Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.
Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.
复制标题
用腺苷和环 AMP 的 8-叠氮衍生物共价标记人胎盘 S-腺苷高半胱氨酸水解酶的配体结合位点。
DOI：
10.1042/bj2320643
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Hershfield,MS
中科院分区：
文献类型：
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作者：
Aiyar,VN;Hershfield,MS
S-腺苷高半胱氨酸水解酶（S-Adenosylhomocysteine hydrolase，简称S-Hcyase）是一种细胞质腺苷和环腺苷酸结合蛋白。为了研究腺苷和环腺苷酸结合位点之间的关系，我们探索了使用8-叠氮类似物的腺苷和环腺苷酸作为光亲和试剂共价标记从人胎盘纯化的Hcyase。8-叠氮腺苷（8-N3-Ado），像腺苷一样，灭活过氧化氢酶，高碘酸氧化大大增加了灭活率。此外，发现8-N3-Ado参与了脱氢酶催化机制的第一步，导致酶结合的NAD+转化为NADH，尽管它不是完全酶催化反应的底物。放射性标记的8-N3-Ado、其高碘酸氧化衍生物和8-叠氮腺苷3′，5′-磷酸（8-N3-cAMP）特异性地结合于腺苷酸半胱氨酸酶上的腺苷结合位点，并在照射后与该酶共价连接。光亲和标记酶可被抗人谷胱甘肽半胱氨酸酶单克隆抗体沉淀。两个观察结果表明，环AMP和腺苷结合到相同的网站上的脱氢酶。首先，环AMP和腺苷各自阻断了放射性标记的8-N3-Ado和8-N3-cAMP的结合，其次，用V8蛋白酶消化产生了来自已用[32 P]8-N3-cAMP和[3 H]8-N3-Ado光标记的脱氢酶的肽的相同模式。环AMP的结合位点对CYP 4 Hcyase被认为是不同的功能和结构上的环AMP结合位点的环AMP依赖性蛋白激酶的R1调节亚基。
S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3′, 5′-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.