Ionophores and weak bases inhibit phagolysosomal proteolysis in Paramecium.
In a recent report we showed that ionophores and weak bases inhibit digestive vacuoles (DV) acidification primarily and lysosome-DV fusion secondarily but have no effect on lysosome-DV fusion when acidification is normal. In this study we attempted 1) to show that fluorescein isothiocyanate (FITC)-albumin taken up by phagocytosis could be used for a sensitive proteolytic assay, 2) to use this assay to determine the effect of ionophores and weak bases on proteolysis and 3) to learn how an inhibition of acidification and/or lysosome-DV fusion would affect proteolysis. When cells were pulsed with FITC-albumin and latex beads for 3 min and chased, the amount of albumin degraded increased linearly from 9 to 27 min, reaching a plateau by 30 min, and was inhibited by leupeptin and pepstatin A by 47 to 89%. These results showed that the degradation of FITC-albumin occurred in the phagolysosomes. When added before acidification had commenced, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), monensin and NH4Cl partially inhibited lysosome-DV fusion (25-50%) and strongly inhibited proteolysis by 64 to 79%. Added between acidification and lysosome-DV fusion, fusion was unaffected while proteolysis was reduced by 40 to 50%. Added after lysosome-DV fusion was completed, proteolysis was still reduced by the same amount. Chloroquine at 0.25 mM had no effect on proteolysis except when added before acidification, it inhibited fusion by 22% and proteolysis by 16%. These data, together with those published recently, showed that 1) ionophores and weak bases inhibited acidification first, lysosome-DV fusion second and proteolysis third, but they also inhibited proteolysis directly and independent of the prior steps and 2) the proteolysis inhibitory effects were additive.
Pacific Biomedical Research Center, University of Hawaii, Honolulu 96822.
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Ueno
D000818:Animals; D002850:Chromatography, Gel; D004063:Digestion / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology*; D008970:Molecular Weight; D010247:Paramecium / Q000187:drug effects / Q000378:metabolism*; D010588:Phagosomes / Q000187:drug effects* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism
D005452:Fluoresceins; D007476:Ionophores; D011506:Proteins; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate