Klingenberg M, Riccio P, Aquila H
Biochim Biophys Acta. 1978 Aug 8;503(2):193-210. doi: 10.1016/0005-2728(78)90182-2.
The procedure for the isolation from mitochondria of the undenatured ADP, ATP carrier is described. The condition of retaining the nativity are elaborated. 1. As indicator for the ADP, ATP carrier (35S)- or (3H) carboxyatractylate were used. By preloading the mitochondria with carboxyatractylate, a stable carboxyatractylate . protein complex could be retained after solubilization with Triton X-100. Among the polyoxyethylene detergents emulphogen is also solubilizing, whereas Brij and Lubrol fail to solubilize. 2. When unloaded mitochondria are solubilized the capacity for binding carboxyatractylate disappears rapidly, particularly at 20 degrees C. 3. When mitochondria are preloaded with atractylate, the binding after solubilization with Triton X-100 is considerably lower than with carboxyatractylate, indicating that the high affinity of carboxyatractylate is required for effectively protecting the protein. 4. For purification hydroxyapatite is most effective. The carboxyatractylate-protein complex appears in the pass-through whereas the bulk of other mitochondrial proteins are retained such that a 7-fold purification is obtained. The nonadsorptivity to hydroxyapatite is dependent on the undenatured state maintained in the carboxyatractylate . protein complex. 5. Subsequent gel filtration on Sepharose results in a 1.5-fold further enrichment of specific carboxyatractylate binding up to 17 mumol/g protein, corresponding to a 10-fold purification from mitochondria. This value cannot be increased with further measures. 6. At the last purification step, in sodium dodecyl sulfate polyacrylamide gel electrophoresis virtually a single band of 30 000 molecular weight is found, confirming the purity at this stage. A molecular weight of 60 000 is calculated from the carboxyatractylate binding, indicating that the carboxyatractylate protein complex consists of two 30 000 subunits. From this the protein share of the ADP, ATP carrier in beef heart mitochondria can be calculated to amount to 9.5%9 7. The intact carboxyatractylate . protein complex is protected against proteolytic degradation. The release of carboxyatractylate ensues a conformational change of protein as assayed by conformation specific antibodies, concomitant with unmasking of proteolytic site as assayed by tryptic digestion. 8. The amino acid composition indicates hydrophobicity (39% polarity) and a high content of basic amino acid such as lysine and arginine. There is 1.5 mol percent cysteine and a blocked N-terminal. 9. From the solubilized complex (35S) carboxyatractylate can be removed by carboxyatractylate, ADP and ATP but not by ITP, etc., indicating the presence of recognizing sites specific fof ADP, ATP and therefore, identity with the ADP, ATP carrier. 10. Other reported procedures for isolating the ADP, ATP carrier are shown to either fail or have lower yield than the present, original procedure.
本文描述了从未变性的线粒体中分离ADP、ATP载体的方法。阐述了保持其天然状态的条件。1. 以(35S)-或(3H)羧基苍术苷作为ADP、ATP载体的指示剂。通过用羧基苍术苷预加载线粒体，在用Triton X-100溶解后可保留稳定的羧基苍术苷-蛋白质复合物。在聚氧乙烯去污剂中，乳化剂也具有增溶作用，而Brij和Lubrol则不能增溶。2. 当未加载的线粒体被溶解时，结合羧基苍术苷的能力迅速消失，特别是在20℃时。3. 当线粒体用苍术苷预加载时，用Triton X-100溶解后的结合能力明显低于用羧基苍术苷时，这表明有效保护蛋白质需要羧基苍术苷的高亲和力。4. 对于纯化来说，羟基磷灰石最有效。羧基苍术苷-蛋白质复合物出现在穿透液中，而大部分其他线粒体蛋白质被保留，从而实现了7倍的纯化。对羟基磷灰石的非吸附性取决于羧基苍术苷-蛋白质复合物中维持的未变性状态。5. 随后在琼脂糖凝胶上进行凝胶过滤，可使特异性羧基苍术苷结合进一步富集1.5倍，达到17μmol/g蛋白质，相当于从线粒体中纯化了10倍。该值不能通过进一步的措施提高。6. 在最后一步纯化中，在十二烷基硫酸钠聚丙烯酰胺凝胶电泳中几乎发现一条分子量为30000的单带，证实了此时的纯度。根据羧基苍术苷结合计算出分子量为60000，表明羧基苍术苷蛋白质复合物由两个30000亚基组成。由此可计算出牛心线粒体中ADP、ATP载体的蛋白质含量为9.5%。7. 完整的羧基苍术苷-蛋白质复合物可防止蛋白水解降解。羧基苍术苷的释放伴随着蛋白质的构象变化，这通过构象特异性抗体检测，同时伴随着胰蛋白酶消化检测到的蛋白水解位点的暴露。8. 氨基酸组成表明其具有疏水性(39%极性)，并且含有高含量的碱性氨基酸，如赖氨酸和精氨酸。有1.5摩尔百分比的半胱氨酸和一个封闭的N端。9. 从溶解的复合物中，(35S)羧基苍术苷可被羧基苍术苷、ADP和ATP去除，但不能被ITP等去除，这表明存在对ADP、ATP特异性识别的位点，因此与ADP、ATP载体一致。10. 其他报道的分离ADP、ATP载体的方法被证明要么失败，要么产率低于目前的原始方法。
