Rafael J, Pampel I, Wang X
Institut für Biochemie I, Universität Heidelberg, Germany.
Eur J Biochem. 1994 Aug 1;223(3):971-80. doi: 10.1111/j.1432-1033.1994.tb19075.x.
Binding of purine nucleotides to the uncoupling protein (UCP) was investigated in membrane particles prepared from brown fat mitochondria of cold-acclimated rats. Mitochondrial membranes were separated from soluble protein with Lubrol WX and treated with 3 M urea at basic pH. The resulting membrane vesicles were permeable to GDP and contained up to 3 nmol UCP/mg protein with unchanged nucleotide binding, as compared to the mitochondria (GDP/UCP ratio = 1.0; pKd GDP = 6.0 at pH 7.0). UCP bound nucleotides to one type of specific binding sites, located exclusively on the cytosolic side of the mitochondrial membrane. The binding affinity of guanine nucleotides was 3-18-times higher than that of the corresponding adenine nucleotides, when measured in membrane particles from cold-acclimated rats, hamsters, and guinea pigs. The pH-dependent binding affinities of GDP and ADP attained a maximum at pH 5.0-6.0 (pKd GDP = 6.8, pKd ADP = 5.8) and were decreased by a factor of 10(2) at pH 4.0 and pH 8.0, respectively, whereas the binding affinity of ATP was maximal at pH 4.0 (pKd = 7.0) and was decreased by a factor of 10(3) at pH 7.5. Participation of the protein binding center in nucleotide interaction with UCP in the membrane was highly pH-dependent. Mg2+ modified the number of binding sites engaged at a given nucleotide concentration by complex binding of nucleotides; the Kd for Mg.GTP2- and Mg.GDP- was 20-50-times lower than that of the free nucleotides.
在由冷适应大鼠的棕色脂肪线粒体制备的膜颗粒中，研究了嘌呤核苷酸与解偶联蛋白（UCP）的结合情况。用Lubrol WX将线粒体膜与可溶性蛋白分离，并在碱性pH下用3M尿素处理。与线粒体相比，所得膜囊泡对GDP具有通透性，并且每毫克蛋白含有高达3 nmol的UCP，核苷酸结合未改变（GDP/UCP比率 = 1.0；在pH 7.0时pKd GDP = 6.0）。UCP将核苷酸结合到一种特定的结合位点上，该位点仅位于线粒体膜的胞质侧。当在冷适应大鼠、仓鼠和豚鼠的膜颗粒中测量时，鸟嘌呤核苷酸的结合亲和力比相应腺嘌呤核苷酸的结合亲和力高3至18倍。GDP和ADP的pH依赖性结合亲和力在pH 5.0 - 6.0时达到最大值（pKd GDP = 6.8，pKd ADP = 5.8），在pH 4.0和pH 8.0时分别降低10²倍，而ATP的结合亲和力在pH 4.0时最大（pKd = 7.0），在pH 7.5时降低10³倍。膜中蛋白质结合中心参与核苷酸与UCP的相互作用高度依赖于pH。Mg²⁺通过核苷酸的复合结合改变了在给定核苷酸浓度下参与结合的位点数量；Mg.GTP²⁻和Mg.GDP⁻的Kd比游离核苷酸的Kd低20至50倍。
