Phospholipid Acyl Chain Diversity Controls the Tissue-Specific Assembly of Mitochondrial Cardiolipins

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Identification of unusual phospholipids from bovine heart mitochondria by HPLC-MS/MS

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.     

Functional Compartmentalization of the Plasma Membrane of Neurons by a Unique Acyl Chain Composition of Phospholipids

In neurons, the plasma membrane is functionally separated into several distinct segments. Neurons form these domains by delivering selected components to and by confining them within each segment of the membrane. Although some mechanisms of the delivery are elucidated, that of the confinement is unclear. We show here that 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), a unique molecular species of phospholipids, is concentrated at the protrusion tips of several neuronal culture cells and the presynaptic area of neuronal synapses of the mouse brain. In PC12 cells, NGF-stimulated neuronal differentiation induces a phospholipase A1 activity at the protrusion tips, which co-localizes with the OPPC domain. Inhibition of the phospholipase A1 activity leads to suppression of phospholipid remodeling in the tip membrane and results in disappearance of the OPPC at the tips. In these cells, confinement of dopamine transporter and Gα_o proteins to the tip was also disrupted. These findings link the lateral distribution of the molecular species of phospholipids to the formation of functional segments in the plasma membrane of neurons and to the mechanism of protein confinement at the synapse.     

Problems of Acyl Migration in Lipase-Catalyzed Enantioselecttve Transformation of Meso-1,3-Diol Systems

In the enantioselective hydrolysis of the di-O-acetyl derivatives of meso-1,3-diol catalyzed by lipases, racemization of the monoacetate products occurs due to non-enzymatic general base-catalyzed acyl migration. The rate of acyl migration increases with increase of pH and buffer concentration. A mechanism of the migration has been proposed to proceed through a six-member ring transition that accounts for the experimental results. The acyl migration, however, was not observed in the enantioselective transesterification of meso-1,3-diols in neutral organic solvents.     

一种水稻酰基辅酶A结合蛋白cDNA的鉴定(英文)

对一水稻ｃＤＮＡ 克隆（Ｒ１９０８） 的分析表明, 其可能编码水稻酰基辅酶Ａ 结合蛋白（ａｃｙｌＣｏＡｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ,ＡＣＢＰ）。Ｓｏｕｔｈｅｒｎ 杂交显示水稻（ Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．） 基因组中仅有一个该基因的拷贝。Ｎｏｒｔｈｅｒｎ 分析表明水稻的ＡＣＢＰ基因在水稻的根、茎、叶、叶鞘、黄化苗和幼穗中皆表达,而以黄化苗的绿苗叶鞘中的表达强度高于绿苗叶片。     

Influence of Acyl Chain Composition on the Degradation of Phosphatidylcholine by Phospholipase D in Carnation Microsomal Membranes

Microsomal membranes isolated from the petals of senescing carnationflowers degrade radiolabelled phosphatidylcholine into phosphatidicacid, diacylglycerol, free fatty acids and fatty acid oxidationproducts. A comparison of the rates of degradation for variousmolecular species of phosphatidylcholine has indicated thathighly perturbing molecular species are preferentially degraded.Two lines of evidence indicate that phosphatidylcholine is actedupon initially by phospholipase D. First, all of the radiolabelledwater soluble products released from choline-labelled phosphatidylcholineare recovered as choline. Second, the temporal patterns in whichradiolabelled degradation products are formed support the contentionthat phosphatidylcholine is degraded by phospholipase D. Thedata have been interpreted as indicating that the differencesin rates of degradation for various molecular species of phosphatidylcholinereflect discrimination of molecular species by phospholipaseD and a preference of the enzyme for highly perturbing molecularspecies. This molecular species preference is exhibited by youngand senescent microsomal membranes and also by membranes thathave been partially solubilized with detergent. Key words: Carnation, membranes, phosphatidylcholine, phospholipase D     

Substrate Selectivity of Glycerol-3-phosphate Acyl Transferase in Rice

Substrate selectivity of glycerol-3-phosphate acyltransferase (EC 2. 3. 1. 15) of rice ( Oryza sativa L.) was explored in a comparative study of acyltransferases from seven plant species. In vitro labeling of acyl carrier protein (ACP) with ¹⁴C or ³H showed that acyltransferase from chill-sensitive plants, such as rice that uses either oleic (18:1) or palmitic acid (16:0) as acyl donor at comparable rates, displays lower selectivity than the enzyme from chill-resistant plants, such as spinach, which preferentially uses oleic acid (18:1) rather than palmitic acid (16:0) as an acyl donor. This may be a result of the size and character of the substrate-binding pocket of acyltransferase. Homology modeling and protein structure-based sequence alignment of acyltransferases revealed that proteins from either chill-sensitive or chill-tolerant plants shared a highly conserved domain containing the proposed substrate-binding pocket. However, the aligned residues surrounding the substrate-binding pocket are highly heterogeneous and may have an influence mainly on the size of the substrate binding pockets of acyltransferases. The substrate selectivity of acyltransferase of rice can be improved by enlarging the substrate-binding pocket using molecular biological methods.     

Spin Trapping of Methyl Radicals by the Acyl Nitroso Compound PH-C (= O)NO Formed in the Photochemical Reaction Between Benzohydroxamic Acid, Dimethyl Sulfoxide and Hydrogen Peroxide. An Epr Study

By the use of EPR spectroscopy, it has been shown that acyl nitroso compounds can act as spin traps for short-lived radicals with the formation of acyl aminoxyl radicals. The reaction was studied for the system benzohydroxamicacid[Ph-C (= O)N(H)] - dimethyl sulfoxide - hydrogen peroxide. The acyl aminoxyl radicals appeared almost immediately when the reaction mixture was irradiated in situ in the EPR cavity with UV light. The trapping reaction involved two photochemical reactions, i.e. the oxidation of the hydroxamic acid to the acyl nitroso compound Ph-C (= O)NO, and the formation of methyl radicals from dimethyl sulfoxide. The EPR spectra are superpositions of the spectra of two species of acyl aminoxyl radicals, i.e. the radicals Ph-C (= O)N(O·)H formed by oxidation of the parent benzohydrox-amic acid, and the radical Ph-C (= O)N(O·)CH₃, formed by trapping of methyl radicals.     

Evaluation of the Sensitivity and Specificity of Polymerase Chain Reaction Test Kit,AMPLICOR Chlamydia trachomatis

The sensitivity and specificity of the polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis, were examined by the use of purified elementary bodies (EBs), cells having inclusions containing reticulate bodies alone and 20 clinical isolates. The numbers of EB and inclusion of C. trachomatis at the detection limit were determined to be approximately 2 to 4 EBs and one inclusion per assay, respectively. No reaction occurred for C. psittaci and C. pneumoniae. All clinical isolates were positively reacted in the PCR assay.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Recovery of human erythrocytes from the echinocyte shape induced by added choline-phospholipids is dependent on the acyl chain length

Addition of an amphiphilic lipid, such as phosphatidylcholine (PC) species with two identical saturated chains or lysophosphatidylcholine (lysoPC) species with one saturated acyl chain of various lengths, into a suspension of intact human erythrocytes resulted in lipid incorporation into the erythrocytes membrane to produce echinocytes (crenated cells). The altered shape gradually reverted on incubation at 37 degrees C until the cells reassumed their normal disc shape. The rate of such recovery of shape increased with decreasing acyl chain length for both PC with C8-C12 acyl chains and lysoPC with a C14-C18 acyl chain, and was strongly influenced by incubation temperature. The identical rate of recovery of shape was observed for cells with normal, decreased or increased ATP content, implying that the metabolic state of the cell had no influence on the recovery process. Recovery of shape is therefore considered to be caused by translocation of the incorporated lipid molecules from the outer to the inner leaflet of the membrane lipid bilayer and the rate of recovery increases with decreasing hydrophobicity of the lipid.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Comparison of Substrate Specificities of Transglutaminases Using Synthetic Peptides as Acyl donors

Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca²⁺-independent microbial transglutaminase (MTGase), and Ca²⁺-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase.     

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid-beta peptide

Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.     

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups and scaffolds. Traditional methods for the preparation of ynones are associated with drawbacks including harsh conditions, multiple purification steps, and the presence of unwanted byproducts. An alternative method for the straightforward preparation of ynones from acyl chlorides and potassium alkynyltrifluoroborate salts is described herein. The adoption of organotrifluoroborate salts as an alternative to organometallic reagents for the formation of new carbon-carbon bonds has a number of advantages. Potassium organotrifluoroborate salts are shelf stable, have good functional group tolerance, low toxicity, and a wide variety are straightforward to prepare. The title reaction proceeds rapidly at ambient temperature in the presence of a Lewis acid without the exclusion of air and moisture. Fair to excellent yields may be obtained via reaction of various aryl and alkyl acid chlorides with alkynyltrifluoroborate salts in the presence of boron trichloride.     

海洋季也蒙毕赤酵母ACP基因的克隆及生物信息学分析

酰基载体蛋白是脂肪酸途径中重要的组件,能够结合脂肪酸代谢途径中各种脂肪酰基中间体,在脂肪酸代谢中是不可或缺的辅因子。以本实验室筛选保存的海洋季也蒙毕赤酵母基因组为模板,PCR扩增酰基载体蛋白基因,获得384 bp的目的片段。生物信息学分析显示,其具有完整的开放阅读框,编码127个氨基酸,有磷酸泛酰巯基乙胺结合位点,为非分泌型亲水性蛋白,不存在信号肽,存在9个潜在的磷酸化位点,二级结构和三级结构主要以α螺旋和无规则卷曲为主,与已知的酰基载体蛋白结构有很高的相似性。     

The Specificity of Peptide Chain Extension by N-Carboxyanhydrides

We have used amino acids activated by carbonyldiimidazole to study the enantiospecificity of peptide elongation in aqueoussolution. Peptide `primers' Glu₁₀ and Ala₃Glu₁₀were elongated with the enantiomers of arginine, glutamic acid,asparagine, phenylalanine, serine and valine. The homochiral addition was always the more efficient reaction; the enantiospecificity was large in some cases but very small in others. In every case Ala₃Glu₁₀ was elongated more efficiently than Glu₁₀.     

Characterization of the cardiac phospholipidome of small cetaceans provides adaptational insight and a foundation for indirect population health screening

The maintenance of a tightly regulated heart lipidome is a general hallmark of mammal health. However, in the case of cetaceans, heart tissue lipid composition remains obscure. This work represents a first in-depth state-of-the-art characterization of the lipid content of small cetacean hearts, namely those of Delphinus delphis, Phocoena phocoena, and Stenella coeruleoalba. Phospholipid classes identified were similar to those found in terrestrial mammals. Regarding fatty acid profiles, they revealed a much higher n-3 fatty acid content than that reported for terrestrial counterparts. Phospholipidome characterization allowed identification of 198 lipid species, including phospholipids, lysophospholipids, and sphingomyelins, generally characterized by a high content of species with esterified n-3 fatty acids. The cardiac lipid profile of the three cetacean species is similar in terms of lipid classes, fatty acids, and even molecular lipid species present. However, quantitative differences suggest a divergence of S. coeruleoalba from the other species in terms of fatty acid and phospholipid profile. The identified differences regarding terrestrial counterparts will contribute to better understanding the details of small cetacean cardiac physiology. Moreover, the characterization of cardiac phospholipidome may represent an opportunity to establish lipidomic-based practical tools to reliably and reproducibly assess small cetacean health population status by remote sampling.