甘三酯立体专一分析的研究：（Ⅱ）甘三酯立体专一分析方法初探

在第一部分综合讨论的基础上对甘三酯立体专一分析方法作了初步探讨。以液体油(菜油)及固体脂(猪脂)为基质,系统地研究了这一分析方法,取得了经验并补充了一些具体验证方法。分析液体油的结果与文献数据相一致,分析猪脂的结果欠佳,还存在一些问题需待进一步研究。     

Link between Lipid Second Messengers and Osmotic Stress in Plants

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.     

磷脂酶用于大豆油脱胶的研究

植物油的酶法脱胶是一种新的大豆油脱胶方法。利用新型微生物磷脂酶Lecitase Ultra进行大豆油脱胶的研究,探讨了若干操作参数对大豆油脱胶效果的影响,确定了该酶较优的反应条件:反应时间200 min,加酶量25 mg/kg,pH值4.8,温度46~48℃,大豆油含磷量能降到4.7 mg/kg。结果表明,Lecitase Ultra应用于植物油脱胶效果好且稳定,是一种更适宜于工业化应用的酶种。     

Hepatic patatin-like phospholipase domain-containing protein 3 sequence,single nucleotide polymorphism presence,protein confirmation,and responsiveness to energy balance in dairy cows

Patatin-like phospholipase domain-containing protein 3 (PNPLA3), commonly known as adiponutrin, is part of a novel subfamily of triglyceride lipase enzymes with potential effects on triglyceride metabolism in adipose and hepatic tissues. The predicted bovine PNPLA3 sequence has been identified, but expression of the gene had not been examined. The objectives of this study were to confirm the predicted bovine PNPLA3 gene sequence, determine expression of the bovine PNPLA3 gene in response to whole-animal energy balance, identify single nucleotide polymorphisms present in dairy cows, and verify the presence of the protein in the liver. Using liver biopsy samples collected from cows at +28 d relative to calving (DRTC), RNA was isolated and used to generate a cDNA template for amplification of the entire predicted coding sequence of PNPLA3 via PCR. To determine if energy balance alters the expression of PNPLA3, RNA was isolated and mRNA expression quantified in liver samples from mid-lactation cows after a 5-d ad libitum period (n = 5) and after a subsequent 5-d 50% feed restriction period (n = 5), and in samples collected from cows at −14, +1, +14, and +28 DRTC (n = 16). The presence of PNPLA3 protein was detected by Western blot in liver protein samples collected at +28 DRTC. Expression of hepatic PNPLA3 was decreased after a period of feed restriction (8.14 vs. 1.08 ± 2.17 arbitrary units, ad libitum vs. fasted). Expression of PNPLA3 mRNA was decreased at +1 and +14 DRTC compared with −14 DRTC (23.35, 7.28, 10.17, and 14.5 ± 4.9 arbitrary units, −14, +1, +14, and +28 DRTC, respectively). The presence of PNPLA3 protein was detected as a 55-kDa band in hepatic protein isolations from liver tissue collected at +28 DRTC. These data confirm the presence and sequence of the bovine hepatic PNPLA3 gene and single nucleotide polymorphisms. Furthermore, these data indicate responsiveness of bovine hepatic PNPLA3 to energy balance.     

An aqueous suspension system for phospholipase D-mediated synthesis of PS without toxic organic solvent

Enzymatic synthesis of PS by phospholipase D (PLD)-mediated transphosphatidylation in an aqueous media was investigated. The purpose of this study was to establish a novel synthetic method where no toxic organic solvents were used. An attempt to react soybean lecithin (simply dispersed in an aqueous buffer) with an aqueous solution of l-serine and PLD was unsuccessful, giving only 20% of PS. By contrast, a suspension of lecithin adsorbed on fine powders such as silica was effectively converted into PS in an aqueous solution of l-serine and PLD. After screening various powders for use as the lecithin adsorbent, calcium sulfate was found to be the best with respect to lecithin conversion. In addition, calcium sulfate did not require prior adsorption of lecithin (i.e., the reaction proceeded effectively simply by adding the powder to an aqueous mixture of lecithin, l-serine, and PLD). With this “aqueous suspension system” of calcium sulfate, up to 180 mg/mL lecithin was completely converted, resulting in more than 80% PS in 24 h. The synthesized PS could easily be recovered from the powder by extracting with a mixture of n-hexane, ethanol, and diluted HCl.     

Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C

The nucleotide sequence of a 4.3 kb fragment downstream of the LIG4 gene of Candida albicans has been determined. This fragment contains two entire ORFs (ORF1 and ORF2) and a truncated one (ORF3). ORF1 (1029 bp; EMBL databank, Accession No. AJ277539) encodes a putative protein of 343 amino acids with a high degree of similarity to phosphatidylinositol-specific phospholipases C (PI-PLC) of bacterial origin and, to a lesser degree, to similar proteins from trypanosome, fly and human. Isolated ORF1 confers PI-PLC activity to Escherichia coli transformants. ORF2 (1572 bp; EMBL databank, Accession No. AJ277538) predicts a protein of 524 amino acids with high similarity along most of the entire length to Ydr393w from Saccharomyces cerevisiae. This protein carries a domain with significant similarity to several cytoskeleton proteins of different origins. YDR393w (SHE9) is an orphan gene whose overexpression compromises cell growth. ORF3 appears to encode the homologue of the well-conserved proteasomal 26S regulatory subunit.     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipaseA₂ in catalysis and substrate binding, using site-directed mutagenesis.A mutant was constructed containing Phe at position 69. Kineticcharacterization revealed that the Phe-69 mutant has retainedenzymatic activity on monomeric and micellar substrates, andthat the mutation has only minor effects on k_cat and K_m. Thisshows that Tyr-69 plays no role in the true catalytic eventsduring substrate hydrolysis. In contrast, the mutation has aprofound influence on the stereospecificity of the enzyme. Whereasthe wild-type phospholipase A₂ is only able to catalyse thedegradation of sn-3 phospholipids, the Phe-69 mutant hydrolysesboth the sn-3 isomers and, at a low (1–2%) rate, the sn-1isomers. Despite the fact that the stereospecificity of themutant phospholipase has been altered, Phe-69 phospholipasestill requires Ca²⁺ ions as a cofactor and also retains itsspecificity for the sn-2 ester bond. Our data suggest that inporcine pancreatic phospholipase A₂ the hydroxyl group of Tyr-69serves to fix and orient the phosphate group of phospholipidmonomers by hydrogen bonding. Because no such interaction canoccur between the Phe-69 side-chain and the phosphate moietyof the substrate monomer, the mutant enzyme loses part of itsstereospecificity but not its positional specificity.     

An Air‐Supported Liquid Crystal System for Real‐Time and Label‐Free Characterization of Phospholipases and Their Inhibitors

A new air‐supported liquid crystal (LC) system for analyzing interfacial phenomena that occur based on the molecular interaction between LCs and adsorbed molecules of interest at the aqueous/LC interface is reported. Compared with existing LC‐based detection systems, the miniature system reported here requires less sample and involves simpler preparation. Using this system, the enzymatic hydrolysis of various phospholipases such as phospholipase A₂ (PLA₂), phospholipase C (PLC) and phospholipase D (PLD) are characterized. The hydrolysis of phospholipid monolayers self‐assembled at aqueous/LC interface induces an orientational response from the LCs. As a result, an optical signal that reflects the spatial and temporal distribution of phospholipids during the enzymatic reaction can be generated in a real‐time manner. When well‐known phospholipase inhibitors are introduced together with respective phospholipases, no orientational response of LCs is observed. In the case of inhibitors MJ33 and compound 48/80, cross‐inhibitions among phospholipases are also observed. This work demonstrates that the air‐supported LC system provides a facile label‐free assay for characterizing phospholipase activities and for screening enzyme inhibitors. It could potentially be useful for different high throughput and cost‐effective enzyme screening assays.     

大麦根磷脂酶提取条件研究

大麦根中含有较高活力的磷脂酶。提取磷脂酶的条件为 1 0倍大麦根重量的水浸提 2 0小时 ,浸提温度 2 0℃ ,p H6.5。温度对磷脂酶提取影响最大 ,依次为 p H和提取时间。Ca2 +对磷脂酶有激活作用 ,当 Ca2 +浓度 0 .0 0 5mol/L,反应温度 40℃时酶活力达最大值 ,65℃以上酶基本失活