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) 在交感神经元兴奋后作为心脏肾上腺素能受体下游第二信使的作用。为了解决这个问题，ADP-核糖基环化酶活性被测量为大鼠心室肌细胞粗膜部分中从[3H]NAD+形成[3H]cADP-核糖的速率。 1 mM 异丙肾上腺素可使心室肌中 ADP-核糖基环化酶活性增加 1.7 倍；这种增加被普萘洛尔抑制。 10 nM GTP 和 10 mM GTP-g-S 模拟对环化酶的刺激作用，而 10 mM GTP 抑制环化酶。霍乱毒素通过异丙肾上腺素和 GTP 阻断环化酶的激活。异丙肾上腺素和 GTP 在心室膜中的上述作用通过环状 GDP-核糖形成荧光测定法得到证实。这些结果证明心室肌细胞中存在从 β-肾上腺素能受体通过 G 蛋白到膜结合 ADP-核糖基环化酶的信号通路，并表明 cADP-核糖合成的增加参与了交感神经刺激对心脏功能的上调。

项目成果

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。

Ishizaka,N.et al.: "Muscarinic acetylcholine receptors subtypes in the human iris." Brain Res.787. 344-347 (1998)

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, Haruhiro, Taketo, Megumi, Takahashi, Hiroto, Yokoyama, Shigeru, and Hashii Minako: "Potential mechanism for bradykinin-activated and inositol tetrakisphosphate-dependent CaィイD12+ィエD1 influx by Ras and GAP1 in fibroblast cells."Immunopharmacol..

Higashida、Haruhiro、Taketo、Megumi、Takahashi、Hiroto、Yokoyama、Shigeru 和 Hashii Minako：“成纤维细胞中由 Ras 和 GAP1 引起的缓激肽激活和肌醇四磷酸依赖性 CaD12+D1 流入的潜在机制。”Immunopharmacol ..

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。