| Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
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Author:
Date:
2017-03-20
[Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following ...
[摘要] 核糖体失活蛋白（RIP）是由于其N-糖基化酶（EC 3.2.2.22）活性而不可逆地灭活核糖体的酶。酶从28S rRNA的腺嘌呤编号4324和大鼠核糖体中的核糖（或来自其他生物体的敏感核糖体中的等同腺嘌呤）切割N-糖苷键。该腺嘌呤位于α-原丝素 - 蓖麻毒素环（SRL）中，这对于在原核生物和真核生物中的mRNA-tRNA易位期间分别在核糖体上锚定延伸因子（EF）G和EF2至关重要。 RIP主要从植物中分离出来，这些蛋白质的实例是蓖麻毒蛋白或者口服抗病毒蛋白（PAP）。这些蛋白质，单独或作为免疫毒素的一部分，是癌症治疗的有用工具。以下方案描述了当通过在聚丙烯酰胺凝胶上电泳在来自兔网织红细胞裂解物的RIP处理的脱嘌呤RNA在酸性苯胺存在下孵育时检测释放的RNA片段的方法。发布的片段（Endo的片段）是RIP的动作的诊断。
Endo和Tsurugi首先在大鼠核糖体中描述了RIP对真核28S rRNA的N-糖基化酶活性，蓖麻毒素（Endo and ...
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| Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
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Author:
Date:
2016-07-20
[Abstract] Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the ...
[摘要] 无机多磷酸盐（polyP）是存在于原核和真核生物中的线性聚合物，由通过磷酸酐键连接的三至数百个正磷酸盐残基制成。 这种分子的生物学作用超越了作为P 1储存或能量源以代替ATP。 例如，在酵母中，polyP水平与应激适应相关，并且该分子已经显示为蛋白质多磷酸化的底物。 在这里，我们描述了从酵母酿酒酵母中纯化polyP的两种不同方法和随后的通过分光光度法测量在酶水解纯化的polyP时产生的Pi来定量polyP水平的方案。 必须注意的是，所使用的纯化方案极大地影响所获得的polyP值。
图1. polyP的酶水解
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