Haylett T, Thilo L
Medical Biochemistry, University of Cape Town Medical School, Observatory, South Africa.
J Biol Chem. 1991 May 5;266(13):8322-7.
Based on an initial study (Dunn, W. A., Hubbard, A. L., and Aronson, Jr., N. N. (1980) J. Biol. Chem. 255, 5971-5978), low temperature is often used to selectively inhibit fusion between endosomes and lysosomes. Here we have tried to characterize the nature of this inhibition. In addition to endocytic contents markers, we have used a covalent membrane marker to measure the interaction between endosomes and lysosomes over extended periods of time at low temperature. Mouse macrophage cells (P388D1) and human skin fibroblasts were enzymatically labeled with radioactive galactose to provide a covalent marker for plasma-membrane glycoconjugates. Subsequent endocytic membrane traffic for 24 h at 16 degrees C resulted in a significant transfer of membrane marker, as well as of endocytic contents marker, to high density lysosomes, as observed by subcellular fractionation. The kinetics of this transfer have been analyzed for macrophages using the membrane marker, horseradish peroxidase as fluid-phase, and iodinated acetyl low density lipoprotein as receptor-mediated endocytic contents marker. Transfer to lysosomes occurred only about 6 h after application of the respective marker at 16 degrees C. When transfer to lysosomes was initiated by 15 min preincubation at 37 degrees C, subsequent cooling to 16 degrees C did not inhibit ongoing transfer which continued with the same kinetics as when observed after the lag phase. These results show that low temperature delays an unidentified pre-fusion step, but does not inhibit endosome-lysosome fusion as such.
基于一项初步研究（邓恩，W.A.，哈伯德，A.L.，以及小阿隆森，N.N.（1980年）《生物化学杂志》255卷，5971 - 5978页），低温常被用于选择性抑制内体与溶酶体之间的融合。在此我们试图描述这种抑制作用的本质。除了内吞内容物标记物外，我们还使用了一种共价膜标记物来测量在低温下长时间内体与溶酶体之间的相互作用。小鼠巨噬细胞（P388D1）和人皮肤成纤维细胞用放射性半乳糖进行酶标记，以提供质膜糖缀合物的共价标记物。随后在16℃下进行24小时的内吞膜运输，通过亚细胞分级分离观察到，膜标记物以及内吞内容物标记物都有显著转移至高密度溶酶体。使用膜标记物、辣根过氧化物酶作为液相标记物以及碘化乙酰低密度脂蛋白作为受体介导的内吞内容物标记物，对巨噬细胞这种转移的动力学进行了分析。在16℃施加各自标记物后约6小时才发生向溶酶体的转移。当通过在37℃预孵育15分钟启动向溶酶体的转移时，随后冷却至16℃并不抑制正在进行的转移，转移继续进行，其动力学与滞后阶段后观察到的相同。这些结果表明，低温延迟了一个未明确的预融合步骤，但并不抑制内体 - 溶酶体融合本身。
