Purification and Some Properties of Membrane Guanylate Cyclase in Rat Brain.

大鼠脑膜鸟苷酸环化酶的纯化及其一些性质。

• 批准号: 01570109
• 负责人: ASAKAWA Takeo
• 金额: $ 1.28万
• 依托单位: Saga Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570109/
• 关键词: Guanylate cyclase; Unsaturated fatty acid; カルシウム; GTP; グアニレ-トシクラ-ゼ; サイクリックGMP; シナプス膜

1. We found a membrane bound type of guanylate cyclase activated by Ca^<2+> and unsaturated fatty acid in synaptic plasma membranes. The activation of the cyclase required co-existence of GTP or its analogues. The cyclase was tried to be purified, under retaining the properties of being activated by Ca^<2+>, GTP and unsaturated fatty acid. After several steps of chromatographies ; ion exchange, GTP-affinity, hydrophobic affinity, WGA-affinity and gel filtration, the highly purified cyclase was obtained. The finally purified enzyme was observed to be stimulated by the activators above, but in less extent than that in the membranes.2. The binding of Ca^<2+>, GTP and unsaturated fatty acid to bands in acrylamide gels from SDS-PAGE with the finally purified enzyme was examined to identify sub-units of the enzyme which participated in the activation.3. Kinetic properties of the activation by GTP, Ca^<2+> and unsaturated fatty acid were analyzed by using computer programs.4. Among several chemical compounds tested, a group of compounds was found to inhibit the cyclase selectively. This finding is useful in elucidating a physiological role of this cyclase in cell function.5. Specificity of unsaturated fatty acid in the activation was studied. Unsaturated fatty acid-peroxide was observed to be ineffective.

1.我们在突触质膜上发现了一种被Ca ^2+和不饱和脂肪酸激活的膜结合型鸟苷酸环化酶。环化酶的激活需要GTP或其类似物的共存。在保留Ca ^<2 +>、GTP和不饱和脂肪酸激活特性的前提下，对环化酶进行了纯化。经离子交换层析、GTP亲和层析、疏水亲和层析、WGA亲和层析和凝胶过滤层析，获得了高纯度的环化酶。最终纯化的酶被上述激活剂激活，但激活程度低于膜中的激活程度.用最终纯化的酶检测Ca ^<2 +>、GTP和不饱和脂肪酸与聚丙烯酰胺凝胶电泳条带的结合，以鉴定参与激活的酶亚基.利用计算机程序分析了GTP、Ca ^<2 +>和不饱和脂肪酸激活的动力学特性.在测试的几种化合物中，发现一组化合物选择性地抑制环化酶。这一发现有助于阐明这种环化酶在细胞功能中的生理作用。研究了不饱和脂肪酸在活化过程中的特异性。观察到不饱和脂肪酸-过氧化物无效。

项目成果

Masuko Takano: "Some properties of the activation of Ca^<2+> and unsaturated fatty acid-dependent guanylate cyclase in synaptic plasma membranes." Jpn. J. Pharmacol.49. 113 (1989)

Masuko Takano：“突触质膜中 Ca^2 和不饱和脂肪酸依赖性鸟苷酸环化酶激活的一些特性。”

Masako Takano: "Some properties of the activation of Ca^<2+> and unsaturated fatty acidーdependent guanylate cyclase in synaptie plasma membranes." Jpn.J.Pharmacol.suppl.49. 113 (1989)

Masako Takano：“突触质膜中Ca 2+ 和不饱和脂肪酸依赖性鸟苷酸环化酶的一些特性。Jpn.J.Pharmacol.suppl.49 (1989)。

Takeo Asakawa: "A evidence for the presence of a GTPーacting site for the activation of Ca^<2+> and unsatarated fatty acidーdependent guanylate cyclase in synaptic plasma membranes." 7th international conference on cyclic nucleotides, calcium, and protein p

Takeo Asakawa：“突触膜血浆中存在激活 Ca^2+ 和不饱和脂肪酸依赖性鸟苷酸环化酶的 GTP 作用位点的证据。” p

高野 正子: "脳シナプス膜グアニレ-トシクラ-ゼと精製酵素の性質について" 日本薬理学雑誌. 97. 19 (1991)

高野正子：“关于脑突触膜鸟苷酸环化酶和纯化酶的特性”日本药理学杂志 97. 19 (1991)。

Takeo Asakawa: "A evidence for the presence of a GTP-acting site for the actirolion of Ca^<2+> and unsaturated fatty acid-dependent guamylate cyclase in syraptic plasma membranes." 7th international conference on cyclie nucleotides,calcium,and protein pho

Takeo Asakawa：“证据表明在触动质膜中存在 Ca^2 的 Actirolion 和不饱和脂肪酸依赖性鸟苷酸环化酶的 GTP 作用位点。”