STIMULATION AND DISORDER OF PHOSPHATIDYLCHOLINE METABOLISM CELL GROWTH

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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.