Kanazawa H, Yabuki M, Miki J, Fudemoto T, Ikeda H, Noumi T, Shin Y
Department of Biotechnology, Faculty of Engineering Sciences, University of Okayama, Japan.
Arch Biochem Biophys. 1995 Mar 10;317(2):348-56. doi: 10.1006/abbi.1995.1174.
Five monoclonal antibodies against the alpha subunit of F1-ATPase from Escherichia coli alpha 104, alpha 105, alpha 107, alpha 109, and alpha 110 were prepared. The monoclonal antibodies alpha 104 and alpha 110 enhanced the F1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while alpha 105 did not. Both antibodies bound to a peptide corresponding to the region between residues 354 and 513. Mutations in this region which caused reduced binding of the alpha subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for alpha 104 and residues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-506 for alpha 110. These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of the alpha subunit. Among the mutations, Leu-456 to Pro and Ser-440 to Pro mutations caused increase of the F1-ATPase activity up to 1.9 and 1.2 times that of the wild-type, respectively, while Leu-471 to Pro mutation caused a defect in assembly of the F1-ATPase on the membrane. The other mutations caused no significant change in ATPase activity. These results suggested that Ser-440 and Leu-456 have an important role in regulating catalysis by the F1-ATPase, but that the neighboring residue Leu-471 has an important role in assembly of the F1-ATPase complex. It was also suggested that binding of the monoclonal antibodies alpha 104 and alpha 110 to residues Ser-440 and Leu-456 caused local conformational changes, leading to enhancing effects on F1-ATPase activity similar to the Ser-440 to Pro and Leu-456 to Pro mutations.
制备了五种针对大肠杆菌F1 - ATP酶α亚基的单克隆抗体，分别为α104、α105、α107、α109和α110。单克隆抗体α104和α110分别将F1 - ATP酶活性最大程度提高至野生型的1.6倍和1.7倍，而α105则没有。这两种抗体均与对应于354至513位残基之间区域的肽段结合。在该区域中，针对α104，位于Ser - 440、Leu - 456、Leu - 471、Leu - 482、Met - 483和Ser - 506位残基处鉴定出导致α亚基与抗体结合减少的突变；针对α110，位于Ser - 440、Leu - 456、Leu - 471、Asp - 476、Leu - 482、Met - 483和Ser - 506位残基处鉴定出此类突变。这些残基可能参与抗体的表位，且在α亚基表面彼此相邻。在这些突变中，Leu - 456突变为Pro以及Ser - 440突变为Pro分别使F1 - ATP酶活性增加至野生型的1.9倍和1.2倍，而Leu - 471突变为Pro导致F1 - ATP酶在膜上组装缺陷。其他突变未引起ATP酶活性的显著变化。这些结果表明，Ser - 440和Leu - 456在F1 - ATP酶催化调节中起重要作用，但相邻残基Leu - 471在F1 - ATP酶复合物组装中起重要作用。还表明单克隆抗体α104和α110与Ser - 440和Leu - 456残基的结合引起局部构象变化，导致对F1 - ATP酶活性产生类似于Ser - 440突变为Pro和Leu - 456突变为Pro的增强作用。
