Analysis of the mechanism of the invasion of the A subunit of heat-labile enterotoxin from enterotoxigenic E coli

在这段时间里，我们可以得到以下数据：1。霍乱弧菌产生的蛋白酶（VC-1）能在产肠毒素大肠杆菌中不带切口的不耐热肠毒素A亚基N端192位（Arg）产生切口。在培养基中加入毒素使中国仓鼠过度生长的细胞产生的蛋白酶能够对未切口的不耐热肠毒素A亚基产生切口.在产肠毒素大肠杆菌的细胞提取物中检测到不与B亚基结合的游离A亚基，并对其进行纯化。它很不稳定，其生物学活性低于正常A亚基.从猪源性大肠杆菌中分离纯化的不耐热肠毒素（LTp）存在异质性。5. A亚基第112位氨基酸由Glu替换为Lys（E112 X）影响其A1鞭毛的ADP-核糖基转移酶活性。因此，提示Glu 112对ADP核糖基转移酶的活性有重要作用. E112 X突变株LT虽然不具有ADP核糖基转移酶活性，但具有ADP核糖基化因子（ARF）与霍乱毒素相互作用的抑制活性。这些数据表明，ARF可以激活霍乱毒素，具有变构效应。我的同事石田博士发现，在加热的牛奶中有两种糖蛋白可以与LT反应，而在未加热的牛奶中则没有。经氨基酸分析，它们分别为α-乳蛋白和β-乳球蛋白。

In this time, we can get the data as followed ;1. Vibrio cholerae produced the protease(this is named as VC-1) that could nick at the position 192(Arg) from the N-terminus of the A subunit of unnicked heat-labile enterotoxins from enterotoxigenic Escherichia coli.2. The Chinese hamster overly cell, which is elongated with the addition of toxin to its culture medium, produced the protease that could nick the unnicked heat-labile enterotoxin A subunit.3. THE free A subunit, which did not bind to B subunit, was detected in the cell extract from enterotoxigenic E.coli and was purified. It was very unstable and its biological activity was lower than that of the normal A subunit.4. There was the heterogeneity among the heat-labile enterotoxins(LTp) purified from the porcine enterotoxigenic E.coli strains. 5. The amino acid substitution at the position 112 of the A subunit from Glu to Lys(E112X) affects the ADP-ribosyltransferase activity of its A1 flagment. Therefore it is suggested that Glu 112 is important for the its ADP-ribosyltransferase activity.6. Though the E112X mutant LT did not have ADP-ribosyltransferase activity, it had the inhibition activity of the interaction between ADP ribosylation factor(ARF) to cholera toxin. These data indicates that the ARF could activate the cholera toxin with the alosteric effect.7. My coworker, Dr.Shida, found that there were two kinds of the glycoprotein, which could react with LT, in heated cow milk, but not in unheated one. By amino acid analyze of them, they were alpha-lactoalbumin and beta-lactoglobulin.