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

新型细胞内信使环状 ADP-核糖测定方法的开发

基本信息

批准号：
06557129
负责人：
KATADA Toshiaki
金额：
$ 7.94万
依托单位：
The University of Tokyo (Faculty of Pharmaceutical Sciences)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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

人细胞表面抗原CD 38是一种单跨膜的II型糖蛋白，其氨基酸序列与拟南芥ADP-核糖基环化酶同源。CD 38不仅催化NAD^+的水解，而且催化环腺苷二磷酸核糖（cADPR）的形成和水解，后者是介导细胞内Ca ^2+库释放Ca^2+的新候选物。通过Ca^2+释放活性的生物测定和/或柱色谱法测定其量，表明在各种类型的细胞中细胞cADPR含量增加。然而，这些测定在特异性和灵敏度方面不足以精确测定细胞cADPR含量。在本研究中，我们首先建立了一种放射免疫分析法（RIA）用于测量细胞cADPR含量。本RIA方法对cADPR表现出合理的特异性和灵敏度，并预先用酶处理样品， ...更多信息 CD 38可能参与了细胞cADPR的形成。1.拟南芥ADP-核糖基环化酶被归类为裂解酶而不是水解酶。2.二硫苏糖醇对CD 38 NAD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彼此不同，尽管它们都裂解NAD^+的N-糖苷键，导致烟酰胺的释放。3. Zn ~（2+）与CD_（38）直接作用，使其催化性质由NAD酶转变为ADP-核糖环化酶，可能是由于Zn ~（2+）阻止了水分子进入酶-底物复合物的中间体。4.视黄酸诱导HL-60细胞分化的同时，细胞cADPR明显升高。5.在其它表达CD 38 mRNA的白血病细胞系中，cADPR的表达水平也较高。因此，最初被鉴定为NAD酶的CD 38似乎负责细胞cADPR的形成。少