Phosphatidylcholine metabolism and intracellular signal transduction

磷脂酰胆碱在细胞信号转导中起重要作用。它的代谢是由激动剂占据膜受体而诱导的，从而产生各种脂质第二信使。在本项目中，我们研究了磷脂酶D和溶血磷脂酶两种主要的磷脂酰胆碱代谢酶。磷脂酶D包括两类，脂肪酸依赖型和G蛋白依赖型。我们之前已经成功地从猪肺微粒体中高度纯化了脂肪酸依赖酶。在这里，我们利用酵母、蓖麻豆和人酶的共识合成了三种不同类型的大鼠磷脂酶D克隆，并在裂殖酵母中进行了表达。有一种酶与人类PLD1完全相同，因此被命名为rPLD1。第二种酶是通过选择性剪接产生的rPLD1的异构体。这两种酶都受到小G蛋白、Art和RhoA以及磷脂酰肌醇4，5-二磷酸的刺激。RPLD2受磷脂酰肌醇4，5-二磷酸的刺激，但不受小G蛋白的影响，溶血磷脂酶是溶血磷脂酰胆碱的水解酶，溶血磷脂酶可以调节蛋白激酶C的活性，因此被认为是细胞信号转导途径的下调因子。在此，我们从大鼠肝脏和猪胃黏膜中获得了四种酶制剂，并进行了抗体制备。利用这些抗体，我们发现至少有三种不同抗原性和组织分布的溶血磷脂酶亚型：I型低分子量型(24kd)、II型低分子量型(23kd)和III型高分子量型(60kd)具有转酰基酶活性。克隆了I型酶，并证明在HL-60早幼粒细胞向粒细胞分化过程中，I型酶在转录后被诱导。

Phosphatidylcholine plays an important role in the signal transduction of cells. Its metabolism is induced upon the occupancy of membrane receptors by agonists, resulting in the generation of various lipid second messengers. In this project, we studied two major enzymes in phosphatidylcholine metabolism, phospholipase D and lysophospholipase.Phospholipase D contains two groups, fatty acid-dependent and G-protein-dependent types. We had previously succeeded in highly purifying the fatty acid-dependent enzyme from pig lung microsomes. Here, we obtained three different types of rat phospholipase D clones using PCR probes synthesized based on the consensus among yeast, castor bean, and human enzymes, and expressed them in the fission yeast Schizosaccharomyces pombe. One enzyme was identical to human PLD1 and thus designated rPLD1. The secondr enzyme was an isoform of rPLD1 generated by alternative splicing. Both enzymes were stimulated by the small G proteins, Art and RhoA,and phosphatidylinositol 4,5-bisphosphate. The third enzyme was a new isoform encoded by a different gene and desinated rPLD2.rPLD2 was stimulated by phosphatidylinositol 4,5-bisphosphate, but was not affected by the small G proteins.Lysophospholipase hydrolyzes lysophosphatidylcholine which is known to modulate the activity of protein kinase C and thus is considered to be a down-regulator of the signal transduction pathway of cells. Here, we obtained four enzyme preparations from rat liver and pig gastric mucosa and then raised antibodies. Using the antibodies we showed that there are at least three isoforms of lysophospholipase with different antigenicity and tissue distribution : type I low molecular weight form (24kd), type II low molecular weight form (23kd), and type III high molecular weight form (60 kd) with transacylase activity. Type I enzyme was cloned and shown to be posttranscriptionally induced during differentiation of HL-60 promyelocytic cells into granulocytes.