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Interaction of biomimetic lipid membranes with detergents with different physicochemical characteristics.
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-31 DOI: 10.1016/j.chemphyslip.2025.105473
Mariana S S Oliveira, Amanda C Caritá, Karin A Riske

Membrane solubilization by detergents is routinely performed to separate membrane components, and to extract and purify membrane proteins. This process depends both on the characteristics of the detergent and properties of the membrane. Here we investigate the interaction of eight detergents with very distinct physicochemical features with model membranes in different biologically relevant phases. The detergents chosen were the non-ionic Triton X-100, Triton X-165, C10E5, octyl glucopyranoside (OG) and dodecyl maltoside (DDM) and the ionic sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Chaps. Three lipid compositions were explored: pure palmitoyl oleoyl phosphatidylcholine (POPC), in the liquid-disordered (Ld) phase, sphingomyelin (SM)/cholesterol 7:3 (chol) in the liquid-ordered (Lo) phase and the biomimetic POPC/SM/chol 2:1:2, which might exhibit Lo/Ld phase separation. Turbidity measurements of small liposomes were performed along the titration with the detergents to obtain the overall solubilization profiles and optical microscopy of giant unilamellar vesicles (GUVs) was used to reveal the mechanism of interaction of the detergents. The presence of cholesterol renders the membranes partly/fully insoluble in all detergents, and the charged detergents are the least effective to solubilize POPC. The non-ionic detergents, with exception of DDM, with the bulkiest headgroup, caused a substantial increase in surface area of POPC, which was quantified directly on single GUVs. The other detergents induced mainly vesicle burst. Detergents that caused some increase in area induced Lo/Ld phase separation in the ternary mixture, with preferential solubilization of the latter. The insoluble area fraction left intact was quantified. Overall, the non-ionic detergents were the most effective in solubilizing lipid membranes.

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引用次数: 0
Broad-based targeted lipidomic analysis of dental fluorosis population in an adult population 成人氟牙症人群的基础广泛靶向脂质组学分析。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-09 DOI: 10.1016/j.chemphyslip.2025.105471
Huiying Kong , Shanshan Liu , Zhenzhen Li , Li Xu , Kai Zhang , Yuanyin Wang
Dental fluorosis, as a common chronic fluoride toxicity oral disease, is mainly caused by long-term excessive intake of fluoride, which seriously affects the aesthetics and function of patients' teeth. In recent years, with the rapid development of metabolomics technology, lipidomics, as an important means to study the changes in lipid metabolism in organisms, has shown great potential in revealing the mechanisms of disease development. As a major component of cell membranes and a signaling molecule, metabolic disorders of lipids are closely related to a variety of diseases, but the specific mechanism of action in dental fluorosis is still unclear. Therefore, the present study aimed to systematically analyze the differences in lipid profiles between dental fluorosis patients and healthy populations by using broad-based targeted lipidomics technology to provide new perspectives on the pathogenesis of dental fluorosis. To this end, the researchers compared the salivary lipidome of healthy participants with the salivary micro lipidome of dental fluorosis patients. Their saliva samples were collected, and advanced broad-based targeted lipidomics technology, combined with a high-performance liquid chromatography-mass spectrometry (LC-MS) system, was used to comprehensively detect and quantify the lipids in the samples. The lipid data were processed and analyzed by bioinformatics to identify the unique patterns of changes in the lipid profiles of dental fluorosis patients and to verify the significance of these changes using statistical methods. Several glycerophospholipids, fatty acyls, and sphingolipids exhibited marked alterations in dental Among these, glycocholic acid, LPA (18:4), taurolithocholic acid-3-sulfate, lithocholic acid-3-sulfate, and taurochenodeoxycholic acid-3-sulfate were observed between dental fluorosis patients and healthy controls. taurochenodeoxycholic acid was significantly decreased, while PA (12:0_12:0) levels were significantly elevated. These findings suggest that These findings suggest that disturbances in lipid metabolism play a crucial role in developing dental fluorosis.
氟斑牙是一种常见的慢性氟毒性口腔疾病,主要是由于长期过量摄入氟化物,严重影响患者牙齿美观和功能所致。近年来,随着代谢组学技术的迅速发展,脂质组学作为研究生物体内脂质代谢变化的重要手段,在揭示疾病发生机制方面显示出巨大的潜力。脂质作为细胞膜的主要成分和信号分子,代谢紊乱与多种疾病密切相关,但在氟斑牙中的具体作用机制尚不清楚。因此,本研究旨在通过广泛的靶向脂质组学技术,系统分析氟牙症患者与健康人群的脂质谱差异,为氟牙症的发病机制提供新的视角。为此,研究人员将健康参与者的唾液脂质组与氟斑牙患者的唾液微脂质组进行了比较。采集唾液样本,采用先进的基础广泛的靶向脂质组学技术,结合高效液相色谱-质谱(LC-MS)系统,对样本中的脂质进行全面检测和定量。对脂质数据进行生物信息学处理和分析,以确定氟牙症患者脂质谱变化的独特模式,并使用统计学方法验证这些变化的意义。几种甘油磷脂、脂肪酰基和鞘脂在牙齿中表现出明显的变化,其中,糖胆酸、LPA(18:4)、牛磺酸-3-硫酸石胆酸、硫酸石胆酸-3-硫酸石胆酸和牛磺酸-3-硫酸去氧胆酸在氟斑牙患者和健康对照组中被观察到。taurochenodeoxycholic acid显著降低,PA(12:0 ~ 12:0)水平显著升高。这些发现表明脂质代谢紊乱在氟斑牙的发生中起着至关重要的作用。
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引用次数: 0
Electrophysiological dissection of the ion channel activity of the Pseudomonas aeruginosa ionophore protein toxin Tse5 铜绿假单胞菌离子载体蛋白毒素Tse5离子通道活性的电生理解剖。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-06 DOI: 10.1016/j.chemphyslip.2025.105472
Jessica Rojas-Palomino , Jon Altuna-Alvarez , Amaia González-Magaña , María Queralt-Martín , David Albesa-Jové , Antonio Alcaraz
We present an in-depth electrophysiological analysis of Tse5, a pore-forming toxin (PFT) delivered by the type VI secretion system (T6SS) of Pseudomonas aeruginosa. The T6SS is a sophisticated bacterial secretion system that injects toxic effector proteins into competing bacteria or host cells, providing a competitive advantage by disabling other microbes and modulating their environment. Our findings highlight the dependency of Tse5 insertion on membrane charge and electrolyte concentration, suggesting an in vivo effect from the periplasmic space. Conductance and selectivity experiments reveal a predominant and reproducible pore architecture of Tse5, characterized by a weak cation selectivity without chemical specificity. pH titration experiments suggest a proteolipidic pore structure influenced by both protein and lipid charges, a hypothesis further supported by experiments involving engineered mutants of Tse5 with altered glycine zippers. These results significantly advance our understanding of Tse5's molecular mechanism of toxicity, paving the way for potential applications in biosensing and macromolecular delivery.
本文对铜绿假单胞菌(Pseudomonas aeruginosa) VI型分泌系统(T6SS)释放的成孔毒素(PFT) Tse5进行了深入的电生理分析。T6SS是一种复杂的细菌分泌系统,它将有毒效应蛋白注射到竞争细菌或宿主细胞中,通过使其他微生物丧失功能并调节其环境来提供竞争优势。我们的研究结果强调了Tse5的插入对膜电荷和电解质浓度的依赖性,这表明在体内有来自质周空间的影响。电导和选择性实验揭示了Tse5的主要和可复制的孔隙结构,其特征是弱阳离子选择性,没有化学特异性。pH滴定实验表明,蛋白脂质孔隙结构受到蛋白和脂质电荷的影响,这一假设进一步得到了具有改变甘氨酸拉链的工程突变体Tse5的实验的支持。这些结果极大地促进了我们对Tse5毒性分子机制的理解,为其在生物传感和大分子传递方面的潜在应用铺平了道路。
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引用次数: 0
Laurdan in living cells: Where do we stand? 活细胞中的劳尔丹:现状如何?
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-01 DOI: 10.1016/j.chemphyslip.2024.105458
L. Stefania Vargas-Velez , Natalia Wilke
Laurdan is a valuable tool for analyzing phase transitions and general behavior in synthetic lipid membranes. Its use is very straightforward, thus, its application in cells has expanded rapidly in recent years. It has been demonstrated that Laurdan is very useful for analyzing membrane trends when cells are subjected to some treatment, or when different cell mutations are compared. However, a deep interpretation of the data is not as straightforward as in synthetic lipid bilayers. In this review, we complied results found in mammalian and bacterial cells and noted that the use of Laurdan could be improved if a comparison between publications could be done. At the moment this is not easy, mainly due to the lack of complete information in the publications, and to the different methodologies employed in the data recording and processing. We conclude that research in cell membrane topics would benefit from a better use of the Laurdan probe.
劳尔丹是分析合成脂膜相变和一般行为的重要工具。它的使用非常简单,因此近年来在细胞中的应用迅速扩大。研究表明,当细胞受到某种处理或比较不同的细胞突变时,Laurdan 对分析膜的趋势非常有用。然而,对数据的深入解读并不像在合成脂质双层膜中那样简单。在这篇综述中,我们汇总了在哺乳动物细胞和细菌细胞中发现的结果,并指出如果能对不同出版物进行比较,劳尔丹的使用将得到改善。目前这并不容易,主要原因是出版物中缺乏完整的信息,以及在数据记录和处理中采用了不同的方法。我们的结论是,更好地使用劳尔丹探针将有利于细胞膜课题的研究。
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引用次数: 0
Effect of hydrophobic proteins in modulating the mechanical properties of lung surfactant membranes 疏水蛋白在调节肺表面活性物质膜力学性能中的作用。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-12-21 DOI: 10.1016/j.chemphyslip.2024.105464
Ainhoa Collada , Johann Mertens , Emma Batllori-Badia , Alberto Galindo , Antonio Cruz , Jesús Pérez-Gil
Pulmonary surfactant is a membranous complex that enables breathing dynamics at the respiratory surface. Extremely low values of surface tension are achieved at end-expiration thanks to a unique mixture of lipids and proteins. In particular, the hydrophobic surfactant proteins, specially the protein SP-B, are crucial for surfactant biophysical function, in order to provide the surfactant lipid matrix with the ability to form membranous multi-layered interfacial films that sustain optimal mechanical properties. To analyse the contribution of the proteins to modulate the resistance to mechanical forces of surfactant membrane-based structures, atomic force microscopy of supported lipid bilayers has been used here to determine quantitative mechanical parameters defining the effect of the presence of proteins SP-B and/or SP-C on phospholipid membranes intended to model at least part of the structures integrated into pulmonary surfactant complexes. The results show clear differences introduced by proteins in membrane thickness, lateral packing and elasticity, providing evidence supporting protein-promoted modulating of the mechanical properties of surfactant membranes. These effects are found consistent with the behaviour of two relevant native materials: whole pulmonary surfactant isolated from porcine bronchoalveolar lavages and freshly produced human pulmonary surfactant isolated from amniotic fluid, where it is transferred from the foetal lung before the respiratory air-liquid interface has been established.
肺表面活性物质是一种膜状复合体,使呼吸表面的呼吸动力学成为可能。由于脂质和蛋白质的独特混合物,在过期时达到极低的表面张力值。特别是疏水表面活性剂蛋白,特别是SP-B蛋白,对于表面活性剂的生物物理功能至关重要,以提供表面活性剂脂质基质形成膜状多层界面膜的能力,保持最佳的机械性能。为了分析蛋白质在调节表面活性剂膜基结构对机械力的抵抗力方面的贡献,本文使用了支撑脂质双层的原子力显微镜来确定定量力学参数,以确定蛋白质SP-B和/或SP-C对磷脂膜的影响,旨在模拟至少部分整合到肺表面活性剂复合物中的结构。结果表明,蛋白质在膜厚度、横向堆积和弹性方面存在明显差异,为蛋白质促进表面活性剂膜力学性能的调节提供了证据。这些影响与两种相关天然物质的行为一致:从猪支气管肺泡灌洗液中分离的整个肺表面活性剂和从羊水中分离的新鲜生产的人肺表面活性剂,在呼吸气液界面建立之前从胎儿肺部转移。
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引用次数: 0
The mechanical properties of nerves, the size of the action potential, and consequences for the brain 神经的机械特性,动作电位的大小,以及对大脑的影响。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-30 DOI: 10.1016/j.chemphyslip.2024.105461
Thomas Heimburg
The action potential is widely regarded as a purely electrical phenomenon. However, one also finds mechanical and thermal changes that can be observed experimentally. In particular, nerve membranes become thicker and axons contract. The spatial length of the action potential can be quite large, ranging from millimeters to many centimeters. This suggests the use of macroscopic thermodynamics methods to understand its properties. The pulse length is several orders of magnitude larger than the synaptic gap, larger than the distance of the nodes of Ranvier and even larger than the size of many neurons such as pyramidal cells or brain stem motor neurons. Here, we review the mechanical changes in nerves, we discuss theoretical possibilities to explain them and implications of a mechanical nerve pulse for neurons and for the brain. In particular, the contraction of nerves leads to the possibility of fast mechanical synapses.
动作电位被广泛认为是一种纯电现象。然而,人们也发现可以通过实验观察到机械和热的变化。特别是神经膜变厚,轴突收缩。动作电位的空间长度可以相当大,从毫米到许多厘米不等。这表明可以使用宏观热力学方法来理解其性质。脉冲长度比突触间隙大几个数量级,比兰维耶结的距离大,甚至比锥体细胞或脑干运动神经元等许多神经元的大小还要大。在这里,我们回顾了神经的机械变化,讨论了解释这些变化的理论可能性,以及机械神经脉冲对神经元和大脑的影响。特别是,神经的收缩使快速机械突触成为可能。
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引用次数: 0
Modifications of biological membranes, fat globules and liposomes promoted by cavitation processes. Consequences and applications 空化过程促进生物膜、脂肪球和脂质体的修饰。结果和应用。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-30 DOI: 10.1016/j.chemphyslip.2024.105462
Santiago Fleite , Miryan Cassanello , María del Pilar Buera
Cavitation-based technologies, such as ultrasound (or acoustic cavitation, AC) and hydrodynamic cavitation (HC), are gaining interest among green processing technologies due to their cost effectiveness in operation, toxic solvent use reduction, and ability to obtain superior processed products, compared to conventional methods. Both AC and HC generate bubbles, but their effects may differ and it is difficult to make comparisons as both are based on different phenomena and are subject to different operational variables. AC is one of the most used techniques in extraction and homogenization processes at the laboratory level. However, upscaling to an industrial level is hard. On the other hand, HC is based on the passage of the liquid through a constriction (orifice plate, Venturi, throttling valve), which causes an increase in liquid velocity at the expense of local pressure, forcing the pressure around the contraction below the threshold pressure that induces the formation of cavities. Some applications of cavitation technologies, such as the production of liposomes or lipid nanoparticles (LNPs) allow the generation of delivery systems for biomedical applications.Many others (inactivation of pathogenic viruses, bacteria and algae for water purification, extraction procedures, third generation of biofuel production, green extractions) are based on the disruption of lipid membranes. There are also applications aimed at the modification of membranes (like the milk fat globule) for the development of innovative products. Process parameters, such as cavitation intensity, duration and temperature define the impact of the process on the physical, chemical, and biological characteristics of the membranes. Thus, the adequate implementation of cavitation processes requires understanding of interactions and synergistic mechanisms in complex systems and of their effects on membranes at the microscopic or molecular level. In the present work, the use of cavitation technologies for the generation of LNPs or nanostructured lipid carriers, and the effects of AC and HC treatments on several types of membrane systems (liposomes, solid lipid nanoparticles, milk fat globules, algae and bacterial membranes) are discussed, focusing on the structural and chemical modifications of lipidic structures under cavitation.
基于空化的技术,如超声(或声空化,AC)和流体动力空化(HC),由于其操作成本效益,减少有毒溶剂的使用以及与传统方法相比能够获得更好的加工产品,正在绿色加工技术中引起人们的兴趣。AC和HC都产生气泡,但它们的效果可能不同,很难进行比较,因为它们基于不同的现象,受不同的操作变量影响。AC是实验室水平萃取和均质过程中最常用的技术之一。然而,升级到工业水平是困难的。另一方面,HC是基于液体通过收缩装置(孔板、文丘里管、节流阀),以牺牲局部压力为代价增加液体速度,迫使收缩周围的压力低于诱发空腔形成的阈值压力。空化技术的一些应用,如脂质体或脂质纳米颗粒(LNPs)的生产,允许产生用于生物医学应用的输送系统。许多其他技术(用于水净化的致病性病毒、细菌和藻类灭活、提取程序、第三代生物燃料生产、绿色提取)都是基于破坏脂质膜。还有一些应用是针对膜的修饰(如乳脂球),以开发创新产品。工艺参数,如空化强度、持续时间和温度,决定了工艺对膜的物理、化学和生物特性的影响。因此,充分实施空化过程需要了解复杂系统中的相互作用和协同机制,以及它们在微观或分子水平上对膜的影响。在本工作中,讨论了利用空化技术生成LNPs或纳米结构脂质载体,以及AC和HC处理对几种膜系统(脂质体、固体脂质纳米颗粒、乳脂球、藻类和细菌膜)的影响,重点讨论了空化条件下脂质结构的结构和化学修饰。
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引用次数: 0
The pleomorphic cholesterol sensing motifs of transmembrane proteins 跨膜蛋白的多形性胆固醇感应基序
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-29 DOI: 10.1016/j.chemphyslip.2024.105460
Francisco J. Barrantes
Millions of years of phylogenetic evolution have shaped the crosstalk between sterols and membrane-embedded proteins. This lengthy process, which began before the appearance of eukaryotic cells, has sculpted the two types of molecules to cover a wide spectrum of structural interconnectedness, ranging from rapid touch-and-go hits of low-affinity between surfaces to stronger lock-and-key type structural contacts. The former usually involve relatively loose contacts between linear amino acid sequences on the membrane-exposed transmembrane domains of the protein, readily accessible to the sterols as they briefly visit clefts between adjacent transmembrane segments while in rapid exchange with the bulk lipid bilayer. This operational mode is probably the most ancestral one, since it was already present in primitive bacteria interacting with hopanoid lipids. At the other end of this spectrum are more complex cholesterol binding sites that have required the acquisition of complex 3D non-sequential segments of the membrane protein to establish stereochemically elaborate 3D designs complementary to the rough and smooth surfaces of the eukaryotic neutral lipid, cholesterol. This short review explores cholesterol-membrane protein interactions using membrane protein paradigms having in common their participation in intercellular communications neurotransmission, hormone signalling, amino acid/neurotransmitter transport- and in cancer.
数百万年的系统进化形成了固醇和膜内蛋白之间的相互作用。这个漫长的过程始于真核细胞出现之前,它塑造了两种类型的分子,覆盖了广泛的结构互联性,从表面之间低亲和力的快速触碰到更强的锁-钥匙型结构接触。前者通常涉及蛋白质跨膜结构域上的线性氨基酸序列之间相对松散的接触,当甾醇在与大量脂质双分子层快速交换时,它们会短暂地访问相邻跨膜段之间的间隙,从而很容易接近。这种操作模式可能是最古老的一种,因为它已经存在于与类hopanoid脂相互作用的原始细菌中。在这个光谱的另一端是更复杂的胆固醇结合位点,需要获得膜蛋白的复杂3D非顺序片段,以建立立体化学上精细的3D设计,以补充真核中性脂质胆固醇的粗糙和光滑表面。这篇简短的综述利用膜蛋白范式探讨了胆固醇与膜蛋白的相互作用,它们共同参与细胞间通讯、神经传递、激素信号、氨基酸/神经递质转运以及癌症。
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引用次数: 0
Studying the interfacial activity and structure of pulmonary surfactant complexes 研究肺表面活性复合物的界面活性和结构。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-23 DOI: 10.1016/j.chemphyslip.2024.105459
Ainhoa Collada, Antonio Cruz, Jesús Pérez-Gil
Pulmonary surfactant (PS) is a membranous complex that coats the respiratory air-liquid interface in air-breathing animal lungs. Its main function is to minimize the surface tension at the end of expiration, what is needed for preventing alveolar collapse. Although the tension reduction capabilities of surfactant depend on the formation of air-exposed phospholipid-enriched monolayers, the interfacial surfactant films are far from simple monolayers. Surfactant surface films are dynamically interconnected to continuously secreted newly synthetized material thanks to the action of a pair of very hydrophobic proteins, termed SP-B and SP-C, which are responsible to modulate the biophysical behavior of the complex. Other proteins in the system, such as the hydrophilic SP-A and SP-D, are integrated into different surfactant structures but participate primarily in the immune defense of the lung. In spite of countless studies on the structure and chemico-physical properties of surfactant membranes, the full complexity of surfactant three-dimensional structure is far from being completely understood. Here we review some of the most useful techniques that have allowed the characterization of the PS system along the years to develop the current models interpreting surfactant structure-function relationships.
肺表面活性物质(PS)是一种膜状复合物,在呼吸空气的动物肺中包裹着呼吸空气-液体界面。它的主要功能是在呼气结束时最大限度地降低表面张力,这是防止肺泡塌陷所必需的。虽然表面活性剂降低张力的能力取决于富含磷脂的空气暴露单层的形成,但界面表面活性剂薄膜远非简单的单层。表面活性剂表面膜与不断分泌的新合成物质之间是动态连接的,这要归功于一对非常疏水的蛋白质(称为 SP-B 和 SP-C),它们负责调节复合物的生物物理行为。该系统中的其他蛋白质,如亲水性的 SP-A 和 SP-D,被整合到不同的表面活性物质结构中,但主要参与肺部的免疫防御。尽管对表面活性物质膜的结构和化学物理性质进行了无数次研究,但人们对表面活性物质三维结构的复杂性还远未完全了解。在此,我们回顾了多年来表征表面活性物质系统的一些最有用的技术,以建立当前解释表面活性物质结构与功能关系的模型。
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引用次数: 0
Use of stable isotope-labeled fatty acids to measure desaturase activities with negative chemical ionization GC-MS 利用负化学电离 GC-MS 测量稳定同位素标记的脂肪酸的去饱和酶活性。
IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-08 DOI: 10.1016/j.chemphyslip.2024.105451
Youenn Launay , Iwan Jan , Vincent Ciesielski , Lydie Hue , Mélodie Succar , Léa Fret , Thomas Guerbette , Karima Begriche , Philippe Legrand , Daniel Catheline , Manuel Vlach , Vincent Rioux
Fatty acid desaturases are key enzymes in lipid metabolism. They introduce double bonds between defined carbons of the fatty acyl chain and catalyze rate-limiting steps in the biosynthesis of polyunsaturated fatty acids. For decades, in vitro desaturase activities have been determined by using radiolabeled fatty acids as substrates, incubated with tissue or cell fractions containing membrane-bound desaturases. However, handling radioactivity is being increasingly complicated due to safety and regulatory concern. Radiolabeled fatty acids are also expensive and many of them are not commercially available. There is therefore a crucial need to develop new methods. Although methods using unlabeled fatty acids as substrates have recently been validated, they are well suited for large tissue samples and did not achieve the same sensitivity as the radioactive ones. Here, we show that negative chemical ionization GC-MS on stable isotope-labeled fatty acids, derivatized to pentafluorobenzyl esters, now offers this opportunity, because of its high sensitivity in the selected ion monitoring mode. By using this simple and affordable improved method, we measured the kinetic parameters of mouse liver Δ6-desaturase for its two main substrates (C18:2 n-6 and C18:3 n-3; 10–13 µM). Moreover, this method enabled to compare Δ5-desaturase apparent Km values (19–22 µM) for its two main substrates (C20:3 n-6 and C20:4 n-3). Finally, we re-evaluated the controversial effect of freezing on desaturase activities by using both frozen rat tissues and cryopreserved human hepatocytes. This safe, reliable and sensitive method may be applied to other enzymatic activities involving fatty acids (elongation, hydroxylation) in miniaturized samples.
脂肪酸去饱和酶是脂质代谢中的关键酶。它们在脂肪酰基链的特定碳之间引入双键,并催化多不饱和脂肪酸生物合成过程中的限速步骤。几十年来,体外去饱和酶活性的测定一直是以放射性标记的脂肪酸为底物,与含有膜结合去饱和酶的组织或细胞组分进行孵育。然而,出于安全和监管方面的考虑,处理放射性越来越复杂。此外,放射性标记的脂肪酸价格昂贵,而且很多都无法在市场上买到。因此,亟需开发新的方法。虽然使用未标记脂肪酸作为底物的方法最近得到了验证,但这些方法非常适合大量组织样本,而且无法达到与放射性方法相同的灵敏度。在这里,我们展示了以五氟苄基衍生化的稳定同位素标记脂肪酸为底物的负化学电离气相色谱-质谱(GC-MS),由于其在选择离子监测模式下的高灵敏度,现在提供了这一机会。利用这种简单而经济的改进方法,我们测量了小鼠肝脏Δ6-去饱和酶对其两种主要底物(C18:2 n-6 和 C18:3 n-3;10-13 µM)的动力学参数。此外,这种方法还能比较Δ5-去饱和酶对其两种主要底物(C20:3 n-6 和 C20:4 n-3)的表观 Km 值(19-22 µM)。最后,我们利用冷冻的大鼠组织和冷冻保存的人类肝细胞,重新评估了冷冻对去饱和酶活性的争议性影响。这种安全、可靠和灵敏的方法可用于微型样本中涉及脂肪酸(伸长、羟基化)的其他酶活性。
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引用次数: 0
期刊
Chemistry and Physics of Lipids
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