D-Amino acid synthetic enzyme of the mammalian origin

D-天冬氨酸（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。本文报道大鼠嗜铬细胞瘤（PC）12细胞产生D-天冬氨酸。虽然细胞中D-Asp的精确合成途径尚不清楚，但微生物和低等动物中的D-Asp被认为是由Asp消旋酶产生的。本研究建立了一种新的高效液相色谱法快速、灵敏地测定天冬氨酸消旋酶的方法。我们研制了一种新型的荧光衍生试剂（BTCC），并建立了全自动高效液相色谱系统。利用该体系可以简便、灵敏地测定超嗜热古菌中的天冬氨酸消旋酶，并对D-Asp外排进行了研究。我们表征了两种外排途径：1）其中一种是自发的、持续的外排途径，需要细胞外Ca^<2+>。它不依赖于胞吐途径，因为SNARE蛋白之一显然不参与该途径。此外，电压依赖性Ca^<2+>通道的抑制剂对外排没有影响，排除了该途径中可能涉及胞吐作用。2)另一个途径依赖于细胞外渗透压。D-Asp由低渗刺激分泌。基于其对抑制剂的敏感性，推测外排是通过一种阴离子通道。适当的激动剂刺激可进一步增加低渗刺激的外排。

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.