ISOLATION OF HEPATOCYTE PLASMA MEMBRANE PROTEINS FROM NORMAL & NEOPLASTIC CELLS

我们之前对从沙门氏菌中分离出的糖蛋白的研究 正常大鼠和肿瘤大鼠肝细胞质膜的研究 双向凝胶电泳(2D-PAGE)揭示了许多 差异，包括质的和量的。的主要目标是 这个项目是分离、提纯和结构特征的 表达明显改变的特异性糖蛋白 在化学诱导的肝癌发生过程中 理解它们作为标记物或因果因素在 细胞转化的过程。到目前为止取得的成果如下 如下：已尝试分离出特定的 糖蛋白(分子量200kD；等电点5.8)，它是 正常大鼠肝脏细胞转化过程中表达下调 通过灌流、匀浆、超速离心法和简化法 超音速。糖蛋白通过进一步纯化。 刀豆蛋白A(ConA)亲和层析及凝胶 用Tris缓冲液洗脱的Superose-12柱的过滤 含有6M的胍类物质。在通过透析法脱盐之后 溶液对抗1.0M醋酸，后续提纯有 通过使用单色S的离子交换层析实现 色谱柱：1.0M氯化钠线性梯度洗脱柱 (氯化钠)在醋酸铵缓冲液(pH 5.0)中。二维图 凝胶电泳法已被用来监测 每一步都对糖蛋白进行纯化。

Our previous investigations of glycoproteins isolated from the plasma membranes of normal and neoplastic rat livers using two- dimensional gel electrophoresis (2D-PAGE) have revealed many differences, both qualitative and quantitative. The main goal of this project is to isolate, purify, and structurally characterize the specific glycoproteins whose expression is markedly altered during chemically induced hepatocarcinogenesis in order to understand their role either as markers or causal agents in the process of cell transformation. Results obtained so far are as follows: Attempts have been made to isolate a specific glycoprotein (molecular weight, 200 KD; pI 5.8), which is downregulated during cell transformation, from normal rat liver by perfusion, homogenization, ultra-centrifugation, and brief sonication. The glycoprotein was further purified by Concanavalin-A (ConA) affinity chromatography and then gel filtration using a Superose-12 column eluted with Tris buffer containing 6M guanidine. Following desalting by dialysis of the solution against 1.0 M acetic acid, subsequent purification has been achieved by ion-exchange chromatography using a Mono S column eluted with a linear gradient of 1.0 M sodium chloride (NaCl) in ammonium acetate buffer (pH 5.0). Two-dimensional gel electrophoresis has been used to monitor the progress of purification of the glycoprotein at each step.