Degradation of surface-labeled hepatoma membrane polypeptides: effect of inhibitors.
When their membrane proteins were labeled with 125I by lactoperoxidase, dividing hepatoma cells lost radioactivity to the medium in a biphasic manner (T1/2 = 16-26 h, greater than 40 h). Lysosomotropic weak bases, chloroquine, and NH4Cl inhibited the rapid phase by 59%. More than 50% of the radioactivity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60-71% inhibited by lysosomotropic agents chloroquine and NH4Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH4Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-L-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by 21-24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 degrees C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lysosomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to agents which disrupt the cytoskeleton.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D007457:Iodine Radioisotopes / Q000302:isolation & purification; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013696:Temperature
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010455:Peptides; D011480:Protease Inhibitors; D000643:Ammonium Chloride; D002738:Chloroquine; D008985:Monensin