Yamato I, Anraku Y
J Biochem. 1977 May;81(5):1517-23.
The properties of the carrier for isoleucine in Escherichia coli were studied using cytoplasmic membrane vesicles (IM vesicles) prepared by the method of Yamato, Anraku, and Hirosawa (J. Biochem. 77, 705 (1975)). The IM vesicles exhibited respiration-dependent isoleucine transport activity which was more than 30-fold higher than that of "Kaback vesicles" prepared by our hand from the same strains of E. coli K12. The isoleucine carrier activity of IM vesicles was inhibited by norleucine but not by threonine. The carrier was driven by proton motive force. Mutants were isolated which had lost the carrier activity for isoleucine, as judged by assay with IM vesicles. Using these mutants, the effects of binding proteins specific for branched chain amino acids on the translocation of substrate in IM vesicles were studied. Leucine-isoleucine-valine-threonine-binding protein (LIVT-binding protein) stimulated the initial rate of isoleucine uptake by IM vesicles only when the vesicles possessed carrier activity and it did not affect the Kt value for entry of substrate. This evidence suggests the partial reconstitution of the osmotic shock-sensitive transport reaction in which the binding protein seems to affect the carrier activity with turnover ability.
利用大和、安楽和広沢（《生物化学杂志》77, 705 (1975)）的方法制备的细胞质膜囊泡（内膜囊泡，IM囊泡），对大肠杆菌中异亮氨酸载体的性质进行了研究。IM囊泡表现出依赖呼吸作用的异亮氨酸转运活性，其活性比我们用相同的大肠杆菌K12菌株亲手制备的“卡巴克囊泡”高出30多倍。IM囊泡的异亮氨酸载体活性受到正亮氨酸的抑制，但不受苏氨酸的抑制。该载体由质子动力驱动。通过用IM囊泡进行测定判断，分离出了丧失异亮氨酸载体活性的突变体。利用这些突变体，研究了支链氨基酸特异性结合蛋白对IM囊泡中底物转运的影响。亮氨酸-异亮氨酸-缬氨酸-苏氨酸结合蛋白（LIVT结合蛋白）仅在囊泡具有载体活性时刺激IM囊泡摄取异亮氨酸的初始速率，并且它不影响底物进入的Kt值。这一证据表明了渗透休克敏感转运反应的部分重构，其中结合蛋白似乎影响具有周转能力的载体活性。
