殺虫剤BHC分解に関与する脱塩素酵素群のグラム陰性細菌細胞内局在性に関する研究

革兰氏阴性菌降解杀虫剂六六六脱氯酶细胞内定位研究

• 批准号: 08660096
• 负责人: 永田 裕二
• 金额: $ 1.34万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660096/
• 关键词: Sphingomonas; γ-BHC; Biodegradation; periplasm; dehalogenase

グラム陰性細菌であるS.paucimobilis UT26のBHC分解に関与する酵素の局在性を検討する第一段階として,まず,同じグラム陰性細菌である大腸菌の細胞内成分の分画法に準じてUT26の細胞内成分をペリプラズム,細胞質,膜の3つの画分に分画し,それぞれの画分のBHC分解関与酵素(LinA,LinB,LinC,LinD,LinE)活性を測定し,その分布を調べた。分画の正当性は,局在性がわかっているphosphatase,glyceroaldehyde 3-phosphate dehydrogenase,pyruvate dehydrogenase等のマーカー酵素の活性を測定するなどして行った。その結果,LinA,LinB,LinDの3種の脱塩素酵素活性の多くは,ペリプラズム画分に,LinC活性は膜画分に,LinE活性は細胞質画分に見出された。さらに,精製したLinA,LinBを用いて,それぞれの抗体を作製し,LinA,LinBのタンパク自体の分布についても検討を加えた結果,両蛋白質の多くはペリプラズム画分に見出された。当初の予定であった免疫電顕法による解析には至らなかったが,LinA,LinBの多くがペリプラズムに局在していることをほぼ示すことが出来た。一方,クローン化したlinA,linB遺伝子を大腸菌に導入し,発現させた際のLinA,LinBの局在性についても検討を加えた結果,大腸菌でもUT26と同様の分布,すなわちペリプラズムへの局在化を示すことが示唆された。以上,今回の申請研究において,既知のシグナル配列を持たない脱塩素酵素LinA,LinBがペリプラズムに局在化するという現象をほぼ抑えることができた。

In the first stage of the study on the activity of enzymes related to BHC decomposition in S.paucimobilis UT26, the intracellular components of E. coli isolated from S.paucimobilis UT26 were analyzed accurately. The activities of enzymes related to BHC decomposition (LinA,LinB,LinC,LinD,LinE) in the intracellular components of UT26 were measured and their distribution was adjusted. The activity of enzymes such as phosphatase, glycoaldehyde 3-phosphate dehydrogenase,pyruvate dehydrogenase, etc. was determined. LinA,LinB and LinD were found to be active in most of the three kinds of dephosphorylase,LinC activity in membrane and LinE activity in cytoplasm. In addition, LinA and LinB were refined and used in the production of antibodies. The distribution of LinA and LinB was studied. As a result, the distribution of proteins was analyzed. In the first place, the analysis of the immune system was carried out in the middle of the analysis,LinA,LinB and many other cases. On the other hand, the LinA,LinB genes were introduced into E. coli, and LinA,LinB genes were found in the presence of E. coli. The results showed that E. coli UT26 was distributed in the same way, and all genes were present in the presence of E. coli. In the present study, we know that the enzyme LinA and LinB are involved in the process of protein synthesis.