Analysis of the Active Site of the A Subunit of LT Produced by Enterotoxigenic E Coli

以产肠毒素大肠杆菌EWD 299为质粒，经羟胺处理后，获得了一种编码热不稳定肠毒素（LT）突变基因的质粒。携带突变质粒pTUH 6A的大肠杆菌HB 101突变株产生了与抗LT抗体交叉反应的低毒LT类似物（突变体LT）。在兔回肠环试验和兔皮肤渗透性试验中，突变体LT活性分别低于正常LT的0.15和0.006%。突变体LT-B亚基的氨基酸组成与正常B亚基相同。突变体LT的A_2片段与正常LT DNA分析一致，但A_1片段在112位有1个氨基酸的差异；也就是说它有赖氨酸而不是谷氨酸来自N端。这些数据表明，A_1片段N端112位的谷氨酸对于A亚基表达其生物活性是重要的。

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, - was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin peameability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A_2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A_1 fragment of the mutant LT differed from the normal A_1 fragment in one amino acid at position 112 ; namely it had lysine instead of glutamic acid from the N terminus.These data suggest that glutamic acid at position 112 from the N terminus of the A_1 fragment is important for the A subunit to express its biological activity.