In vitro Measurement of CMP-Sialic Acid Transporter Activity in Reconstituted Proteoliposomes
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Author:
Date:
2020-03-20
[Abstract] Nucleotide-sugar transporters (NSTs) facilitate eukaryotic cellular glycosylation by transporting nucleotide-sugar conjugates into the Golgi lumen and endoplasmic reticulum for use by glycosyltransferases, while also transferring nucleotide monophosphate byproducts to the cytoplasm. Mutations in this family of proteins can cause a number of significant cellular pathologies, and wild type members can act as virulence factors for many parasites and fungi. Here, we describe an in vitro assay to measure the transport activity of the CMP-sialic acid transporter (CST), one of seven NSTs found in mammals. While in vitro transport assays have been previously described for CST, these studies failed to account for the fact that 1) commercially available stocks of CMP-sialic acid ...
[摘要] [ 摘要] 核苷酸糖转运蛋白(NST)通过将核苷酸糖结合物转运到高尔基腔和内质网供糖基转移酶使用,同时将核苷酸单磷酸副产物转移到细胞质中,从而促进了真核细胞的糖基化。该蛋白质家族的突变可引起许多重要的细胞病理,野生型成员可充当许多寄生虫和真菌的致病因子。在这里,我们描述了一种体外测定法,以测量CMP-唾液酸转运蛋白(CST)的转运活性,CMP-唾液酸转运蛋白(CST)是在哺乳动物中发现的七个NST之一。虽然在体外 以前已经针对CST进行了转运分析,但这些研究未能说明以下事实:1 )CMP- 唾液酸(CMP-Sia )的商业库存由约10%的高亲和力CMP组成,以及2)CMP- SIA 是水解d 到CMP和在水溶液中的唾液酸。在这里,我们描述了一种用非选择性磷酸酶南极磷酸酶治疗CMP-Sia的方法,以将所有游离CMP转化为胞苷。这使我们能够准确地测量重组为蛋白脂质体的纯化CST的底物亲和力和运输动力学。
[ 背景技术]一旦在细胞质或细胞核合成,核苷酸偶联的糖被输送到内质网的内腔(ER)由核苷酸-糖转运蛋白(NSTS)或高尔基体(青木等人,2003)。在这些亚细胞区室中,糖基转移酶利用糖部分糖基化脂质和蛋白质,产生副产物核苷酸单磷酸酯(NMP)(Capasso和Hirschberg,1984 ;Milla和Hirschberg ,1989; ...
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Self-organization Assay for Min Proteins of Escherichia coli in Micro-droplets Covered with Lipids
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Author:
Date:
2020-03-20
[Abstract] The Min system determines the cell division plane of bacteria. As a cue of spatiotemporal regulation, the Min system uses wave propagation of MinD protein (Min wave). Therefore, the reconstitution of the Min wave in cell-sized closed space will lead to the creation of artificial cells capable of cell division. The Min waves emerge via coupling between the reactions among MinD, MinE, and ATP and the differences in diffusion rate on the cell membrane and in the cytoplasm. Because Min waves appear only under the balanced condition of the reaction-diffusion coupling, special attentions are needed towards several technical points for the reconstitution of Min waves in artificial cells. This protocol describes a technical method for stably generating Min waves in artificial cells.
[摘要] [摘要 ] Min系统确定细菌的细胞分裂平面。作为时空调节的提示,Min系统使用MinD 蛋白的波传播(Min wave)。因此,Min波在细胞大小的封闭空间中的重构将导致能够分裂细胞的人造细胞的产生。闵波出现经由耦合之间反应小号中MinD的,的MinE ,和ATP 和所述differenc ES 在细胞膜上的扩散速度和在细胞质中。因为最小波仅在反应扩散耦合的平衡条件下出现, 特别关注,需要对几个技术要点为闽波在人造细胞重建。该协议描述了一种在人造细胞中稳定产生Min波的技术方法。
[背景 ] 敏系统,它决定了细胞分ER 对称细胞分裂,是在细菌细胞内的组织系统的最显着的例子之一(Rothfield 等人,2005;和罗利特马戈林,2013年)。敏系统使用图案形成在细胞内的时间依赖性蛋白梯度的公知的作为敏波(宽松等人,2008; Halatek和Frey,2012;邦尼等人,2013; Zieske 。等人,2016 ; Kohyama 。等人, 2019 )。Min波是由两种蛋白MinD 和MinE 的反应扩散耦合产生的。通过与ATP结合,MinD 形成二聚体并附着在膜上。的MinE 被招募到的ATP MinD的和诱导ATP酶的活性MinD的。通过MinE ,ATP- MinD 变为ADP- MinD ,并从膜上脱离。ADP- MinD的被转换回ATP- ...
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