Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate.
Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate.
复制标题
2-叠氮腺苷 5-[α-32P]二磷酸对线粒体三磷酸腺苷酶进行光亲和标记。
作者：
F. Boulay;P. Dalbon;P. Vignais
制备了α位32P标记的2-叠氮腺苷-5‘-二磷酸(2-叠氮腺苷-ADP)，并用于光标记牛心线粒体F1-ATPase的核苷酸结合部位。原生F1由Knowles和Penefsky的程序准备[Knowles，A.F.，&Penefsky，H.S.(1972)J.Biol.化学。247,6617-6623]平均每摩尔酶含有2.9mol紧密结合的ADP和ATP。将F1与微摩尔浓度的[α-32P]-2-叠氮基-ADP在添加镁离子的黑暗中短期孵育，可迅速与F1的3摩尔标记/摩尔结合，这与每个F1存在6个核苷酸结合位点一致。[α-32P]-2-叠氮基-ADP与线粒体F1在黑暗中可逆结合的Kd在氯化镁存在下为5微米，在乙二胺四乙酸存在下为30微米。F_1的失活百分率与[α-32P]-2-叠氮-ADP共价光标记的程度呈线性关系，失活百分率高达90%，外推为F_1的共价结合的[α-32P]-2-叠氮-ADP/摩尔。在这些条件下，只有β亚基被光标记。每个β亚基一个光标记物的共价结合是通过根据电荷差异对标记的和未标记的β亚基进行电泳法分离，并通过图谱研究显示每个光标记β亚基有一个主要的放射性多肽片段来确定的。
2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
2-叠氮腺苷 [β-32P]二磷酸与完整人血小板上的受体结合，抑制腺苷酸环化酶。
影响因子：
2.9
作者：
Macfarlane,DE;Mills,DC;Srivastava,PC
通讯作者：
Srivastava,PC