Brooker R J
Department of Genetics & Cell Biology, University of Minnesota, St. Paul 55108.
J Biol Chem. 1990 Mar 5;265(7):4155-60.
The double mutant, Val-177/Asn-322, was investigated with regard to its ability to transport H+ and galactosides. In downhill lactose transport assays, the wild-type strain had a Km value for lactose uptake of 0.9 mM and a Vmax of 0.65 mumol lactose/min.mg protein while the mutant had a significantly higher Km value of 1.9 mM but a similar Vmax of 0.49 mumol/min.mg protein. In spite of its moderate ability to transport lactose downhill, the Val-177/Asn-322 mutant exhibited the striking property of being completely defective in the uphill accumulation of lactose or methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport, however, showed that the mutant's defect in active accumulation is not due to a defect in the ability to transport H+ with lactose or methyl-beta-D-thiogalactopyranoside. The Val-177/Asn-322 mutant strain had a H+:lactose stoichiometry of 0.84 which was similar to that measured in the wild-type strain (0.68). These results are discussed with regard to the role His-322 plays in H+ transport, active accumulation of sugars, and sugar recognition.
对双突变体Val-177/Asn-322转运H⁺和半乳糖苷的能力进行了研究。在乳糖下坡转运试验中，野生型菌株摄取乳糖的Km值为0.9 mM，Vmax为0.65 μmol乳糖/分钟·毫克蛋白，而突变体的Km值显著更高，为1.9 mM，但Vmax相似，为0.49 μmol/分钟·毫克蛋白。尽管Val-177/Asn-322突变体向下转运乳糖的能力中等，但其在乳糖或甲基-β-D-硫代半乳糖吡喃糖苷的上坡积累方面表现出完全缺陷的显著特性。然而，对H⁺转运的直接测量表明，该突变体在主动积累方面的缺陷并非由于其与乳糖或甲基-β-D-硫代半乳糖吡喃糖苷一起转运H⁺的能力存在缺陷。Val-177/Asn-322突变体菌株的H⁺:乳糖化学计量比为0.84，与野生型菌株中测得的化学计量比（0.68）相似。结合组氨酸-322在H⁺转运、糖的主动积累和糖识别中所起的作用对这些结果进行了讨论。
