of another basic protein, lysozyme added  at a concentration of 0.2 mg/ml to the culture medium, is shown to induce the same type of effects as does the dimer of ribonuclease; the half-life of endocytosed peroxidase increased from 5 to 15 h after 2 h exposure of HTC cells to dimerized lysozyme. The effect of both dimers on intracellular protein processing can be reversed by addition of 100 mm-galactose to the culture medium, up to 5 h after pretreatment of the cells. The dimers of ribonuclease A or of lysozyme have thus probably the same mechanism of action. Evidence that the two dimers share the same binding sites on the cells is presented.
D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects*; D005690:Galactose / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009113:Muramidase / Q000494:pharmacology*; D009195:Peroxidase; D010544:Peroxidases / Q000378:metabolism*; D051381:Rats; D012260:Ribonucleases / Q000494:pharmacology*
D007527:Isoenzymes; D046911:Macromolecular Substances; D010544:Peroxidases; D009195:Peroxidase; D012260:Ribonucleases; D009113:Muramidase; D005690:Galactose