Omoto E, Tavassoli M
University of Mississippi Medical Center, Jackson.
Arch Biochem Biophys. 1990 Oct;282(1):34-8. doi: 10.1016/0003-9861(90)90083-b.
Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.
铜蓝蛋白（CP）是一种循环糖蛋白，以其铜转运功能而闻名。最近，其活性谱已扩大到包括多种酶功能。CP与肝内皮细胞结合，并通过一种涉及受体介导的内吞作用的机制穿过细胞。为了分离CP受体，我们获得了大鼠肝内皮细胞的纯化制剂。然后通过超速离心分离膜，并在Triton X-100中溶解。膜蛋白用125I标记，并通过一个亲和柱，其中CP与琼脂糖4B共价连接。大部分放射性在前5天用缓冲液洗脱。当不再用缓冲液洗脱放射性时，用过量的冷CP或1 M NaCl进行竞争性洗脱。通过这种技术，获得了一个尖锐的单一放射性峰，并在还原和非还原条件下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。在这两种条件下，受体均表现为一条单一的带，Mr为35000，含3%的碳水化合物，等电点为5.2。
