Nuclear magnetic resonance studies of the nucleotide binding sites of porcine adenylate kinase.
Nuclear magnetic resonance studies of the nucleotide binding sites of porcine adenylate kinase.
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猪腺苷酸激酶核苷酸结合位点的核磁共振研究。
DOI：
10.1021/bi00267a014
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Mildvan,AS
中科院分区：
文献类型：
--
作者：
Smith,GM;Mildvan,AS
材料与方法猪腺苷酸激酶购自Sigma。通过聚丙烯酰胺凝胶电泳发现商业制剂的纯度约为60%。在用50 mM Tris-HCl，1 pH 7.5平衡的Sephadex G-50（1.5 × 100 cm）上通过凝胶过滤色谱法除去主要污染物。分子量约为60 000的污染物在空隙体积中洗脱为尖锐条带，腺苷酸激酶[Mr 21 700（Noda，1973）]出现在包含的体积中。纯化的酶的浓度通过其在280 nm处的吸光度[emg/mL= 0.52（Noda，1973）]确定。腺苷酸激酶活性通过如Price等人所述的丙酮酸激酶-乳酸脱氢酶偶联测定法以电化学计量法测量。（1973年）。
Materials and MethodsPorcine adenylate kinase was purchased from Sigma. The commercial preparation was found to be about 60% pure by polyacrylamide gel electrophoresis. Themajor contaminant was removed by gel filtration chromatography on Sephadex G-50 (1.5 X 100 cm) equilibrated with 50 mM Tris-HCl, 1 pH 7.5. The contaminant, which had a molecular weight of~ 60 000, was eluted as a sharp band in the void volume, and adenylate kinase [Mr 21 700 (Noda, 1973)] appeared in the included volume. The concentration of the purified enzyme was determined by its absorbance at 280 nm[eimg/mL= 0.52 (Noda, 1973)]. Adenylate kinase activity was measured spectrophotometrically by the pyruvate kinase-lactate de-hydrogenase coupled assay as described by Price et al.(1973).