谷胱甘肽

谷胱甘肽 Glutathione is a tripeptide with a gamma peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain. It is an antioxidant, preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides. Thiol groups are reducing agents, existing at a concentration of approximately 5 mM in animal cells. Glutathione reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor. In the process, glutathione is converted to its oxidized form glutathione disulfide (GSSG), also called L(-)-Glutathione. Once oxidized, glutathione can be reduced back by glutathione reductase, using NADPH as an electron donor. The ratio of reduced glutathione to oxidized glutathione within cells is often used as a measure of cellular toxicity. Biosynthesis Glutathione is not an essential nutrient, it can be synthesized in the body from the amino acids L-cysteine, L-glutamic acid, and glycine. The sulfhydryl (thiol) group (SH) of cysteine serves as a proton donor and is responsible for the biological activity of glutathione. Cysteine is the rate-limiting factor in cellular glutathione synthesis, since this amino acid is relatively rare in foodstuffs. Glutathione is synthesized in two adenosine triphosphate-dependent steps: , First, gamma-glutamylcysteine is synthesized from L-glutamate and cysteine via the enzyme gamma-glutamylcysteine synthetase. This reaction is the rate-limiting step in glutathione synthesis. , Second, glycine is added to the C-terminal of gamma-glutamylcysteine via the enzyme glutathione synthetase. Function Glutathione exists in reduced (GSH) and oxidized (GSSG) states. In the reduced state, the thiol +-group of cysteine is able to donate a reducing equivalent (H+ e) to other unstable molecules, such as reactive oxygen species. In donating an electron, glutathione itself becomes reactive, but readily reacts with another reactive glutathione to form glutathione disulfide (GSSG). Such a reaction is probable due to the relatively high concentration of glutathione in cells (up to 5 mM in the liver). GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR): NADPH reduces FAD present in GSR to produce a transient FADH-anion. This anion then quickly breaks a disulfide bond (Cys58 - Cys63) and leads to Cys63's nucleophilically attacking the nearest sulfide unit in the GSSG molecule (promoted by His467), which creates a mixed disulfide bond (GS-Cys58) and a GS-anion. His467 of GSR then protonates the GS-anion to form the first GSH. Next, Cys63 nucleophilically attacks the sulfide of Cys58, releasing a GS-anion, which, in turn, picks up a solvent proton and is released from the enzyme, thereby creating the second GSH. So, for every GSSG and NADPH, two reduced GSH molecules are gained, which can again act as antioxidants scavenging reactive oxygen species in the cell. In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH) and less than 10% exists in the disulfide form (GSSG). An increased GSSG-to-GSH ratio is considered indicative of oxidative stress. Glutathione has multiple functions: , It is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds, as well as maintaining exogenous antioxidants such as vitamins C and E in their reduced (active) forms. , Regulation of the nitric oxide cycle, which is critical for life but can be problematic if unregulated , It is used in metabolic and biochemical reactions such as DNA synthesis and repair, protein synthesis, prostaglandin synthesis, amino acid transport, and enzyme activation. Thus, every system in the body can be affected by the state of the glutathione system, especially the immune system, the nervous system, the gastrointestinal system and the lungs. , It has a vital function in iron metabolism. Yeast cells depleted of or containing toxic levels of GSH show an intense iron starvation-like response and impairment of the activity of extra-mitochondrial ISC enzymes, followed by death. Methods to determine glutathione Reduced glutathione may be visualized using Ellman's reagent or bimane derivates such as monobromobimane. The monobromobimane method is more sensitive. In this procedure, cells are lysed and thiols extracted using a HCl buffer. The thiols are then reduced with dithiothreitol (DTT) and labelled by monobromobimane. Monobromobimane becomes fluorescent after binding to GSH. The thiols are then separated by HPLC and the fluorescence quantified with a fluorescence detector. Bimane may also be used to quantify glutathione in vivo. The quantification is done by confocal laser scanning microscopy after application of the dye to living cells. Another approach, which allows to measure the glutathione redox potential at a high spatial and temporal resolution in living cells is based on redox imaging using the redox-sensitive green fluorescent protein (roGFP)or redox sensitive yellow fluorescent protein (rxYFP) 谷胱甘肽是一种三肽，由谷氨酸、甘氨酸及半胱氨酸组成。一个肽键由半胱氨酸的羧基与甘氨酸的氨基形成，另外一个肽键与普通的肽键不同，是由谷氨酸的γ-羧基与半胱氨酸的氨基组成的 。它是一种抗氧化剂，可以防止诸如自由基、过氧化物等氧化剂对细胞重要成分造成伤害。 谷胱甘肽的硫醇部分是一种还原剂，在动物细胞中浓度大约为5mmol/L。还原型谷胱甘肽(GSH)可以作为电子给予体来消除细胞质蛋白与半胱氨酸之间的二硫键，在这个过程中，还原型谷胱甘肽被转化为氧化型谷胱甘肽(GSSG)，也被称为L(-)-谷胱甘肽。 一旦被氧化,谷胱甘肽还原酶又可以利用NADPH作为电子给予体将GSSG还原为GSH。因此，常常将细胞中GSSG与GSH量的比例作为细胞毒性的量度。 谷胱甘肽的生物合成 谷胱甘肽不是必需营养物，它可以在体内由L-半胱氨酸、L-谷氨酸和甘氨酸自主合成。半胱氨酸的硫醇部分作为质子给予体保持谷胱甘肽的生物活性。由于在食品中半胱氨酸的含量相对较少，因此半胱氨酸在一定程度上会限制谷胱甘肽的合成。 谷胱甘肽的两个腺苷三磷酸依赖性合成步骤: 第一步:在γ-谷酰胺半胱氨酸连接酶的催化下，L-谷氨酸和半胱氨酸合成 γ-谷酰胺半胱氨酸。由于半胱氨酸的参与，因此第一步是整个过程的限速步骤。 第二步:谷胱氨酸合成酶将甘氨酸连接到γ-谷酰胺半胱氨酸的碳末端。 谷胱甘肽的生物功能 谷胱甘肽有还原型(GSH)和氧化型(GSSG)两种形式。还原态中，其硫醇部分可以提供一个当量的电子来增强一些如活性氧化物等不稳定分子的稳定性，而GSH则被转化为一种活性中间体，并很快和另外一个同样具有活性的中间体合成GSSG。反应之所以发生，很有可能是因为细胞中谷光氨肽的浓度相对较高(肝脏中最多可以达到5mmmol/L). 利用谷胱甘肽还原酶(GSR)将GSSG再生为GSH:NADPH将GSR中的FAD分子还原成阴离子。FAD阴离子会迅速打断Cys58 - Cys63之间的二硫键，形成的Cys63’s残基会亲核性地攻击离它最近距离的GSSG分子(与酶中的His467结合)中的硫化基团，结果形成了一分子复杂的二硫键(GS-Cys58)和一分子GS阴离子。这时，GSR中的His467使GS阴离子质子化从而形成一分子GSH。另外，Cys63亲核性地攻击GS-Cys58，释放一分子GS阴离子，该阴离子从溶剂中得到一分子质子并从酶中释放出来从而形成另一分子GSH。所以，每分子的GSSG和NADPH可以还原得到两分子的GSH，从而保证有足够的GSH来清除细胞中的活性氧化物。 在健康的细胞和组织中，超过90%的谷胱甘肽以GSH形式存在，而只有不足10%的以二硫化物GSSG存在。细胞中GSSG-GSH比例的升高往往作为氧化应激的象征。 谷胱甘肽具有多重生物功能: -- 它是由细胞产生的主要内源型抗氧化剂并直接参与自由基和活性氧化物的中和作 用，同时维持维生素C和E等其它外源型抗氧化剂的活性还原状态。 -- 维持氧化一氮周期的稳定，这对生命非常重要，一旦氧化一氮不稳定，机体就很有可能出现病症。 -- 参与一些代谢和生化反应，如:DNA的合成与修饰、蛋白质合成、前列腺素合成、氨基酸转运、酶活化等。因此，机体的每一个系统都会受到谷胱甘肽系统状态的影响，尤其是免疫系统、神经系统、消化系统和肺部。 -- 在细胞铁代谢中起着重要作用。如酵母菌消耗完GSH或者GSH含量不足时会现出铁饥饿反应和线粒体ISC酶活性降低，继而死亡。 谷胱甘肽的鉴定 利用Ellman试剂或者bimane衍生物(如荧光试剂)可以将还原型谷胱甘肽直观地检测出来。荧光试剂发更灵敏，这种方法中，用盐酸缓冲液将细胞溶解并提取出硫醇;硫醇和二硫苏糖醇共沉淀并被荧光试剂标记。和GSH结合后，荧光试剂表现出荧光性。用HPLC可以将硫醇分离出来，用荧光检测仪可以检测荧光亮度。Bimane也被用于活体内谷胱甘肽的定量检测，在对活体细胞进行染色后用共聚焦激光扫描显微镜进行观察。另外一个方法是:利用对氧化还原反应敏感的绿色或黄色蛋白氧化还原成像性，在一个高时空分辨率的条件下，通过观测氧化还原图像的特点来测定谷胱甘肽氧化还原反应的电动势，从而进一步测得谷胱甘肽的含量。