D-Amino acid synthetic enzyme of the mammalian origin

D-天冬氨酸(D-Aspartate，D-Asp)存在于哺乳动物的神经内分泌和内分泌组织中，目前已知参与激素的产生和分泌。我们已报道D-天冬氨酸是在大鼠嗜铬细胞瘤(PC)12细胞中产生的。虽然细胞中D-天冬氨酸的确切合成途径尚不清楚，但微生物和低等动物中的D-天冬氨酸可能是由天冬氨酸外消旋酶产生的。在本研究中，我们建立了一种快速、灵敏地测定天冬氨酸外消旋酶的高效液相色谱体系。我们制备了一种新型的荧光衍生化试剂(BTCC)，并开发了一套全自动高效液相色谱分析系统。利用该体系可以方便、灵敏地测定高温古生菌中的天冬氨酸外消旋酶，并对D-天冬氨酸的外流进行了表征。我们描述了两种外流途径：1)一种是自发的、持续的外流途径，需要细胞外钙离子和钙离子。它不依赖于胞吐途径，因为其中一种SNARE蛋白显然不参与这一途径。此外，电压依赖性钙通道的抑制剂对外流没有影响，排除了胞吐可能参与这一途径的可能性。2)另一种途径依赖于细胞外渗透压。D-天冬氨酸是由低血压刺激分泌的。根据其对抑制剂的敏感性，推测外流是通过一种阴离子通道进行的。适当的激动剂刺激可进一步增加低血压刺激引起的外流。

D-Aspartate (D-Asp) is found in the mammalian neuroendocrine and endocrine tissues, and in now known to be involved in hormonal production and secretion. We have reported that D-Asp is produced in rat pheochromocytoma (PC) 12 cells. Although the precise synthetic pathway remains to be known in the cells, D-Asp in the microorganisms and the lower animals is presumed to be produced by Asp racemase. In this study, we developed a new HPLC system for rapid and sensitive assay of Asp racemase. We prepared a novel fluorogenic derivatization reagent (named BTCC), and developed a fully automated HPLC system. With this system, Asp racemase in hyperthermophilc archaea was easily and sensitively assayed.The D-Asp efflux was also characterized in this study. We characterized two efflux pathways : 1) One of them is a spontaneous and continuous efflux pathway that requires extracellular Ca^<2+>. It is independent on the exocytotic pathway, since one of SNARE proteins is apparently not involved in this pathway. In addition, an inhibitor of voltage-dependent Ca^<2+> channel has no effect on the efflux, excluding the possible involvement of exocytosis in this pathway. 2) The other pathway is dependent on extracellular osmolarity. D-Asp is secreted by hyposmolarity stimuli. The efflux is presumed to be through a type of anion channel, based on its sensitivity to inhibitors. The efflux by hyposmolarity stimuli is further increased by an appropriate agonist stimulation.