Deckers-Hebestreit G, Altendorf K
Eur J Biochem. 1986 Nov 17;161(1):225-31. doi: 10.1111/j.1432-1033.1986.tb10146.x.
Antisera have been raised against denatured and non-denatured subunits a, b and c of the F0 complex of the ATP synthase from Escherichia coli. The subunit specificity of the antibodies has been established with immunoblot analysis or enzyme-linked immunosorbent assay (ELISA). In inside-out oriented membrane vesicles the binding avidities of both sets of antisera, against denatured and non-denatured subunits of F0, were similar in the presence as well as in the absence of the F1 part. F1-depleted everted membrane vesicles always produced an efficient binding of the different antisera. In the presence of F1 no antibody recognition could be observed with the anti-a antisera, while anti-b and anti-c antisera showed strong binding. However, a higher membrane protein concentration was necessary for the same antibody binding as in F1-stripped vesicles. In membrane vesicles with right-side-out orientation the recognition of the three F0 subunits was dependent on the antisera set used. Antisera raised against denatured subunits showed no binding to the membrane vesicles, only in case of anti-(dodecylsulfate-denatured b) antiserum could a slight affinity be detected. An antigen-antibody recognition with all three F0 subunits occurred when the antisera against non-denatured subunits were incubated with membrane vesicles of right-side-out orientation. The membrane protein concentration which was necessary to produce a significant binding was 10-100-fold higher compared to that of F1-depleted everted membrane vesicles.
已制备出针对大肠杆菌ATP合酶F0复合物变性和未变性的a、b和c亚基的抗血清。通过免疫印迹分析或酶联免疫吸附测定（ELISA）确定了抗体的亚基特异性。在内外翻转的膜囊泡中，针对F0变性和未变性亚基的两组抗血清的结合亲和力在有或没有F1部分的情况下都相似。去除F1的外翻膜囊泡总能有效地结合不同的抗血清。在有F1存在的情况下，抗a抗血清未观察到抗体识别，而抗b和抗c抗血清显示出强结合。然而，与去除F1的囊泡相比，相同抗体结合需要更高的膜蛋白浓度。在右侧向外取向的膜囊泡中，对F0三个亚基的识别取决于所用的抗血清组。针对变性亚基产生的抗血清与膜囊泡无结合，仅在抗（十二烷基硫酸钠变性b）抗血清的情况下可检测到轻微亲和力。当针对未变性亚基的抗血清与右侧向外取向的膜囊泡孵育时，会发生与F0所有三个亚基的抗原-抗体识别。产生显著结合所需的膜蛋白浓度比去除F1的外翻膜囊泡高10 - 100倍。
