Fok A K, Ueno M S
Pacific Biomedical Research Center, University of Hawaii, Honolulu 96822.
Eur J Cell Biol. 1987 Dec;45(1):145-50.
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.
在最近的一份报告中，我们表明离子载体和弱碱主要抑制消化泡（DV）酸化，其次抑制溶酶体 - DV融合，但在酸化正常时对溶酶体 - DV融合没有影响。在本研究中，我们试图：1）证明通过吞噬作用摄取的异硫氰酸荧光素（FITC） - 白蛋白可用于灵敏的蛋白水解测定；2）使用该测定法确定离子载体和弱碱对蛋白水解的影响；3）了解酸化和/或溶酶体 - DV融合的抑制如何影响蛋白水解。当用FITC - 白蛋白和乳胶珠对细胞进行3分钟脉冲并进行追踪时，降解的白蛋白量在9至27分钟内呈线性增加，到30分钟时达到平台期，并被亮抑酶肽和胃蛋白酶抑制剂A抑制47%至89%。这些结果表明FITC - 白蛋白的降解发生在吞噬溶酶体中。在酸化开始前添加时，羰基氰化物对 - 三氟甲氧基苯腙（FCCP）、莫能菌素和氯化铵部分抑制溶酶体 - DV融合（25 - 50%），并强烈抑制蛋白水解64%至79%。在酸化和溶酶体 - DV融合之间添加时，融合不受影响，而蛋白水解减少40%至50%。在溶酶体 - DV融合完成后添加时，蛋白水解仍减少相同的量。0.25 mM的氯喹对蛋白水解没有影响，除非在酸化前添加，此时它抑制融合22%，抑制蛋白水解16%。这些数据与最近发表的数据一起表明：1）离子载体和弱碱首先抑制酸化，其次抑制溶酶体 - DV融合，第三抑制蛋白水解，但它们也直接抑制蛋白水解，且独立于先前步骤；2）蛋白水解抑制作用是累加的。
