Graeff R M, Walseth T F, Fryxell K, Branton W D, Lee H C
Department of Physiology, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1994 Dec 2;269(48):30260-7.
Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.
环核苷酸如cAMP和cGMP是介导各种信号通路的第二信使。环ADP - 核糖（cADPR）是该家族最近发现的成员，由NAD +衍生而来，是各种细胞系统中Ca2 +动员的介质。cADPR的合成和降解分别由ADP - 核糖基环化酶和cADPR水解酶催化。CD38是B淋巴细胞的分化抗原，最近已被证明是一种双功能酶，催化cADPR的形成和水解。CD38催化的总体反应是由NAD +形成ADP - 核糖和烟酰胺，这与NAD酶催化的反应相同。检测cADPR形成的困难导致CD38经常被鉴定为经典的NAD酶。在本研究中，我们表明ADP - 核糖基环化酶和CD38，而不是NAD酶，可以使烟酰胺鸟嘌呤二核苷酸（NGD +）环化产生一种新的核苷酸。高效液相色谱和质谱分析表明，产物是环鸟苷二磷酸核糖（cGDPR），其结构与cADPR相似，只是鸟嘌呤取代了腺嘌呤。与cADPR相比，cGDPR是一种更稳定的化合物，对热诱导水解的抗性高2.8倍。这些结果与CD38的催化机制一致，即底物的环化先于水解反应。光谱分析表明，cGDPR具有荧光性，其吸收光谱与NGD +和GDP - 核糖均不同，这为监测其酶促形成提供了一种非常方便的方法。发现使用NGD +作为底物测定环化反应适用于纯酶以及粗组织提取物，使其成为区分CD38样酶和降解性NAD酶的有用诊断工具。
