标记的腺苷（5'）四磷酸（5'）腺苷（Ap4A）和腺苷（5'）四磷酸（5'）核苷（Ap4N）。用萤火虫荧光素酶进行合成。

Labeled adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')tetraphospho(5')nucleoside (Ap4N). Synthesis with firefly luciferase.

作者信息

Garrido S, Zaera E, Torrecilla A, Sillero A, Günther Sillero M A

机构信息

Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.

出版信息

J Biochem Biophys Methods. 1995 Jun;30(2-3):191-8. doi: 10.1016/0165-022x(95)00007-e.

DOI:10.1016/0165-022x(95)00007-e

PMID:7494096

Abstract

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5')tetraphospho(5')adenosine (Ap4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap4A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [beta, gamma-32P]ATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap4A is obtained. Radioactive adenosine(5')tetraphospho(5')nucleoside (Ap4N) can also be synthesized from ATP gamma S and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive ApnA and pnA (n > 4) can also be approached with luciferase.

摘要

尽管标记的二核苷多磷酸是代谢调节中的重要分子，但它们并无商业供应。萤火虫荧光素酶（EC 1.13.12.7）是合成腺苷（5'）四磷酸（5'）腺苷（Ap4A）的有用酶。由于荧光素酶在低ATP浓度下表现为核苷酸酶，因此应使用适当浓度（高于0.1 mM ATP）以获得高产率的标记Ap4A。[32P]Ap4A也已由ATP和[32P]PPi合成。第一步，在荧光素酶催化的ATP-[32P]PPi交换反应中生成[β，γ-32P]ATP。第二步，向反应中加入焦磷酸酶，得到32P标记的Ap4A。放射性腺苷（5'）四磷酸（5'）核苷（Ap4N）也可由ATPγS和标记的NTP合成，或由低浓度标记ATP和高浓度冷NTP合成。放射性ApnA和pnA（n>4）的合成也可通过荧光素酶实现。