Mechanism of cyclic ADP-ribose hydrolysis in mammalian cells.

1.我们从人胰岛素瘤中分离出CD38基因，并在COS-7细胞中表达。CD38是一种人类白细胞抗原。当质膜与NAD~+组分或环状ADP-核糖(CADPR)孵育后，用高效液相色谱(HPLC)分析反应产物，检测cADPR(ADP-核糖环化酶和cADPR水解酶活性)的形成和水解。此外，我们还发现β细胞在葡萄糖代谢过程中产生的2-10 mM的ATP抑制了cADPR的水解活性，导致cADPR的积聚。2.从朗格汉斯胰岛中提取大鼠CD38基因，并测定其一级结构。大鼠CD38由303个氨基酸组成，与人CD38有89%的同源性。导入大鼠CD38表达载体的COS-7细胞膜部分具有ADP-核糖环化酶和cADPR水解酶活性。大鼠CD38mRNA在包括胰岛在内的各种组织中表达，但在RINm5F细胞中不表达。3.We p…更多的转基因小鼠在胰岛β细胞中高表达人CD38。转基因胰岛中CD38的酶活性显著提高，而ATP有效地抑制了cADPR水解酶的活性。葡萄糖和异丙酮酸(和葡萄糖一样，在代谢过程中产生三磷酸腺苷)诱导而不是甲苯丁胺或KCI诱导的转基因胰岛的胰岛素分泌是对照的1.7-2.3倍。在糖耐量试验中，转基因小鼠血清胰岛素水平高于对照。因此，cADPR-CD38信号系统在葡萄糖诱导的胰岛β细胞胰岛素分泌中具有调节作用，这表明不仅细胞外来源的Ca^lt；2+gt；通过葡萄糖诱导的细胞膜去极化引起的电压依赖性钙通道内流，而且细胞内储存的Ca^lt；2+&gt；也释放。4.我们对CD38进行了定点突变，发现C119K-和/或C202E-CD38只表现出ADP-核糖环化酶活性。此外，我们引入突变K95C和E176C的海兔ADP-核糖环化酶不仅具有ADP-核糖环化酶活性，而且具有cADPR水解酶活性。5.我们分离了人CD38基因，并对其结构进行了测定。CD38基因由8个外显子组成，在人类基因组上延伸-100kbp，并通过荧光原位杂交定位于人类染色体4p15。较少

1.We isolated a cDNA for CD38, which has been reported to be a human leukocyte atigen, from a human insulinoma and expressed the cDNA in COS-7 cells. When we incubated the plasma membrane with fraction NAD^+ or cyclic ADP-ribose (cADPR) and analyzed the reaction products by HPLC,the formation and hydrolysis of cADPR (ADP-ribosyl cyclase and cADPR hydrolase activities) were detected. Moreover, we found that ATP (2-10 mM), generated in the glucose metabolism in beta-cells, inhibited the cADPR-hydrolyzing activity, resulting in the accumulation of cADPR.2.We isolated rat CD38 cDNA from islets of Langerhans and determined its primary structure. Rat CD38 is composed of 303 amino acids and shares 89% homology with human CD38. The membrane fraction of the COS-7 cells into which rat CD38 expression vector had been introduced exhibited both ADP-ribosyl cyclase and cADPR hydrolase activities. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.3.We p … More roduced transgenic mice overexpressing human CD38 in pancreatic beta cells. The enzymatic activity of CD38 in transgenic islets was greatly increased, and ATP efficiently inhibited the cADPR hydrolase activity. Glucose- and ketoisocaproate (which, like glucose, generates ATP during the metabolism) -induced but not tolbutamide- nor KCI-induced insulin secretion from transgenic islets were 1.7-2.3-fold higher than that of control. In glucose-tolerance tests, the transgenic serum insulin level was higher than that of control. Thus, the cADPR-CD38 signaling system has a regulatory role in insulin secretion by glucose in beta-cells, suggesting that not only Ca^<2+> from extracellular sources (Ca^<2+> influx through voltage-dependent Ca^<2+> channels evoked by glucose-induced cell membrane depolarization) but also Ca^<2+> release from intracellular stores (cADPR-induced Ca^<2+> release from the endoplasmic reticulum) play important roles in insulin secretion.4.We introduced site-directed mutations to CD38 and found that C119K- and/or C202E-CD38 exhibited only ADP-ribosyl cyclase activity. Furthermore, Aplysia ADP-ribosyl cyclase into which we introduced the mutations K95C and E176C,which correspond to residues 119 and 201 of human CD38, exhibited not only ADP-ribosyl cyclase activity but also cADPR hydrolase.5.We isolated human CD38 gene and determined its structure. The CD38 is consists of 8 exons that extending -100 kbp on the human genome, and is mapped to human chromosome 4p 15 by fluorescent in situ hybridization. Less