ATP-dependent proteolysis in mitochondria

线粒体中 ATP 依赖性蛋白水解作用

• 批准号: 13640683
• 负责人: WATABE Shoji
• 金额: $ 2.3万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640683/
• 关键词: mitochondria; protease; gene expression; adrenal cortex; proteolysis; GFP; ATP依存性プロテアーゼ; チオレドキシン; 活性酸素

We have investigated about ATP-dependent proteolytic system in mitochondria. Although there are some ATP-dependent proteases in animal mitochondria, we focused our studies on ATP-dependent LON protease. We purified the protease from bovine adrenal cortex and characterized it. In the present research we found that the enzyme was stimulated by peptides or proteins and that the stimulation was due to the stimulation of release of ADP (one of the reaction products) from the enzyme. We determined nucleotide sequence of the cDNA encoding the protease. The open reading frame of the cDNA contained 960 amino acids including presequence. To determine the precise length of the presequence we determined N-terminal amino acid sequence of the mature enzyme. We revealed that the enzyme has heterogeneous N-terminal starting from an amino acid between Trp-69 and Asn-73. To determine whether the presequence contained the information for transporting the protein into mitochondria, we expressed a fusion protein composed of the presequence and Green Fluorescent Protein (GFP) in Chinese hamster ovary cells and found that the green fluorescence was incorporated into granules which were assumed to be mitochondria.

我们对线粒体中依赖于ATP的蛋白分解系统进行了研究。虽然动物线粒体中存在一些依赖于ATP的蛋白水解酶，但我们的研究主要集中在依赖于ATP的LON蛋白。我们从牛肾上腺皮质中提纯了该酶，并对其进行了鉴定。在目前的研究中，我们发现该酶受到多肽或蛋白质的刺激，这种刺激是由于酶刺激反应产物之一ADP的释放所致。我们测定了编码该酶的基因的核苷酸序列。该基因的开放阅读框包含960个氨基酸，包括前序列。为了确定前序列的准确长度，我们测定了成熟酶的N-末端氨基酸序列。我们发现该酶具有异源N-末端，起始于Trp-69和ASN-73之间的一个氨基酸。为了确定前序列是否包含将蛋白质转运到线粒体的信息，我们在中国仓鼠卵巢细胞中表达了由前序列和绿色荧光蛋白(GFP)组成的融合蛋白，发现绿色荧光被掺入到假定为线粒体的颗粒中。

项目成果

渡部省二: "副腎皮質ミトコンドリアにおけるチオレドキシン・ペルオキシダーゼ酵素系"山口医学. 51・2. 29-38 (2002)

Shoji Watanabe：“肾上腺皮质线粒体中的硫氧还蛋白过氧化物酶系统”山口医学51・2（2002）。

Yamahama Y., Uto N., Tamotsu S., Miyata I., Yamamoto Y., Watabe S., Takahashi S. Y.: "Invivo activation of pro-form Bombix cysteine proteinase (BCP) in silkworm eggs: localization of yolk proteins and BCP, and acidification of yolk granules"J. Insect Phys

Yamahama Y.、Uto N.、Tamotsu S.、Miyata I.、Yamamoto Y.、Watabe S.、Takahashi S. Y.：“蚕卵中原型 Bombix 半胱氨酸蛋白酶 (BCP) 的体内激活：卵黄蛋白和 BCP 的定位

Yamamoto, Y., Kurata, M., Watabe, S., Murakami, R., Takahashi, S.Y.: "Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases"Current Protein and Peptide Science. 3・2. 231-238 (2002)

山本 Y.、仓田 M.、渡边 S.、村上 R.、高桥 S.Y.：“与半胱氨酸蛋白酶前区同源的新型半胱氨酸蛋白酶抑制剂”当前蛋白质和肽科学 3・2。 231-238（2002）

Watabe S., Hara M., Yamamoto M., Yamamoto,Y., Takahashi S.Y: "Activation of mitochondrial ATP-dependent protease by peptides and proteins"Tohoku J. Exp. Med.. 195. 153-161 (2001)

Watabe S.、Hara M.、Yamamoto M.、Yamamoto,Y.、Takahashi S.Y：“肽和蛋白质激活线粒体 ATP 依赖性蛋白酶”Tohoku J. Exp。

Watabe S.: "Thioredoxin peroxidase system in adrenal cortex"Yamaguchi Med. J.. 51. 29-38 (2002)

Watabe S.：“肾上腺皮质中的硫氧还蛋白过氧化物酶系统”Yamaguchi Med。