Roy K, Mandal A K, Sen P C
Department of Chemistry, Bose Institute, Calcutta, India.
Eur J Biochem. 1999 Apr;261(1):84-8. doi: 10.1046/j.1432-1327.1999.00212.x.
A Na+,K+-ATPase inhibitor protein has been purified to homogeneity from rat brain cytosol by ammonium sulphate precipitation, DEAE anion-exchange chromatography and hydroxyapatite adsorption column chromatography. The purified protein migrates as a single polypeptide band of 75 kDa on 7.5% SDS/PAGE. Amino acid composition data shows the presence of a high number of acidic amino acids in the molecule in relation to the pI value of 4.6. The inhibitor binds Na+,K+-ATPase reversibly and blocks ATP binding sites at micromolar concentrations with an I50 of approximately 700 nm. As a result, formation of the phosphorylated intermediate of Na+,K+-ATPase is hindered in the presence of the inhibitor. It does not affect p-nitrophenylphosphatase activity. Tryptophan fluorescence studies and CD analysis suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor.
通过硫酸铵沉淀、DEAE阴离子交换色谱和羟基磷灰石吸附柱色谱，从大鼠脑细胞溶胶中纯化出一种钠钾ATP酶抑制蛋白，且达到了均一性。纯化后的蛋白在7.5%的SDS/PAGE上迁移为一条75 kDa的单一条带。氨基酸组成数据显示，该分子中酸性氨基酸的数量较多，其pI值为4.6。该抑制剂与钠钾ATP酶可逆结合，并在微摩尔浓度下阻断ATP结合位点，I50约为700 nM。因此，在存在抑制剂的情况下，钠钾ATP酶磷酸化中间体的形成受到阻碍。它不影响对硝基苯磷酸酶的活性。色氨酸荧光研究和圆二色性分析表明，钠钾ATP酶与抑制剂结合后发生了构象变化。
