Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis.
Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis.
复制标题
枯草芽孢杆菌 Ca2/H 逆向转运蛋白的纯化和表征。
DOI：
10.1111/j.1432-1033.1986.tb09553.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Iwao Kusaka
中科院分区：
文献类型：
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作者：
Taku Matsushita;Teruko Ueda;Iwao Kusaka
以NADH为能量源时，枯草芽孢杆菌胞膜囊内出现了Ca 2+的积累。Δ pH（酸性内部）也可以驱动膜囊泡中的Ca 2+积累，并且这种积累被羰基氰化物对三氟甲氧基苯腙和尼日利亚菌素加K+抑制。这些结果表明在这种生物体中存在Ca 2 +/H+反向转运蛋白（交换器）。通过羟基磷灰石、二乙基氨基乙基（DEAE）-Toyopolymer 650 M和丁基-Toyopolymer 650 M层析，从膜蛋白中分离并纯化反向转运蛋白至均一。纯化的反向转运蛋白具有约45000道尔顿的分子量和5.0的等电点。通过纯化的反向转运蛋白在蛋白脂质体中积累Ca 2+期间，花青染料（碘化3，3 '-二丙基硫代二碳花青[diS-C3-（5）]）的荧光猝灭显示膜电位（内部负）的产生，表明转运中的H+/Ca 2+化学计量比高于2。这也支持的结果，K+-扩散电位，内部正，刺激的Ca 2+的摄取，在存在一个Δ pH值。的表观Km为Ca 2+的反向转运蛋白是约40 μ M和La 3+抑制运输。纯化的反向转运蛋白的氨基酸分析表明存在大量的谷氨酸和天冬氨酸和少量的组氨酸，赖氨酸和精氨酸。这与蛋白质的低等电点（约5.0）一致。
Ca2+ was accumulated in inside-out membrane vesicles of Bacillus subtilis when NADH was used as an energy source. A delta pH (acid interior) could also drive Ca2+ accumulation in the membrane vesicles and the accumulation was inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin plus K+. These results indicate the presence of a Ca2+/H+ antiporter (exchanger) in this organism. The antiporter was isolated and purified to homogeneity from the membrane proteins by chromatography on hydroxyapatite, diethylaminoethyl(DEAE)-Toyopearl 650 M and butyl-Toyopearl 650 M. The purified antiporter has a molecular mass of about 45 000 daltons and an isoelectric point of 5.0. The fluorescence quenching of a cyanine dye (3,3'-dipropylthiodicarbocyanine iodide [diS-C3-(5)] during Ca2+ accumulation in proteoliposomes by the purified antiporter showed the generation of a membrane potential (interior negative) suggesting a H+/Ca2+ stoichiometry above 2 in the transport. This was also supported by the result that the K+-diffusion potential, interior positive, stimulated the Ca2+ uptake in the presence of a delta pH. The apparent Km for Ca2+ of the antiporter was about 40 microM and La3+ inhibited the transport. Amino acid analysis of the purified antiporter indicated the presence of large amounts of glutamic and aspartic acids and small amounts of histidine, lysine and arginine. This is consistent with the low isoelectric point (about 5.0) of the protein.