STIMULATION AND DISORDER OF PHOSPHATIDYLCHOLINE METABOLISM CELL GROWTH

磷脂酰胆碱与细胞的信号转导和生长密切相关。我们感兴趣的是磷脂酰胆碱代谢在细胞增殖中的作用。本研究对磷脂酰胆碱代谢中磷脂酶基因的纯化和克隆进行了研究。主要结果如下：磷脂酶D被认为在细胞生长信号通路中起核心作用，通过柱层析从猪肝微粒体中纯化了2,200倍的磷脂酶D。经十二烷基硫酸钠凝胶电泳法测得其相对分子质量为19万。作用于溶血磷脂酰胆碱的溶血磷脂酶是从大鼠肝脏和猪胃黏膜中纯化得到的。从肝脏中分离到两种亚型，其分子质量分别为24和60kd。后一种酶不仅具有水解酶活性，还具有转酰基酶活性。从胃粘膜中还分离到两种溶血磷脂酶，其相对分子质量分别为22和23kd。抗肝24kd和胃22kd和23kd酶的抗体。Western印迹分析表明，肝24-kd酶与胃22-kd酶免疫相关，而与肝60-kd和胃23-kd溶血磷脂酶无关。磷脂酸和溶血磷脂酸对这些溶血磷脂酶均有不同程度的抑制作用。用该抗体从Lambda GTL-1肝脏cDNA文库中获得了肝脏24-kd溶血磷脂酶的cDNA克隆。该克隆含有与纯化的肝脏24-kd溶血磷脂酶的部分氨基酸序列相对应的核苷酸序列。

Phosphatidylcholine is profoundly involved in the signal transduction and growth of cells. We are interested in the roles of phosphatidylcholine metabolism in cell proliferation. The presen study dealt with the purification, and cloning of cDNA of phospholipases in hosphatidylcholine metabolism. The major results can be summarized as follows.Phospholipase D,believed to play a central role in the cell growth signaling pathway, was purified 2,200-fold to homogeneity from pig liver microsomes by a combination of column chromatographies. The molecular weight was determined to be 190,000 by SDS electrophoresis. Lysophospholipases acting on lysophosphatidylcholine were purified to homogeneity from rat liver and pig gastric mucosa. From liver, two isoforms with molecular masses of 24 and 60 kd were obtained. The latter enzyme exhibited not only hydrolase activity, but also transacylase activity. From gastric mucosa, two lysophospholipases were also obtained with molecular masses of 22 and 23 kd. Antibodies were raised against hepatic 24-kd and gastric 22- and 23-kd enzymes. Western blot analysis showed that the liver 24-kd enzyme was immunologically related to gastric 22-kd enzyme, but not to liver 60-kd and gastric 23-kd lysophospholipases. All these lysophospholipases were inhibited by phosphatidic acid and lysophosphatidic acid to varying extents. We obtained cDNA clone for hepatic 24-kd lysophospholipase from lambda gtl 1 liver cDNA library using the antibody. The cDNA clone contained the nucleotide sequence corresponding to the partial amino acid sequences determined for the purified hepatic 24-kd lysophospholipase.