Viitanen P V, Menick D R, Sarkar H K, Trumble W R, Kaback H R
Biochemistry. 1985 Dec 17;24(26):7628-35. doi: 10.1021/bi00347a020.
By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue [Trumble, W. R., Viitanen, P. V., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860]. Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E. coli T184. Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl alpha-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease. In contrast, the Vmax for lactose transport in vesicles containing mutant permease is significantly decreased. Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl alpha-D-galactopyranoside with high affinity. Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity. Importantly, beta-galactosyl 1-thio-beta-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant. The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease. Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl alpha-D-galactopyranoside from wild-type permease, does not affect binding in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
通过对大肠杆菌的乳糖操纵子y基因进行寡核苷酸定向的位点特异性诱变，乳糖通透酶中的半胱氨酸148已被甘氨酸残基取代[特朗布尔，W.R.，维塔宁，P.V.，萨卡尔，H.K.，波尼安，M.S.，&卡巴克，H.R.（1984年）《生物化学与生物物理研究通讯》119，860]。构建了携带野生型或突变型乳糖操纵子y的重组质粒，并用于转化大肠杆菌T184。在含有野生型或突变型通透酶的外翻囊泡中，乳糖积累的稳态水平、在有能量条件下乳糖的表观Km以及对硝基苯基α-D-吡喃半乳糖苷的KD相当。相比之下，含有突变型通透酶的囊泡中乳糖转运的Vmax显著降低。尽管抗体结合研究表明，来自突变体的囊泡所含的通透酶几乎与野生型囊泡一样多，但令人惊讶的是，只有约四分之一的改变分子能高亲和力结合对硝基苯基α-D-吡喃半乳糖苷。突变型通透酶对N-乙基马来酰亚胺失活的敏感性较低，尽管该烷基化剂仍能完全抑制转运活性。重要的是，β-半乳糖基1-硫代-β-D-吡喃半乳糖苷能完全保护野生型通透酶免受N-乙基马来酰亚胺的影响，但对突变体没有任何保护作用。N-乙基马来酰亚胺使野生型和突变型通透酶失活的速率在碱性pH下以及存在质子电化学梯度（内部为负且呈碱性）时会增加，并且这些现象在含有突变型通透酶的囊泡中更为明显。最后，对（氯汞）苯磺酸盐能完全从野生型通透酶中置换结合的对硝基苯基α-D-吡喃半乳糖苷，但对突变体中的结合没有影响。（摘要截短至250字）
