ISOLATION OF HEPATOCYTE PLASMA MEMBRANE PROTEINS FROM NORMAL & NEOPLASTIC CELLS

我们以前的研究分离的糖蛋白， 正常和肿瘤大鼠肝脏的质膜，使用两种- 二维凝胶电泳（2D-PAGE）揭示了许多 质量和数量上的差异。 的主要目标 这个项目是分离、纯化和结构表征 其表达显著改变的特异性糖蛋白 在化学诱导的肝癌发生过程中， 理解它们在疾病中作为标志物或致病因子的作用， 细胞转化的过程。 到目前为止取得的成果是， 以下：已尝试分离特定的 糖蛋白（分子量，200 KD; pI 5.8）， 在正常大鼠肝脏的细胞转化过程中下调 通过灌注、均质化、超离心和简短的 超声波处理 糖蛋白进一步纯化， 伴刀豆球蛋白-A（ConA）亲和层析，然后凝胶 使用Superose-12柱过滤，用Tris缓冲液洗脱 含有6 M胍。 在通过透析进行透析后， 溶液对1.0 M乙酸，随后的纯化具有 通过离子交换色谱法，使用Mono S 用1.0 M氯化钠线性梯度洗脱的色谱柱 （NaCl）在乙酸铵缓冲液（pH 5.0）中的溶液。 二维 凝胶电泳已被用于监测的进展， 在每个步骤中纯化糖蛋白。

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.