Hermolin J, Fillingame R H
Department of Physiological Chemistry, University of Wisconsin Medical School, Madison 53706.
J Biol Chem. 1989 Mar 5;264(7):3896-903.
Dicyclohexylcarbodiimide (DCCD) specifically inhibits the F1F0-H+-ATP synthase complex of Escherichia coli by covalently modifying a proteolipid subunit that is embedded in the membrane. Multiple copies of the DCCD-reactive protein, also known as subunit c, are found in the F1F0 complex. In order to determine the minimum stoichiometry of reaction, we have treated E. coli membranes with DCCD, at varying concentrations and for varying times, and correlated inhibition of ATPase activity with the degree of modification of subunit c. Subunit c was purified from the membrane, and the degree of modification was determined by two methods. In the "specific radioactivity" method, the moles of [14C]DCCD per total mole of subunit c was calculated from the radioactivity incorporated per mg of protein, and conversion of mg of protein to mol of protein based upon amino acid analysis. In the "high performance liquid chromatography (HPLC) peak area" method, the DCCD-modified subunit c was separated from unmodified subunit c on an anion exchange AX300 HPLC column, and the areas of the peaks from the chromatogram quantitated. The shape of the modification versus inhibition curve indicated that modification of a single subunit c per F0 was sufficient to abolish ATPase activity. The titration data were fit by nonlinear regression analysis to a single hit mathematical model, A = Un(1 - r) + r, where A is the relative activity, U is the ratio of unmodified/total subunit c, n is the number of subunit c per F0, and r is a residual fraction of ATPase activity that was resistant to inhibition by DCCD. The two methods gave values for n equal to 10 by the specific radioactivity method and 14 by the HPLC peak area method, and values for r of 0.28 and 0.30, respectively. Most of the r value was accounted for by the observed dissociation of 15-20% of the F1-ATPase from the membrane under ATPase assay conditions. When the minimal, experimentally justified value of r = 0.15 was used in the equation above, the calculated values of n were reduced to 8 and 11, respectively. The value of n determined here, with a probable range of uncertainty of 8-14, is consistent with, and provides an independent type of experimental support for, the suggested stoichiometry of 10 +/- 1 subunit c per F1F0, which was determined by a more precise radiolabeling method (Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015).
二环己基碳二亚胺（DCCD）通过共价修饰嵌入膜中的一种蛋白脂质亚基，特异性抑制大肠杆菌的F1F0 - H⁺ - ATP合酶复合体。在F1F0复合体中发现了多个DCCD反应性蛋白的拷贝，该蛋白也称为亚基c。为了确定反应的最小化学计量比，我们用不同浓度的DCCD处理大肠杆菌膜，并处理不同时间，将ATP酶活性的抑制与亚基c的修饰程度相关联。从膜中纯化出亚基c，并通过两种方法确定修饰程度。在“比放射性”方法中，根据每毫克蛋白质掺入的放射性以及基于氨基酸分析将蛋白质毫克数转换为蛋白质摩尔数，计算每摩尔亚基c中[¹⁴C]DCCD的摩尔数。在“高效液相色谱（HPLC）峰面积”方法中，在阴离子交换AX300 HPLC柱上，将DCCD修饰的亚基c与未修饰的亚基c分离，并对色谱图中峰的面积进行定量。修饰与抑制曲线的形状表明，每个F0中单个亚基c的修饰足以消除ATP酶活性。滴定数据通过非线性回归分析拟合到单打击数学模型A = Un(1 - r) + r，其中A是相对活性，U是未修饰/总亚基c的比率，n是每个F0中亚基c的数量，r是对DCCD抑制有抗性的ATP酶活性的残留分数。两种方法得出的n值，通过比放射性方法为10，通过HPLC峰面积方法为14，r值分别为0.28和0.30。大部分r值是由于在ATP酶测定条件下观察到15 - 20%的F1 - ATP酶从膜上解离所致。当在上述方程中使用实验合理的最小r值0.15时，计算出的n值分别降至8和11。此处确定的n值，可能的不确定范围为8 - 14，与通过更精确的放射性标记方法确定的每个F1F0有10 ± 1个亚基c的化学计量比一致，并提供了一种独立的实验支持类型（福斯特，D. L.，和菲林盖姆，R. H.（1982年）《生物化学杂志》257，2009 - 2015）。
