Development of Assay Methods for a Novel Intracellular Messenger, Cyclic ADP-ribose

人细胞表面抗原CD38是一种单跨膜型II型糖蛋白，其氨基酸序列与Aplysia ADP-核糖环化酶同源。CD38不仅催化NAD^+的水解，还催化环腺苷二磷核糖(CADPR)的形成和水解，cADPR是一种新的候选物质，可介导细胞内钙离子的释放。通过生物测定Ca~(2+)释放活性和/或柱层析测定其数量，表明不同类型细胞的细胞内cADPR含量增加。然而，就特异性和敏感性而言，这些检测方法不足以准确地确定细胞内cADPR的含量。在本研究中，我们首先建立了一种测定细胞内cADPR含量的放射免疫测定法(RIA)。本发明的放射免疫分析方法，在样品用酶处理的情况下，对cadpr表现出合理的特异性和敏感性，被应用于…的研究。CD38可能参与了细胞内cADPR的形成。1.ADP-核糖环化酶属于裂解酶而不是水解酶。2.二硫苏糖醇对CD38NAD酶活性有抑制作用，但对ADP-核糖环化酶活性无抑制作用。这些结果表明，这两种酶催化的酶反应是不同的，虽然它们都断裂了NAD^+的N-糖苷键，导致烟酰胺的释放。3.锌离子直接与CD38相互作用，使其从NAD酶转化为ADP-核糖环化酶，这可能是由于阻止了水分子进入酶-底物复合体的中间体。4.在维甲酸诱导HL-60细胞分化的过程中，细胞内cADPR的表达显著增加。5.此外，在其他白血病细胞系中也观察到高水平的细胞cADPR，其中表达CD38 mRNA。因此，最初被鉴定为NAD酶的CD38似乎负责细胞内cADPR的形成。较少

The human cell surface antigen CD38, which has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase, is a single-transmembrane type II glycoprotein. CD38 catalyzes not only the hydrolysis of NAD^+, but also the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR), which is a novel candidate that mediates Ca^<2+> release from intracellular Ca^<2+> stores. An increase in the cellular cADPR content was suggested in various types of cells by bioassays of a Ca^<2+>-releasing activity and/or the measurement of its amount on column chromatography. However, these assays are not sufficient for precise determination of the cellular cADPR content in terms of specificity and sensitivity. In the present study, we first developed a radioimmunoassay (RIA) for the measurement of the cellular cADPR content. The present RIA method, which exhibits reasonable specificity for and sensitivity to cADPR with prior treatment of the sample with enzymes, was applied to studies on the … More possible involvement of CD38 in the formation of cellular cADPR.1.Aplysia ADP-ribosyl cyclase was categorized as a lyase rather than hydrolase. 2.The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol. These results indicated that enzyme reactions catalyzed by the two enzymes were different from each other, though both cleaved the N-glycoside bond of NAD^+ resulting in the liberation of nicotinamide. 3.However, Zn^<2+> directly interacted with CD38 to convert its catalytic properties from NADase to ADP-ribosyl cyclase, probably due to prevention of the access of water molecule to an intermediate of the enzyme-substrate complex. 4.A marked increase in cellular cADPR was accompanied by retinoic acid-induced differentiation of HL-60 cells. 5.Moreover, a high level of cellular cADPR was observed in other leukemic cell lines, in which CD38 mRNA was expressed. Thus, CD38, which was initially identified as an NADase, appeared to be responsible for the formation of cellular cADPR. Less