葡萄果肉亚细胞组分的分离和质膜的纯化

Separation of Subcellular Components and Purification of Plasma Membrane from Mesocarp of Grape Berries

利用差速离心法从葡萄果肉组织中分离了微粒体和胞质组分，将微粒体进一步利用两相分配法进行了质膜和细胞内膜的分离。结果表明，微粒体分离是整个实验技术的难点；研磨缓冲液成分是影响微粒体分离和微粒体在两相体系中分配（质膜纯化）的关键因素；排除果肉组织中有机酸、酚类和果胶物质的干扰是确定研磨缓冲液的原则；从设计的８种研磨缓冲液中选出了最佳配方。用最佳研磨缓冲液制备出高得率和高质量的微粒体。两相分配后的标志酶活性检测表明，上相富含质膜，内膜污染较少，质膜纯度较高；而下相富含内膜。证明了ＰＥＧ－Ｄｅｘｔｒａｎ两相分配法对于葡萄果肉组织质膜的纯化也是适用的。

The microsome and cytosol fractions were prepared from the mesocarp of grape berries by differential centrifugation, and the plasma and inner membrane fractions were separated from the microsomes by aqueous polymer twophase partitioning which was composed of 6.2% PEG 3350 and 6.2% Dextran T 500. The composition of homogenizing buffer played a key role in the preparation of microsomes and purification of plasma membrane. Optimum homogenizing buffer was compoed of 250mmol/L sucrose, 3mmol/L EDTA, 10% glycerol, 4g/L BSA, 4g/L casein, 1mmol/L PMSF, 1.5g/L PVP, 5mmol/L DTT, 10mmol/L Vc, 80mmol/L Tris, 5mmol/L BTP-Mes, pH 8.9. A high yield of microsomes with good quality was obtained with this homogenizing buffer. The validity of the polyethylene glycoldextran partition was proved by the distribution between two phases of marker enzyme activities assayed under optimized conditions for this fruit material. The upper phase fraction showed highly plasmalemma-enriched, and the lower phase fraction abounded with inner membranes. 