Moskaug J O, Sandvig K, Olsnes S
Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
J Biol Chem. 1988 Feb 15;263(5):2518-25.
When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH.
当表面结合有125I标记的、带切口的白喉毒素的Vero细胞暴露于pH 4.5时，两种分子量分别为20,000和25,000的多肽受到保护，免受外部施加的链霉蛋白酶E的作用。20 kDa的多肽似乎是毒素A片段，而25 kDa的多肽必定源自B片段。用皂苷使细胞透化，会使20 kDa片段发生外流，而大部分25 kDa多肽仍与细胞结合。一些保护细胞免受白喉毒素作用的化合物和条件，会阻止对链霉蛋白酶E的保护作用。即使通过胞质溶胶酸化使跨膜质子梯度（ΔpH）消散，25 kDa多肽的保护作用仍会发生，而在这些条件下，A片段的保护和释放则会受到阻止。细胞的电去极化和ATP耗竭并不抑制A片段的保护和释放。数据表明，A片段转移至胞质溶胶需要ΔpH，而即使在没有ΔpH的情况下，B片段的一部分在低pH时也会插入膜中。
