| Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana
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Author:
Date:
2018-06-20
[Abstract] As a fundamental metabolic pathway to degrade and recycle cellular cargos, autophagy is highly induced upon stress, starvation and senescence conditions in plants. A double-membrane structure named autophagosome will form during this process for cargo sequestration and delivery into the vacuole.
A number of regulators have been characterized in plants, including the autophagy-related (ATG) proteins and other plant-specific proteins. Among them, ATG8 will undergo a lipidation process to become a membrane-bound ATG8-phosphatidylethanolamine form and mark the growing autophagosomal membrane as well as the completed autophagosome. Therefore, ATG8 has been regarded as a marker for autophagosomes; and biochemical detection of the membrane-associated form of ATG8 is used as one of ...
[摘要] 作为降解和回收细胞货物的基本代谢途径，自噬在植物的胁迫，饥饿和衰老条件下被高度诱导。 在这个过程中，将形成一个称为自噬体的双膜结构，用于货物隔离和输送到液泡中。
已经在植物中表征了许多调控因子，包括自噬相关（ATG）蛋白和其他植物特异性蛋白。 其中，ATG8将经历脂化过程以成为膜结合的ATG8-磷脂酰乙醇胺形式并标记日益增长的自噬体膜以及完成的自噬体。 因此，ATG8被认为是自噬体的标志; 并且膜结合形式的ATG8的生物化学检测被用作测量自噬活性的主要方法之一。 在这里，我们描述了使用拟南芥幼苗检测ATG8-PE形式的ATG8脂化测定法。
【背景】自噬是调节受损细胞器的大量降解和不需要的细胞内容物的基本代谢过程。在自噬过程中，称为自噬体的双膜结构将形成并将货物递送到液泡中以降解。自噬相关蛋白（ATG）需要调节自噬活性（Liu and ...
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| Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)
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Author:
Date:
2018-03-20
[Abstract] Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the ...
[摘要] 肽聚糖包裹细菌细胞质膜以保护细胞免于因膨胀而导致的溶解。 肽聚糖合成的最后步骤需要称为脂质II的膜锚定底物，其中肽聚糖亚基通过焦磷酸部分连接至载体脂质十一碳烯醇。 脂质II是糖肽抗生素和几种抗微生物肽的靶标，并且通过参与细菌竞争的“攻击”酶来降解以诱导裂解。 在这里，我们分别描述了两种使用薄层色谱法（TLC）和高压液相色谱法（HPLC）的方案来测定磷脂酶如Colicin M或来自中间链球菌的LXG毒素蛋白TelC对脂质II的消化，的。 TLC方法也可以监测十一异戊二烯基（pyro）磷酸盐的消化，而HPLC方法允许分离脂质II的二 - ，单 - 或非磷酸化二糖五肽产物。
【背景】肽聚糖（PG）球囊是一种必需的细菌大分子，它可以保护细胞免受由于其膨胀引起的破裂并保持细胞的形状（Vollmer和Bertsche，2008; Typas等人，2012）。 PG由通过短肽连接的聚糖链组成。来自不同物种的PG在肽的结构和二级修饰的存在方面有所不同（Vollmer等人，2008）。 ...
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