Genetic study on a active center of amino acid/Na^+ symport carriers

1)研究了质子和钠离子对Pro载体在大肠杆菌中的结合和转运活性的依赖关系。载体高产菌株细胞质膜囊泡的结合活性完全依赖于Na+的存在，但不一定需要载体质子化。胞质膜小泡的膜转运活性的表观米氏常数(Kt)与低浓度的Na~(++)有关，K_&lt；Na&gt；值估计为25um。膜泡和完整细胞中的脯氨酸转运活性受H~(++)浓度的调节，表现出质子的抑制作用，pKa=6。在此基础上，提出了一个膜/Na~+转运机制的模型，其中一个质子被认为是转运活性的调节因子。2)大肠杆菌Pro/Na~(++)转运载体的Pro结合活性被一个超…抑制更多的羟基试剂NEM。Pro及其类似物以Na~+依赖的方式保护载体免受NEM的抑制。分别用丝氨酸残基取代Cys-281、Cys-344和Cys-349的突变体载体CS281、CS344和CS349对阳离子依赖的Pro结合和NEM敏感性进行了分析。CS281和CS344的Pro结合活性对NEM几乎完全抗性，而CS349则不完全抗性。CS344的Pro结合活性显著低于野生型、CS281和CS349载体。我们推测Cys-344是一个半胱氨酸残基，功能上参与与Na~+和Pro的高亲和力结合。3)通过诱变获得阳离子专一性改变的Pro载体突变体。两株携带pMOP4135和pMOP4141的突变菌株只有在Na~+浓度升高时才能有效地转运脯氨酸。在这些突变载体中，脯氨酸结合的pH依赖性也发生了改变。DNA测序发现，pMOP4141和pMOP4135的299位和656位分别由G碱基变为A碱基，对应的氨基酸分别由Gly-22变为谷氨酸，Cys-141变为酪氨酸。4)测定了编码大肠杆菌谷氨酸/钠离子共载体的gltS基因的核苷酸序列，并推导了该载体的氨基酸序列。预测的谷氨酸载体由401个氨基酸组成，分子量为42,455。预测的蛋白质是非常疏水的，从其疏水性图谱来看，该蛋白质由12个跨膜片段组成。序列同源性搜索发现5个氨基酸残基Gly-42-Ala-82-Leu-87-Gly-91-Arg-92保守排列，它们普遍存在于4种Na~(++)载体蛋白、大肠杆菌的谷氨酸载体和Pro载体以及兔和人肠道的葡萄糖转运蛋白中。较少

1)The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier in Escherichia coli were investigated in detail. The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain was absolutely dependent on the presence of Na^+, but did not necessarily require protonation of the carrier. The apparent Michaelis constant of proline (Kt) of the transport, activity in the cytoplasmic membrane vesicles showed dependence on a low concentration of Na^+ and the K_<Na> value was estimated to be 25 uM. The proline transport activities in membrane vesicles and intact cells were modulated by H^+ concentration, showing the inhibitory effect of protons, pKa = 6. Based on these observations a model of the proline/Na^+ symport mechanism is proposed, inwhich a proton is postulated to be a regulatory factor of the transport activity.2)Proline binding activity of the Escherichia coli proline/Na^+ symport carrier is inhibited by a sul … More fhydryl reagent NEM. Proline and its analogs protected the carrier against the NEM-inhibition in a Na^+-dependent manner. Mutant proline carrier, CS281, CS344 and CS349, which have a serine residue in place of Cys-281, Cys-344 and Cys-349, respectively, were analyzed for cation dependent proline binding and NEM-sensitivity. Proline binding activities of CS281 and CS344 were almost completely resistant to NEM, whereas that of CS349 was not. The proline binding activity of CS344 was remarkably lower than those of the wild-type, CS281 and CS349 carriers. We propose that Cys-344 is a cysteine residue functionally involved in the high affinity binding for Na^+ and proline.3)Proline carrier mutants with altered cation specificity were obtained by mutagenesis. Two mutants strains harboring plasmid pMOP4135 and pMOP4141 could transport proline efficiently only in the presence of an increased concentration of Na^+. The pH dependence of proline binding was also changed in these mutant carriers. DNA sequencing revealed one base alteration of G to A at nucleotides 299 and 656 in pMOP4141 and pMOP4135, respectively, which corresponded to amino acid changes from Gly-22 to glutamic acid and Cys-141 to tyrosine, respectively.4)The nucleotide sequence of the gltS gene coding for an glutamate/Na^+ symport carrier of Escherichia coli was determined and the amino acid sequence of the carrier was deduced. The predicred glutamate carrier consists of 401 amino acids with a molecular weight of 42, 455. The predicted protein is very hydrophobic, and judging from its hydropathy profile, the protein is composed of 12 membrane-spanning segments. A conserved alignment of 5 amino acid residues Gly-42---Ala-82---Leu-87---Gly-91-Arg-92 was uncovered by sequence homology search, which commonly exists in four Na^+-symport carrier proteins, the glutamate carrier and the proline carrier of E. coli, and the glucose transporters of rabbit and human intestine. Less