Purification and cloning of brain isoform of ADP-ribosyl cyclase

ADP-核糖基环化酶脑亚型的纯化和克隆

• 批准号: 09480221
• 负责人: HIGASHIDA Haruhiro
• 金额: $ 6.4万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480221/
• 关键词: ADP-ribose; cyclic ADP ribose; cyclase; heart; ryanodine receptor; NAD; ADP環状化酵素; 細胞膜

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [3H]cADP-ribose formation from [3H]NAD+ in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 mM increased ADP-ribosyl cyclase activity by 1.7-fold in venticular muscle ; This increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 mM GTP-g-S, while 10 mM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from b-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells, and suggest that increased cADP-ribose synthesis is involved in upregulation of cardiac function by sympathetic stimulation.

我们研究了环ADP-核糖（cADP-核糖）作为第二信使下游的肾上腺素能受体在心脏交感神经元兴奋后的作用。为了解决这个问题，ADP-核糖基环化酶活性被测量为在大鼠心室肌细胞的粗膜部分中从[3 H]NAD+形成[3 H] cADP-核糖的速率。1 mM的异丙肾上腺素使心室肌中ADP-核糖基环化酶活性增加1.7倍;普萘洛尔可抑制这种增加。10 nM GTP和10 mM GTP-g-S模拟了对环化酶的刺激作用，而10 mM GTP抑制环化酶。霍乱毒素阻断环化酶的激活异丙肾上腺素和GTP。异丙肾上腺素和GTP在心室膜中的上述作用通过环GDP-核糖形成荧光测定法证实。这些结果表明，在心室肌细胞中存在从β-肾上腺素能受体到膜结合ADP-核糖基环化酶的信号通路，并表明增加cADP-核糖合成参与交感神经刺激上调心脏功能。

项目成果

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Ishizaka,N.et al.：“人类虹膜中的毒蕈碱乙酰胆碱受体亚型。”

Higashida, Haruhiro, Zhang, Jia-Sheng and Takeda Yoshiyu: "Angiotensin II cyclac ADP-ribose formation in rat cardiac myocytes."Biochem. J.. (Submitted).

Higashida、Haruhiro、Zhang、Jia-Sheng 和 Takeda Yoshiyu：“大鼠心肌细胞中血管紧张素 II 环酸 ADP-核糖的形成。”Biochem。

Higashida, H. et al.: "Slow Synaptic Responses Modulation"Slow synaptic responses in neuronal tumor cells : dual regulation of ADP-ribosyl cyclase and inhibition of M-current by muscarinic receptor stimulation. 35-41 (1999)

Higashida，H.等人：“慢速突触反应调节”神经元肿瘤细胞中的慢速突触反应：ADP-核糖基环化酶的双重调节和毒蕈碱受体刺激对M-电流的抑制。

Takanaga, Hitomi, Maeda, Hiroshi, Yabuchi, Hikaru, Tamai, Ikumi, Higashida, Haruhiro and Tsuji, Akira: "Nicotinic acid transport mediated by pH-dependent anion antiporter and protein cotransporter in rabbit intestinal brush-border membrane."J. Pharm. Phar

Takanaga，Hitomi，Maeda，Hiroshi，Yabuchi，Hikaru，Tamai，Ikumi，Higashida，Haruhiro 和 Tsuji，Akira：“兔肠刷状缘膜中 pH 依赖性阴离子逆向转运蛋白和蛋白质协同转运蛋白介导的烟酸转运。”J。

Taketo, Megumi, Yokoyama, Shigeru, Fukuda, Mitsunori, Mikoshiba, Katsuhiko and Higashida, Haruhiro: "Inositol-1,3,4,5-tetrakisphosphate binding sites in control and ras-transformed NIH/3T3 fibroblasts."Biochem. Biophys. Res. Commun.. 239. 349-352 (1997)

Taketo、Megumi、Yokoyama、Shigeru、Fukuda、Mitsunori、Mikoshiba、Katsuhiko 和 Higashida、Haruhiro：“对照和 ras 转化的 NIH/3T3 成纤维细胞中的肌醇-1,3,4,5-四磷酸结合位点。”Biochem。