Detecting ricin: sensitive luminescent assay for ricin A-chain ribosome depurination kinetics.
Detecting ricin: sensitive luminescent assay for ricin A-chain ribosome depurination kinetics.
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DOI：
10.1021/ac8026433
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发表时间：
2009-04-15
影响因子：
7.4
通讯作者：
Schramm, Vern L.
中科院分区：
文献类型：
--
作者：
Sturm, Matthew B.;Schramm, Vern L.
蓖麻毒素是植物中发现的致死性核糖体失活蛋白（RIP）家族成员。蓖麻毒素A链（RTA）是蓖麻毒素A链的一种，它能催化真核生物核糖体RNA的GAGA四环上的一个碱基水解脱嘌呤，从而从肌毒蛋白-蓖麻毒素环（SRL）中释放出一个腺嘌呤。蛋白质合成被延伸因子结合的丧失抑制，导致细胞死亡。我们报告了一个灵敏的耦合分析测量腺嘌呤释放的核糖体或小茎环RNA的RTA催化。腺嘌呤磷酸核糖转移酶（APRTase）和丙酮酸正磷酸二激酶（PPDK）将腺嘌呤转化为ATP，用于萤火虫荧光素酶的定量。所产生的AMP循环为ATP，以产生与腺嘌呤浓度成比例的持续发光。亚皮摩尔腺嘌呤定量允许RTA对真核核糖体的作用在连续的高通量测定中进行。RIP催化活性的简易分析将在植物毒素检测、抑制剂筛选、寡核苷酸和完整核糖体上的脱嘌呤剂的机理分析以及癌症免疫化疗中具有应用。RTA对兔网织红细胞80 S核糖体的催化作用的动力学分析确定了2.6 × 108 M− 1 s −1的催化效率，这是一种扩散限制反应，表明即使使用大的反应物也能进行完美的催化。
Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic ribosomal RNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of elongation factor binding resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Sub-picomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 × 108 M−1s−1, a diffusion limited reaction indicating catalytic perfection even with large reactants.
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