Satre M, Bof M, Issartel J P, Vignais P V
Biochemistry. 1982 Sep 14;21(19):4772-6. doi: 10.1021/bi00262a038.
N,N'-Dicyclohexylcarbodiimide (DCCD) covalently binds to the beta subunit of Escherichia coli F1-ATPase (BF1). The ATPase activity is fully inhibited when 1 mol of DCCD is bound/mol of BF1, in spite of the fact that BF1 contains several beta subunits [Satre, M., Lunardi, J., Pougeois, R., & Vignais, P.V. (1979) Biochemistry 18, 3134-3140]. Advantage was taken of the reactivity of DCCD with respect to BF1 to determine the exact stoichiometry of the beta subunits in BF1. Two methods were used. The first one was based on the fact that modification of the beta subunit by DCCD results in the disappearance of one negative charge, due to the binding of DCCD to a carboxyl group of the beta subunit. The nonmodified and the modified beta subunits were separated by electrofocusing, and the percentage of modified beta subunits was assessed as a function of the percentage of ATPase inactivation. The second method relied on direct comparison, after inactivation of BF1 by [14C]DCCD, of the specific radioactivities of the whole BF1 and the isolated beta subunits. Both methods indicate that each molecule of BF1 contains three beta subunits.
N,N'-二环己基碳二亚胺（DCCD）与大肠杆菌F1-ATP酶（BF1）的β亚基共价结合。当每摩尔BF1结合1摩尔DCCD时，ATP酶活性被完全抑制，尽管BF1含有几个β亚基[Satre, M., Lunardi, J., Pougeois, R., & Vignais, P.V. (1979) Biochemistry 18, 3134 - 3140]。利用DCCD与BF1的反应性来确定BF1中β亚基的确切化学计量。使用了两种方法。第一种方法基于这样一个事实，即DCCD对β亚基的修饰会导致一个负电荷消失，这是由于DCCD与β亚基的一个羧基结合。通过等电聚焦分离未修饰和修饰的β亚基，并根据ATP酶失活的百分比评估修饰的β亚基的百分比。第二种方法依赖于用[14C]DCCD使BF1失活后，直接比较整个BF1和分离的β亚基的比放射性。两种方法都表明每个BF1分子含有三个β亚基。
