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.
Fok
A K
AK
Pacific Biomedical Research Center, University of Hawaii, Honolulu 96822.
Ueno
M S
MS
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