Herrin D, Michaels A
Arch Biochem Biophys. 1985 Feb 15;237(1):224-36. doi: 10.1016/0003-9861(85)90273-5.
Bispecific antisera were prepared to a mixture of thylakoid membrane polypeptides 4.1 and 4.2. The identity of these polypeptides as the alpha and beta subunits of coupling factor (CF1) was established based on the cross-reactivity of the antisera toward CF1 from peas and by an analysis of the thm-24 mutant of Chlamydomonas which lacks the CF1 ATPase. Photochemical labeling of thylakoid membranes with hydrophobic and hydrophilic fluorescent probes indicated that these polypeptides did not significantly penetrate the membrane bilayer. Immunoprecipitation of the translation products of thylakoid-bound and soluble ribosomes showed the thylakoids to be the major site of synthesis of the polypeptides. Immunoprecipitation of the products of translation of total cellular RNA in a reticulocyte lysate showed no evidence for substantially higher molecular weight precursors. Further analysis of the thylakoid-bound synthesis of alpha and beta revealed that some of the in vitro synthesized polypeptides had been incorporated into the CF0-CF1 complex based on their release from membranes with trypsin and copurification with the CF0-CF1 ATPase.
制备了针对类囊体膜多肽4.1和4.2混合物的双特异性抗血清。基于抗血清对豌豆CF1的交叉反应性以及对缺乏CF1 ATP酶的衣藻thm - 24突变体的分析，确定了这些多肽为偶联因子（CF1）的α和β亚基。用疏水和亲水荧光探针对类囊体膜进行光化学标记表明，这些多肽并未显著穿透膜双层。对类囊体结合核糖体和可溶性核糖体翻译产物的免疫沉淀显示，类囊体是这些多肽的主要合成位点。对网织红细胞裂解物中总细胞RNA翻译产物的免疫沉淀未显示出存在分子量明显更高的前体的证据。对α和β亚基类囊体结合合成的进一步分析表明，基于体外合成的一些多肽在经胰蛋白酶处理后从膜上释放并与CF0 - CF1 ATP酶共纯化，它们已被整合到CF0 - CF1复合物中。
