STRUCTURE AND FUNCTION OF NOVEL AMINOPEPTIDASES BELONGING TO OXYTOCINASE SUB-FAMILY

众所周知，胎盘亮氨酸氨基肽酶(P-LAP)/催产素/催产素通过抑制催产素的作用，在妊娠期间增加母体血清中的含量，维持正常妊娠。我们首次克隆了编码P-LAP的基因，证明该酶是一种II型跨膜蛋白。在本研究中，我们进一步详细分析了Eazyme的病理生理特性。我们构建了利用CHO细胞大规模生产重组P-LAP的系统，并对该酶的酶学性质进行了分析。研究发现，除了催产素和加压素外，酶还能降解甲硫氨酸脑啡肽和强啡肽A等神经肽。由于酶在神经细胞中表达，并在神经元分化过程中表达增加，因此认为P-LAP在脑神经细胞的调节中起重要作用。由于抗利尿激素加压素是P-LAP的良好底物，它调节水…肾集合管主细胞重吸收较多，肾组织P-LAP表达增加，分析了激素对细胞内囊泡膜转位的影响。用加压素处理NRK52E细胞后，发现有明显的快速易位现象。上述结果提示，肾脏存在P-LAP介导的加压素作用负反馈机制。此外，我们还对P-LAP基因的启动子功能进行了分析，发现AP-2和Ikaros两个转录因子在该基因的调控中起着重要的作用。详细的结构分析表明，P-LAP和A-LAP应亚脂地归类为氧合酶。构建大规模重组A-LAP生产系统后的酶分析表明，该酶通过失活血管紧张素11和/或肾脏中缓激肽的产生而发挥调节血压的作用。

It is well known that placental leucine aminopeptidase (P-LAP)/oxytocinase increases in matemal serum during pregnancy and maintaihs normal pregnancy by preventing the action of oxytocin. We have cloned the cDNA encoding P-LAP for the first time and shown that the enzyme is a type II membrane- spanning protein. In this study we have further analysed pathological/physiological properties of the eazyme in detail.We have constructed a large-scale production system of recombinant P-LAP employing CHO cells and analysed the enzymatic properties of the enzyme. It was found that in addition to oxytocin and vasopressin, the enayme degraded some neuronal peptides such as Met-enkephalin and dynorphin A. Since the eazyme was expressed in neuronal cells and increased in its expression during neuronal differentiation, it is considered that P-LAP plays an important role in the regulation of neuronal cells in the brain. Since antidiuretic hormone vasopressin, a good substrate for P-LAP, regulates wate … More r reabsorption in renal collecting duct principal cells and expression of P-LAP in the kidne was detected, we have analysed the effect of the hormone on the translocation of the eazyme rom intracellular vesicle to plasma membrane. It was found that after treatment of NRK52E cells with vasopressin, rapid translocation was clearly detected. These results suggested the existence of the P-LAP mediated negative feedback mechabism of the vasopressin action in the kidney. Moreover we have analysed the promoter function of P-LAP gene and found that twotranscription factors, AP-2 and ikaros, were important for the regulation of the gene.By searching a database, we have cloned a novel enzyme tenned adipocyte-derived leucine aminopeptidase (A-LAP). Detailed structural analysis indicated that P-LAP and A-LAP should be classified into oxyiocinase sub-fatnily. Enzymatic analysis after construcdon of a large-scale production system of recombinant A-LAP suggested that the enzyme played a role in the regulation of blood pressure through the inactivation of angiotensin 11 and/or the generation of bradyki' nin in the kidney., Less