Garboczi D N, Hullihen J H, Pedersen P L
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1988 Oct 25;263(30):15694-8.
The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.
大鼠肝脏ATP合酶β亚基的C端三分之二已在碱性磷酸酶（phoA）启动子和前导肽的指导下在大肠杆菌周质中过表达并输出。加工后的可溶性蛋白包含从谷氨酸122到大鼠肝脏β亚基C端丝氨酸479的358个氨基酸，包括化学和基因修饰研究预测参与核苷酸、磷酸根离子和镁离子结合的所有区域。通过Tris/EDTA/溶菌酶处理、渗透裂解和碱性pH洗涤的简单步骤，可制备纯度大于95%、产量大于20mg/升培养物的加工后的β亚基片段。它与2'（3'）-O-（2,4,6-三硝基苯基）腺苷5'-三磷酸（TNP-ATP）相互作用，TNP-ATP结合时荧光强烈增强。与TNP-ADP相互作用时也观察到类似的增强。预先添加ATP或ADP会使两种TNP-核苷酸观察到的增强显著降低，而ATP合酶抑制剂7-氯-4-硝基苯并-2-恶唑-1,3-二氮唑几乎完全阻止这种增强。TNP-ATP和TNP-ADP均以约1摩尔核苷酸/摩尔β亚基片段的化学计量比结合。在相同条件下，TNP-AMP未表现出荧光增强。这项工作表明，在不与其他ATP合酶亚基相互作用的情况下，从谷氨酸122到C端的大鼠肝脏β亚基序列表现出不超过一个易于检测的核苷酸结合结构域。成功生产出“功能性”β亚基片段对于开展聚焦于大鼠肝脏ATP合酶β亚基结构和功能的遗传学和物理学研究具有重要意义。
