Pessin J E, Tillotson L G, Isselbacher K J, Czech M P
Fed Proc. 1984 May 15;43(8):2258-61.
A method has been developed for photoaffinity labeling components of the hexose transport system with [3H]cytochalasin B. We have demonstrated that UV photoirradiation of intact human erythrocytes or ghost membranes with 0.5 microM [3H]cytochalasin B yielded a broad peak with an Mr of 44,000-70,000. Labeling that was insensitive to the presence of 0.5 M D-sorbitol was substantially inhibited by 0.5 M D-glucose. Approximately 80% of labeling in the region of Mr = 49,000-70,000 was inhibited by the presence of 0.5 M D-glucose, whereas labeling of the 44,000- to 49,000-dalton region was inhibited only 30%. Somewhat different results were obtained from photoaffinity labeling of plasma membranes from chicken embryo fibroblasts (CEF). The [3H]cytochalasin B-labeling patterns had two relatively sharp and discrete peaks at 46,000 and 52,000 daltons. Comparison of plasma membranes from glucose-fed and starved CEF revealed that the total D-glucose-sensitive labeling increased approximately 12-fold in the starved cell membranes. Labeling of the 52,000-dalton polypeptide was more sensitive than that of the 46,000-dalton polypeptide to inhibition by the presence of D-glucose. These results indicate that [3H]cytochalasin B photoaffinity labeling has wide applicability for identifying and covalently binding components of the facilitated hexose transport system.
已经开发出一种用[3H]细胞松弛素B对己糖转运系统的组分进行光亲和标记的方法。我们已经证明，用0.5微摩尔[3H]细胞松弛素B对完整的人红细胞或血影膜进行紫外线光照射会产生一个宽峰，其分子量为44,000 - 70,000。对0.5M D - 山梨醇存在不敏感的标记被0.5M D - 葡萄糖显著抑制。在分子量为49,000 - 70,000区域，约80%的标记被0.5M D - 葡萄糖的存在所抑制，而在44,000 - 49,000道尔顿区域的标记仅被抑制30%。从鸡胚成纤维细胞（CEF）的质膜光亲和标记中获得了有些不同的结果。[3H]细胞松弛素B标记模式在46,000和52,000道尔顿处有两个相对尖锐且离散的峰。对葡萄糖喂养和饥饿的CEF的质膜进行比较发现，饥饿细胞膜中总的D - 葡萄糖敏感标记增加了约12倍。52,000道尔顿多肽的标记比46,000道尔顿多肽的标记对D - 葡萄糖存在的抑制更敏感。这些结果表明，[3H]细胞松弛素B光亲和标记在鉴定和共价结合易化己糖转运系统的组分方面具有广泛的适用性。
