Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate
Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate
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DOI：
10.1074/jbc.273.1.118
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发表时间：
1998-01-02
影响因子：
4.8
通讯作者：
Lee, HC
中科院分区：
文献类型：
--
作者：
Graeff, RM;Franco, L;Lee, HC
环腺苷二磷酸核糖（cADPR）和烟酸腺嘌呤二核苷酸磷酸（NAADP）可通过完全独立的机制动员细胞内钙库，这与三磷酸肌醇激活的机制截然不同。虽然cADPR和NAADP在结构和功能上不同，但它们可以由具有ADP-核糖环化酶活性的单一酶合成。使用三种不同的测定来测量海胆卵匀浆中cADPR的代谢，包括放射免疫测定、Ca2+释放测定和薄层色谱测定。鉴定了可溶性和膜结合的ADP-核糖基环化酶，并且两者都环化NAD以产生cADPR。可溶性环化酶被5.3 μ M cGMP刺激一半最大，而不受cAMP的影响，而膜结合型环化酶不受cGMP的影响。两种环化酶对NAD的鸟嘌呤类似物烟酰胺鸟嘌呤二核苷酸（NGD）作为底物的pH依赖性也不同，表明它们是两种独立的酶，cGMP的刺激作用需要ATP或ATP γ S（腺苷5'-O-（3-硫代三磷酸））和cGMP依赖性激酶活性显示存在于可溶性级分中。cADPR降解为ADP-核糖由cADPR水解酶催化，发现其主要与膜相关。与膜结合环化酶类似，cADPR水解酶活性也不依赖于cGMP。可溶性组分和膜组分都能通过烟酸（NA）与NADP的烟酰胺基团交换而催化NAADP的合成，其碱交换活性与cGMP无关，所需NADP和NA的半最大浓度分别约为0.2mM和10mM。交换反应表现出的酸性pH值的偏好，与中性pH值的环化酶活性的最佳对比。本研究中表征的复杂代谢途径表明，可能存在多种调节机制来控制cADPR和NAADP的内源性浓度。
Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate, Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity, In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ release assay, and a thin layer chromatographic assay, Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR, The soluble cyclase was half-maximally stimulated by 5.3 mu M cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP, The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes, The stimulatory effect of cGMP required ATP or ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction, The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA), The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mM and 10 mM, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.