Decker R S, Decker M L, Thomas V, Fuseler J W
J Cell Sci. 1985 Mar;74:119-35. doi: 10.1242/jcs.74.1.119.
Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to 'lysosomotropic' weak bases (chloroquine, methylamine, and NH4Cl) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4Cl, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.
溶酶体已用吖啶橙预先标记的心肌细胞分别暴露于L-氨基酸甲酯（L-亮氨酸或甲硫氨酸甲酯）或“溶酶体亲和性”弱碱（氯喹、甲胺和氯化铵）中1小时。两种类型的干预均使溶酶体同等程度地扩张，并不同程度地抑制蛋白水解。弱碱对吖啶橙染色无明显改变，而甲基化氨基酸则导致荧光染料从溶酶体显著重新分布到肌质中，这表明它们可能引发了溶酶体膜通透性的改变。短暂暴露于弱碱未能增强酸性蛋白酶分泌到培养基中，但明显使细胞组织蛋白酶B活性失活。相比之下，甲基化氨基酸未引起酸性蛋白酶活性或两种蛋白水解酶细胞分布的改变。最后，用荧光素葡聚糖测量发现，弱碱显著提高了溶酶体内pH值，而氨基酸甲酯处理后仅观察到适度升高。目前的观察结果表明，氨基酸甲酯代表了一类作用明显不同于氯喹和氯化铵的新试剂，它们可能为研究细胞培养中的次级溶酶体功能提供一种独特且有价值的手段。
