Davidson L A, McOrmond S L, Harris E D
Graduate Faculty of Nutrition, Kleberg Center, Texas A&M University, College Station 77843-2174.
Biochim Biophys Acta. 1994 Mar 10;1221(1):1-6. doi: 10.1016/0167-4889(94)90208-9.
More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.
在用(67Cu -)铜蓝蛋白短暂孵育(K562)细胞后，从细胞中回收的(67Cu)超过一半存在于细胞的颗粒部分。在Percoll中的各部分密度在(1.040)至(1.060 g/dl)之间。早在(5)分钟时，就可辨别出两个部分，密度分别为(1.051)和(1.056)。(1.051)部分的成分经检测，网格蛋白和过氧化氢酶呈阳性。(1.056)部分的成分沉淀在溶酶体标志物附近。两个部分中的(67Cu)对乙二胺四乙酸、次氮基三乙酸、(\alpha,\alpha'-联吡啶)、肝素酶和抗坏血酸的处理具有稳定性，但在用链霉蛋白酶、胰蛋白酶或十二烷基硫酸钠处理时会解离。用(67Cu -)铜蓝蛋白持续孵育会增强(1.051)和(1.056)部分中的(67Cu)活性。用(125I -)转铁蛋白孵育的细胞主要在(1.051)部分显示标记。持续孵育会增强标记，但与(67Cu)不同的是，它不会转移到较轻或较重的部分。(1.051)部分的电子显微镜照片显示，视野主要由膜结构主导，其中一些是封闭的。全细胞的显微镜照片显示，有许多内陷类似于被膜小窝，在膜表面及其下方有密封结构，这表明膜参与了相当广泛的内吞作用。这些数据提供了证据，表明来自铜蓝蛋白的大部分铜进入(K562)细胞时与膜样小泡结合，其中一部分是封闭的且被网格蛋白包被。这种颗粒途径占进入细胞的大部分铜。
