Shida K, Takamizawa K, Nagaoka M, Kushiro A, Osawa T, Tsuji T
Yakult Central Institute for Microbiological Research, Tokyo, Japan.
J Dairy Sci. 1994 Apr;77(4):930-9. doi: 10.3168/jds.s0022-0302(94)77028-4.
The binding of Escherichia coli heat-labile enterotoxin to caseins, whey proteins, milk fat globule membrane, and proteose-peptone fraction from bovine milk was studied by using the Western blot technique. Two toxin-binding glycoproteins, pp16k and pp20k, with molecular weights of 15,500 and 20,000, respectively, were detected only in a proteose-peptone fraction. These glycoproteins were partially purified by ammonium sulfate precipitation and Toyopearl HW 55 gel filtration chromatography. The binding ability to the toxin was destroyed by periodate treatment or beta-galactosidase treatment, indicating that a carbohydrate moiety, particularly a terminal galactosyl residue, was essential for the binding of the toxin. In contrast, the binding ability was not changed by mild acid treatment, and these glycoproteins did not bind cholera toxin, which can bind to ganglioside GM1, suggesting that the carbohydrate structure of the glycoproteins is different from that of GM1. The N-terminal amino acid sequence and immunoblot analysis indicated that the protein moieties of pp16k and pp20k are identical to alpha-lactalbumin and beta-lactoglobulin, respectively. These toxin-binding glycoproteins were not detected in whey proteins isolated from unheated skim milk, suggesting that they are newly generated during heat treatment of skim milk before the preparation of a proteose-peptone fraction.
采用蛋白质印迹技术研究了大肠杆菌热不稳定肠毒素与酪蛋白、乳清蛋白、乳脂肪球膜及牛乳蛋白胨部分的结合情况。仅在蛋白胨部分检测到两种毒素结合糖蛋白，即分子量分别为15500和20000的pp16k和pp20k。通过硫酸铵沉淀和Toyopearl HW 55凝胶过滤色谱对这些糖蛋白进行了部分纯化。高碘酸盐处理或β-半乳糖苷酶处理会破坏其与毒素的结合能力，这表明碳水化合物部分，尤其是末端半乳糖基残基，对于毒素的结合至关重要。相比之下，温和酸处理不会改变其结合能力，并且这些糖蛋白不结合可与神经节苷脂GM1结合的霍乱毒素，这表明这些糖蛋白的碳水化合物结构与GM1不同。N端氨基酸序列和免疫印迹分析表明，pp16k和pp20k的蛋白质部分分别与α-乳白蛋白和β-乳球蛋白相同。在从未加热的脱脂乳中分离出的乳清蛋白中未检测到这些毒素结合糖蛋白，这表明它们是在制备蛋白胨部分之前对脱脂乳进行热处理的过程中新生的。
