New Type of Particulate Guanylate Cyclase, ATP/DTT-Stimulated Guanylate Cyclase : Some Proparties and the Regulation of its Activity, and Pathophysiological Roles

新型颗粒鸟苷酸环化酶，ATP/DTT 刺激的鸟苷酸环化酶：一些 Proparties 及其活性调节和病理生理作用

• 批准号: 11670091
• 负责人: ASAKAWA Takeo
• 金额: $ 1.92万
• 依托单位: Saga Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670091/
• 关键词: quanylate cyclase; cyclic GMP; ATP; signal transduction; cyclic nucleotide; 環状ヌクリオチド

1) Novel ATP/DTT-Stimulated GC; ATP/DTT-stimulated guanylate cyclass (GC) in lung membrane was stimulated 18-fold by ATP and DTT, and both its activity and ANP-stimulated GC activity were observed to be additive. ATP/DTT-stimulated GC was solubilized by octyl glucoside (OG) to examine the mechanism of ATP/DTT-stimulation. GC in OG-extracts was stimulated maximally 2.5-fold by both ATP, ATPγS or AMPPNP, and DTT. Preincubation of OG-extracts at 10ーC with AMPPNP and DTT (Ist-preincubation) converted GC to an insensitive state to stimulation by both ATP and DTT, and this conversion was partly inhibited by a proteinphosphatase-1 inhibitor (10-1000 nM okadaic acid). On the other hand, ANP-stimulated GC was not converted to an insensitive state to ANP/ATP-stimulation by the 1st-preincubation. And, subsequent preincubation of OG-extracts at 10℃ with both DTT and, ATP or ATPγS but not AMPPNP converted GC to a state sensitive to ATP/DTT-stimulation, and this conversion was partly inhibited by in … More hibitors of Ca^<2+>/calmodulin-dependent protein kinase II (KN 62 and KN-93). In contrast, the preincubation with KN-62 and KN-93 had no effect on ANP-stimulated GC activity. The results suggested that phosphorylation was involved in the regulation of ATP/DTT-stimulated GC sensitivity to ATP/DTT-stimulation, and that ATP/DTT-stimulated GC activity was likely to be a different type from ANP-stimulated GC activity.2) Measurement of Membrane Protein; Accurate determination of low levels of protein in samples containing large amounts of interfering substances is rather difficult. In this report, we described an improved method, the DOC-TCA-washing-BCA method, which enabled us to perform rapid and efficient removal of many interfering substances, and to allow proteins to be detected above 0.05μg.3) New variant of β2 Subunit of Soluble GC (β2b) ; We isolated two distinct cDNA fragments for the β2 subunit of soluble GC (β2a and β2b) from a rat brain cDNA library. The deduced amino acid sequence of β2b is C-terminally shorter by 46 amino acids and thus does not contain a consensus sequence for isoprenylation / carboxymethylation. RT-PCR analysis demonstrated that both variants are expressed in various tissues, including kidney, liver, and brain. Less

1）新型ATP/DTT刺激GC； ATP/DTT刺激的肺膜鸟苷酸环化(GC)被ATP和DTT刺激18倍，并且观察到其活性和ANP刺激的GC活性是相加的。用辛基葡萄糖苷 (OG) 溶解 ATP/DTT 刺激的 GC，以检查 ATP/DTT 刺激的机制。 ATP、ATPγS 或 AMPPNP 和 DTT 最大刺激 OG 提取物中的 GC 2.5 倍。 OG 提取物在 10°C 下与 AMPPNP 和 DTT 预孵育（Ist 预孵育）可将 GC 转变为对 ATP 和 DTT 刺激不敏感的状态，并且这种转变被蛋白磷酸酶 1 抑制剂（10-1000 nM 冈田酸）部分抑制。另一方面，ANP刺激的GC并未通过第一次预孵育转变成对ANP/ATP刺激不敏感的状态。并且，随后在 10℃ 下将 OG 提取物与 DTT 和 ATP 或 ATPγS（而非 AMPPNP）预温育，将 GC 转化为对 ATP/DTT 刺激敏感的状态，并且这种转化受到 Ca^<2+>/钙调蛋白依赖性蛋白激酶 II（KN 62 和 KN-93）抑制剂的部分抑制。相反，用 KN-62 和 KN-93 预孵育对 ANP 刺激的 GC 活性没有影响。结果表明，磷酸化参与了ATP/DTT刺激的GC对ATP/DTT刺激的敏感性的调节，并且ATP/DTT刺激的GC活性很可能与ANP刺激的GC活性不同。2)膜蛋白的测量；准确测定含有大量干扰物质的样品中的低含量蛋白质是相当困难的。在本报告中，我们描述了一种改进的方法，即DOC-TCA-washing-BCA方法，该方法使我们能够快速有效地去除许多干扰物质，并允许检测到0.05μg以上的蛋白质。3）可溶性GC的β2亚基的新变体（β2b）；我们从大鼠脑 cDNA 文库中分离出可溶性 GC β2 亚基的两个不同 cDNA 片段（β2a 和 β2b）。 β2b 的推导氨基酸序列在 C 端短了 46 个氨基酸，因此不包含异戊二烯化/羧甲基化的共有序列。 RT-PCR 分析表明，这两种变体均在多种组织中表达，包括肾脏、肝脏和大脑。较少的

项目成果

H. Okamoto and T. Asakawa: "Molecular cloning and expression analysis of a novel splice variant of rat sGC β2 subuit."The Japanese Journal of Pharmacology. 85・Sup.I. 196 (2001)

H. Okamoto 和 T. Asakawa：“大鼠 sGC β2 亚群的新型剪接变体的分子克隆和表达分析”，《日本药理学杂志》85·Sup.I。

高野正子: "CFG-GC(膜結合型グアニル酸シクラーゼ)の活性化におけるGTP要求性について"生化学. 72・8. 992-992 (2000)

高野正子：“CFG-GC（膜结合鸟苷酸环化酶）激活的 GTP 要求”生物化学 72・8（2000）。

Shiwen Luo: "Purification and some properties of the activation of membrane-bound guanylate cyclase, SAT-GC of rat lung"The Japanese Journal of Pharmacology. 85・Sup.I. 181 (2001)

罗世文：“大鼠肺膜结合鸟苷酸环化酶 SAT-GC 的纯化和活化的一些特性”日本药理学杂志 85・Sup.I.181（2001）。

Shiwen Luo: "新しいタイプの膜結合型グアニル酸シクラーゼ,SAT-GCの可溶化部分精製と,それに伴う膜タンパク質の微量定量法"日本薬理学雑誌. 117・3. 75 (2001)

Shiwen Luo：“新型膜结合鸟苷酸环化酶 SAT-GC 的溶解部分纯化以及膜蛋白的相关微定量”《日本药理学杂志》117・3.75（2001）。

Shiwen Luo: "Purification and some properties of the activation of membrane-bound guanylate cyclase, SAT-GC of rat lung."The Japanese Journal of Pharmacology. 85・SuppI. 181-181 (2001)

罗世文：“大鼠肺膜结合鸟苷酸环化酶 SAT-GC 的纯化和一些特性。”日本药理学杂志 85·SuppI 181-181（2001）。