Characteristics of Spinach Chloroplast Envelope, Thylakoid and Stroma Polypeptides as Revealed by Triton X-114 Phase Partition

摘要

Comparison of the SDS-PAGE profiles of the spinach chloroplast stroma, thylakoid and envelope membranes shows that several polypeptides have the same electrophoretic mobility. To simplify these somewhat complex electrophoretic profiles and to verify whether the polypeptides having similar electrophoretic mobility are identical, we used Triton X-114 phase partition to obtain a separation of the polypeptides according to their relative hydrophobicity. The stroma polypeptides partitioned essentially in the aqueous phase. About half of the thylakoid and envelope membrane polypeptides were exclusively recovered in either one of the two phases. Therefore, the phase partitioning of membrane polypeptides proved to be useful, as the organic phase contained true intrinsic polypeptides, while the aqueous phase was composed of peripheral ones and stroma components. Particularly interesting was the release of the RubisCO large subunit known to copurify with the envelope membranes. Additional experimental approaches were used (immunology, proteosynthesisin organello)to further characterize proteins which had apparent ambiguous phase partitioning. Here, we show that Triton X-l 14 is an excellent tool to unmask polypeptides having identical electrophoretic mobility but different behaviour towards this detergent; its use leads to a clarification of the polypeptide SDS-PAGE profiles of chloroplast membranes.