Molecular mechanism of Energy conversion on the biomembrane

生物膜能量转换的分子机制

• 批准号: 06045012
• 负责人: YOSHIDA Masasuke
• 金额: $ 3.2万
• 依托单位: TOKYO Inst.Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06045012/
• 关键词: ATPase; F1-ATPase; FoF1-ATPase; ATP synthase; H^+-ATP synthase; Reconstitutioin; Single-site catalysis; Chase-promotioin; ユニサイト活定; F1-ATPase; 光親和性標識; 触媒部位; エネルギー共役; 光親和性修飾; アジドATP; アジドADP; ATP水解; 触媒カルボキシル基; 部位特異的変異; ATPase; F1-ATPase; FoF1-ATPase; ATP synthase; H^+-ATP synthase; Reconstitutioin; Single-site catalysis; Chase-promotioin; ユニサイト活定; F1-ATPase; 光親和性標識; 触媒部位; エネルギー共役; 光親和性修飾; アジドATP; アジドADP; ATP水解; 触媒カルボキシル基; 部位特異的変異

In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method to isolate homogeneous prepartion of the alpha3beta3gamma complex with 1,2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu・Tag) were linked to C-terminus of the catalytically incompetent beta (E190Q) subunit. Glu・Tag itself did not affect ATPase activity of the complexes. Two kinds of alpha3beta3gamma complexes, one containing beta (wild-type) and the other Glu・Tag-linked beta (E190Q), were mixed, urea-denatured, dialyzed, and alpha3beta3gamma complexes were reconstituted. Each of the complexes containing different number of Glu・Tag-linked beta (E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady-state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Re-scrambling of subunits between complexes could occur when the complex was aged under some condition and this might be one of the reasons of the previous contradictory result (Miwa et al. (1989) J.Biochem. (Tokyo) 106,730-734).

为了了解来自嗜热芽孢杆菌PS3的F1-ATPase的ATPase活性需要多少功能催化位点，这是一种新方法，用于分离α3Beta3bamma复合物具有1,2或3个无能的催化位点的Alpha3beta3gamma复合物的均匀制备。十个谷氨酸（GLU・TAG）与催化无能的β（E190Q）亚基的C端相关。 GLU ・TAG本身不会影响复合物的ATPase活性。将两种含有β（野生型）和另一种含有beta的betaβ连锁β（e190q）的α3Bempecteres混合，尿素化，透析，透析和alpha3beta3beta3gamma复合物。每种包含不同数量的GLU ・TAG连锁β（E190Q）的复合物通过阴离子交换色谱分离并进行了分析。结果如下。 1）正常的稳态ATPase活性需要三个完整的催化位点。 2）Chase-Acceleration是一种催化协调，至少需要两个完整的催化位点。 3）单位催化可以由单个完整的催化位点介导。当复合物在某种条件下老化时，可能会发生复合物之间的亚基，这可能是先前矛盾结果的原因之一（Miwa等人（1989）J。Biochem。（东京）106,730-734）。