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Sample records for diacylglycerol lipase inhibitor

  1. Comprehensive Analysis of Structure Activity Relationships of α-Ketoheterocycles as sn-1-Diacylglycerol Lipase α Inhibitors

    PubMed Central

    Janssen, Freek J.; Baggelaar, Marc P.; Hummel, Jessica J. A.; Overkleeft, Herman S.; Cravatt, Benjamin F.; Boger, Dale L.; van der Stelt, Mario

    2015-01-01

    Diacylglycerol lipase α (DAGLα) is responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα inhibitors are required to study the physiological role of 2-AG. Previously, we identified the α-ketoheterocycles as potent and highly selective DAGLα inhibitors. Here, we present the first comprehensive structure-activity relationship study of α-ketoheterocycles as DAGLα inhibitors. Our findings indicate that the active site of DAGLα is remarkably sensitive to the type of heterocyclic scaffold with oxazolo-4N-pyridines as the most active framework. We uncovered a fundamental substituent effect in which electron-withdrawing meta-oxazole substituents increased inhibitor potency. (C6-C9)-acyl chains with a distal phenyl group proved to be the most potent inhibitors. The integrated SAR data was consistent with the proposed binding pose in a DAGLα homology model. Altogether our results may guide the design of future DAGLα inhibitors as leads for molecular therapies to treat neuroinflammation, obesity and related metabolic disorders. PMID:26584396

  2. Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

    SciTech Connect

    Cabot, M.C.

    1984-08-30

    Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56/sup 0/C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83-86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2-1.0 ..mu..M range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the (/sup 14/C)dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of (/sup 14/)dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never before documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols. 44 references, 3 figures, 1 table.

  3. Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

    PubMed Central

    Buczynski, Matthew W.; Herman, Melissa A.; Natividad, Luis A.; Irimia, Cristina; Polis, Ilham Y.; Pugh, Holly; Chang, Jae Won; Niphakis, Micah J.; Cravatt, Benjamin F.; Roberto, Marisa; Parsons, Loren H.

    2016-01-01

    Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE. PMID:26755579

  4. Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

    PubMed

    Buczynski, Matthew W; Herman, Melissa A; Hsu, Ku-Lung; Natividad, Luis A; Irimia, Cristina; Polis, Ilham Y; Pugh, Holly; Chang, Jae Won; Niphakis, Micah J; Cravatt, Benjamin F; Roberto, Marisa; Parsons, Loren H

    2016-01-26

    Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

  5. Discrimination against diacylglycerol ethers in lipase-catalysed ethanolysis of shark liver oil.

    PubMed

    Fernández, Óscar; Vázquez, Luis; Reglero, Guillermo; Torres, Carlos F

    2013-01-15

    Lipase-catalysed ethanolysis of squalene-free shark liver oil was investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to 10 different lipases were compared. The ratio of oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed, although poor discrimination against diacylglycerol ether was observed. The best results in terms of selectivity and stability were obtained with immobilised lipase from Candida antarctica (Novozym 435). Ethanolysis reaction after 24h in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacylglycerol ethers (10.6%), monoacylglycerol ethers (32.9%) and fatty acid ethyl esters (46.0%). In addition, when an excess of ethanol was used, diacylglycerol ethers completely disappeared after 15 h, giving a final product mainly composed of monoacylglycerol ethers (36.6%) and fatty acid ethyl esters (46.4%).

  6. Biochemical properties of a new cold-active mono- and diacylglycerol lipase from marine member Janibacter sp. strain HTCC2649.

    PubMed

    Yuan, Dongjuan; Lan, Dongming; Xin, Ruipu; Yang, Bo; Wang, Yonghua

    2014-06-12

    Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.

  7. Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases.

    PubMed

    Rodriguez, J A; Mendoza, L D; Pezzotti, F; Vanthuyne, N; Leclaire, J; Verger, R; Buono, G; Carriere, F; Fotiadu, F

    2008-04-15

    In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.

  8. Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats.

    PubMed

    Shimizu, Takahiro; Yamaguchi, Naoko; Okada, Shoshiro; Lu, Lianyi; Sasaki, Tsuyoshi; Yokotani, Kunihiko

    2007-10-01

    Recently, we reported that intracerebroventricularly (i.c.v.) administered histamine evokes the secretion of noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid cascade in the histamine-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by phospholipase A2 (PLA2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, histamine (27 nmol/animal, i.c.v.) -induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.), ET-18-OCH3 (phosphatidylinositol-specific PLC inhibitor) (10 and 30 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.). However, mepacrine (PLA2 inhibitor) (1.1 and 2.2 micromol/animal, i.c.v.) and D609 (phosphatidylcholine-specific PLC inhibitor) (30, 100 and 300 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain phosphatidylinositol-specific PLC and diacylglycerol lipase in the centrally administered histamine-induced activation of the adrenomedullary outflow in rats.

  9. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase

    PubMed Central

    Sahonero-Canavesi, Diana X.; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M.; Geiger, Otto

    2016-01-01

    Summary Phospholipids are well known for their membrane forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth. PMID:25711932

  10. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

    PubMed

    Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M; Geiger, Otto

    2015-09-01

    Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth.

  11. Brain phospholipase C-diacylglycerol lipase pathway is involved in vasopressin-induced release of noradrenaline and adrenaline from adrenal medulla in rats.

    PubMed

    Shimizu, Takahiro; Okada, Shoshiro; Yamaguchi-Shima, Naoko; Yokotani, Kunihiko

    2004-09-19

    Recently, we reported that intracerebroventricularly (i.c.v.) administered arginine-vasopressin evokes the release of noradrenaline and adrenaline from adrenal medulla by brain thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid in the vasopressin-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by two pathways: phospholipase A2 (PLA2)-dependent pathway; phospholipase C (PLC)- and diacylglycerol lipase-dependent pathway. In the present study, therefore, we attempted to identify which pathway is involved in the vasopressin-induced release of both catecholamines from adrenal medulla using urethane-anesthetized rats. Vasopressin (0.2 nmol/animal, i.c.v.)-induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by neomycin [0.28 and 0.55 micromol (250 and 500 microg)/animal, i.c.v.] and 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) [5 and 10 nmol (2.3 and 4.6 microg)/animal, i.c.v.] (inhibitors of PLC), and also by 1,6-bis(cyclohexyloximinocarbonylamino)hexane (RHC-80267) [1.3 and 2.6 micromol (500 and 1000 microg)/animal, i.c.v.] (an inhibitor of diacylglycerol lipase). On the other hand, mepacrine [1.1 and 2.2 micromol (500 and 1000 microg)/animal, i.c.v.] (an inhibitor of PLA2) was largely ineffective on the vasopressin-induced elevation of plasma catecholamines. These results suggest that vasopressin evokes the release of noradrenaline and adrenaline from adrenal medulla by the brain PLC- and diacylglycerol lipase-dependent mechanisms in rats.

  12. Identification of diacylglycerol acyltransferase inhibitors from Rosa centifolia petals.

    PubMed

    Kondo, Hidehiko; Hashizume, Kohjiro; Shibuya, Yusuke; Hase, Tadashi; Murase, Takatoshi

    2011-08-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final step of triacylglycerol (TAG) synthesis, and is considered as a potential target to control hypertriglyceridemia or other metabolic disorders. In this study, we found that the extract of rose petals suppressed TAG synthesis in cultured cells, and that the extract showed DGAT inhibitory action in a dose-dependent manner. Fractionation of the rose extract revealed that the DGAT inhibitory substances in the extract were ellagitannins; among them rugosin B, and D, and eusupinin A inhibited DGAT activity by 96, 82, and 84% respectively, at 10 μM. These substances did not inhibit the activities of other hepatic microsomal enzymes, glucose-6-phosphatase and HMG-CoA reductase, or pancreatic lipase, suggesting that ellagitannins inhibit DGAT preferentially. In an oral fat load test using mice, postprandial plasma TAG increase was suppressed by rose extract; TAG levels 2 h after the fat load were significantly lower in mice administered a fat emulsion containing rose extract than in control mice (446.3 ± 33.1 vs 345.3 ± 25.0 mg/dL, control vs rose extract group; P < 0.05). These results suggest that rose ellagitannins or rose extract could be beneficial in controlling lipid metabolism and used to improve metabolic disorders.

  13. Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans.

    PubMed

    Lin, Yen-Hung; Chen, Yi-Chun; Kao, Tzu-Yu; Lin, Yi-Chun; Hsu, Tzu-En; Wu, Yi-Chun; Ja, William W; Brummel, Theodore J; Kapahi, Pankaj; Yuh, Chiou-Hwa; Yu, Lin-Kwei; Lin, Zhi-Han; You, Ru-Jing; Jhong, Yi-Ting; Wang, Horng-Dar

    2014-08-01

    Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). Here, we report that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.

  14. Myeloid-Specific Deletion of Diacylglycerol Lipase α Inhibits Atherogenesis in ApoE-Deficient Mice

    PubMed Central

    Schöne, Benedikt; Pfeifer, Philipp; Schild, Katharina; Jenniches, Imke; Bindila, Laura; Lutz, Beat; Lütjohann, Dieter; Zimmer, Andreas; Nickenig, Georg

    2016-01-01

    Background The endocannabinoid 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Despite its high concentration in vascular tissue, the role of 2-AG in atherogenesis has not yet been examined. Methods ApoE-deficient mice were sublethally irradiated and reconstituted with bone marrow from mice with a myeloid-specific knockout of the 2-AG synthesising enzyme diacylglycerol lipase α (Dagla) or control bone marrow with an intact 2-AG biosynthesis. After a cholesterol-rich diet for 8 weeks, plaque size and plaque morphology were examined in chimeric mice. Circulating inflammatory cells were assessed by flow cytometry. Aortic tissue and plasma levels of endocannabinoids were measured using liquid chromatography-multiple reaction monitoring. Results Mice with Dagla-deficient bone marrow and circulating myeloid cells showed a significantly reduced plaque burden compared to controls. The reduction in plaque size was accompanied by a significantly diminished accumulation of both neutrophil granulocytes and macrophages in atherosclerotic lesions of Dagla-deficient mice. Moreover, CB2 expression and the amount of oxidised LDL within atherosclerotic lesions was significantly reduced. FACS analyses revealed that levels of circulating inflammatory cells were unaltered in Dagla-deficient mice. Conclusions Myeloid synthesis of the endocannabinoid 2-AG appears to promote vascular inflammation and atherogenesis. Thus, myeloid-specific disruption of 2-AG synthesis may represent a potential novel therapeutic strategy against atherosclerosis. PMID:26731274

  15. Polyphenolic Compounds as Pancreatic Lipase Inhibitors.

    PubMed

    Buchholz, Tina; Melzig, Matthias F

    2015-07-01

    Obesity and its associated diseases such as diabetes mellitus and coronary heart diseases are a major challenge for our society. An important target for the treatment of obesity includes the development of inhibitors of nutrient digestion and absorption. Inhibition of pancreatic lipase and the associated reduction of lipid absorption is an attractive approach for the discovery of potent agents. Currently, the only clinically approved pharmacologic agent as pancreatic lipase inhibitor is Orlistat. However, its usage is compromised by unpleasant gastrointestinal adverse reactions (oily stools, oily spotting, flatulence). The use of botanical materials as a potential source of new drugs is of increasing importance and application. Natural products that are interesting for obesity treatment are generally considered to have less toxic and side effects than totally synthetic drugs. One of the most important sources of potential pancreatic lipase inhibitors represents the class of polyphenols. This article summarizes most studied subclasses of polyphenols including flavonoids, hydroxycinnamic acids, hydroxybenzoic acids and lignans with pancreatic lipase inhibitory effects. A structural comparison of potent inhibitors shows an increased inhibitory effect depending on number and position of phenolic hydroxyl groups, degree of polymerization and elimination of glycosylation during digestion.

  16. Studies on the Substrate and Stereo/Regioselectivity of Adipose Triglyceride Lipase, Hormone-sensitive Lipase, and Diacylglycerol-O-acyltransferases*

    PubMed Central

    Eichmann, Thomas O.; Kumari, Manju; Haas, Joel T.; Farese, Robert V.; Zimmermann, Robert; Lass, Achim; Zechner, Rudolf

    2012-01-01

    Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization. PMID:23066022

  17. Therapeutic potential of monoacylglycerol lipase inhibitors.

    PubMed

    Mulvihill, Melinda M; Nomura, Daniel K

    2013-03-19

    Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.

  18. Inhibition of diacylglycerol lipase (DAGL) in the lateral hypothalamus of rats prevents the increase in REMS and food ingestion induced by PAR1 stimulation.

    PubMed

    Pérez-Morales, Marcel; López-Colomé, Ana María; Méndez-Díaz, Mónica; Ruiz-Contreras, Alejandra E; Prospéro-García, Oscar

    2014-08-22

    Stimulation of the protease-activated receptor 1 (PAR1) in vitro, was shown to induce synaptic retrograde signaling through the endocannabinoid 2-arachidonoylglycerol (2-AG) synthesis and activation of the cannabinoid receptor type 1 (CB1R). The activation of PAR1 by the agonist S1820 in the lateral hypothalamus (LH) increases rapid eye movement sleep (REMS) and food intake in rats, and both effects are prevented by the CB1R inverse agonist AM251. In the present study, we implanted rats with electrodes and with cannulae aimed bilaterally to the LH. We administered tetrahydrolipstatin (THL), an inhibitor of the diacylglycerol lipase (DAGL), the enzyme responsible for 2-AG synthesis, to evaluate the sleep-wake cycle and food ingestion. THL in the LH readily prevented the increase in REMS and food intake induced by PAR1 stimulation, further supporting 2-AG as an upstream activator of PAR1. Our results demonstrate that the effect of PAR1 on REMS and food intake is blocked by the inhibition of DAGL, further suggesting that PAR1 stimulation in the lateral hypothalamus of rats induces an increase in sleep and food intake through 2-AG.

  19. Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity.

    PubMed

    Xu, Tingting; Liu, Lu; Hou, Shulin; Xu, Jinxin; Yang, Bo; Wang, Yonghua; Liu, Jinsong

    2012-06-01

    Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 Å resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.

  20. Refined homology model of monoacylglycerol lipase: toward a selective inhibitor

    NASA Astrophysics Data System (ADS)

    Bowman, Anna L.; Makriyannis, Alexandros

    2009-11-01

    Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue 2-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.

  1. Molecular recognition between pancreatic lipase and natural and synthetic inhibitors.

    PubMed

    Bello, Martiniano; Basilio-Antonio, Lucia; Fragoso-Vázquez, Jonathan; Avalos-Soriano, Anaguiven; Correa-Basurto, José

    2017-05-01

    Pancreatic lipase (PL) is a primary lipase critical for triacylglyceride digestion in humans and is considered as a promising target for the treatment of obesity. Although the current synthetic drugs available for treating obesity have been demonstrated to be effective in inhibiting PL, their prolonged usage results in severe side effects. Based on this argument, in this study, we evaluated the structural and energetic features linked to molecular recognition between two well-known PL inhibitors, orlistat (ORL, synthetic inhibitor) and (-)-epigallocatechin gallate (EGCG, natural inhibitor) and PL through molecular dynamics simulations and free energy calculations of ORL and EGCG at the PL binding site when it is isolated (PL) from the heterodimer complex, forming the heterodimer complex with colipase (PLCL) and lacking structural calcium. Our study showed that the binding free energy of ORL and EGCG to the target correlates with their experimental affinity tendency. The presence of the heterodimer PLCL state, the presence of structural calcium and the type of inhibitor resulted in differences in structural stability and in the map of protein-ligand and protein-protein interactions. Overall, our results suggest that the heterodimer complex and structural calcium are linked to the binding properties of PL.

  2. Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures.

    PubMed

    Wang, Haibo; Zhao, Xiaoping; Wang, Shufang; Tao, Shan; Ai, Ni; Wang, Yi

    2015-05-01

    Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography-mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96 μM, respectively. The ligand-enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures.

  3. Development of a bioautographic method for the detection of lipase inhibitors.

    PubMed

    Bayineni, Venkata Krishna; Suresh, Sukrutha; Singh, Gurmeet; Kadeppagari, Ravi-Kumar

    2014-10-31

    An autobiographic method based on the thin layer chromatogram was developed by using the chemical system that comprises p-Nitrophenyl butyrate and bromothymol blue for detecting the lipase inhibitor. Lipase inhibitory zones were visualized as blue spots against the greenish yellow background. This method could able to detect the well known lipase inhibitor, orlistat up to the concentration of 1ng which is better than the earlier method. This method could also able to detect the lipase inhibition activities from the un-explored species of Streptomyces. The developed method can be used not only for the screening of unknown samples for the lipase inhibitors but also for the purification of the lipase inhibitors from the unknown samples.

  4. A mechanistic study into the epoxidation of carboxylic acid and alkene in a mono, di-acylglycerol lipase.

    PubMed

    Wang, Xuping; Tang, Qingyun; Popowicz, Grzegorz Maria; Yang, Bo; Wang, Yonghua

    2015-05-01

    More and more industrial chemistry reactions rely on green technologies. Enzymes are finding increasing use in diverse chemical processes. Epoxidized vegetable oils have recently found applications as plasticizers and additives for PVC production. We report here an unusual activity of the Malassezia globosa lipase (SMG1) that is able to catalyze epoxidation of alkenes. SMG1 catalyzes formation of peroxides from long chain carboxylic acids that subsequently react with double bonds of alkenes to produce epoxides. The SMG1 is selective towards carboxylic acids and active also as a mutant lacking hydrolase activity. Moreover we present previously unobserved mechanism of catalysis that does not rely on acyl-substrate complex nor tetrahedral intermediate. Since SMG1 lipase is activated by allosteric change upon binding to the lipophilic-hydrophilic phase interface we reason that it can be used to drive the epoxidation in the lipophilic phase exclusively.

  5. 13C NMR quantification of mono and diacylglycerols obtained through the solvent-free lipase-catalyzed esterification of saturated fatty acids.

    PubMed

    Fernandes, Jane Luiza Nogueira; de Souza, Rodrigo Octavio Mendonça Alves; de Vasconcellos Azeredo, Rodrigo Bagueira

    2012-06-01

    In the present investigation, we studied the enzymatic synthesis of monoacylglycerols (MAG) and diacylglycerols (DAG) via the esterification of saturated fatty acids (stearic, palmitic and an industrial residue containing 87% palmitic acid) and glycerol in a solvent-free system. Three immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) and different reaction conditions were evaluated. Under the optimal reaction conditions, esterifications catalyzed by Lipozyme RM IM resulted in a mixture of MAG and DAG at high conversion rates for all of the substrates. In addition, except for the reaction of industrial residue at atmospheric pressure, all of these products met the World Health Organization and European Union directives for acylglycerol mixtures for use in food applications. The products were quantified by (13)C NMR, with the aid of an external reference signal which was generated from a sealed coaxial tube filled with acetonitrile-d3. After calibrating the area of this signal using the classical external reference method, the same coaxial tube was used repeatedly to quantify the reaction products.

  6. Pancreatic lipase inhibitors from natural sources: unexplored potential.

    PubMed

    Birari, Rahul B; Bhutani, Kamlesh K

    2007-10-01

    The prevalence of obesity is increasing at an alarming rate, but, unfortunately, only a few medications are currently on the market. Obesity is primarily regarded as a disorder of lipid metabolism and the enzymes involved in this process could be selectively targeted to develop antiobesity drugs. Recently, newer approaches for the treatment of obesity have involved inhibition of dietary triglyceride absorption via inhibition of pancreatic lipase (PL) as this is the major source of excess calories. Natural products provide a vast pool of PL inhibitors that can possibly be developed into clinical products. This article reviews various extracts and secondary metabolites from plants and microbial origin with PL inhibitory activity that can be focused for drug development programs.

  7. Screening of lipase inhibitors from Scutellaria baicalensis extract using lipase immobilized on magnetic nanoparticles and study on the inhibitory mechanism.

    PubMed

    Wan, Li-Hong; Jiang, Xiao-Lan; Liu, Yi-Ming; Hu, Jin-Jie; Liang, Jian; Liao, Xun

    2016-03-01

    Scutellaria baicalensis is a traditional Chinese medicinal plant possessing a wide variety of biological activities. In this work, lipase immobilized on magnetic nanoparticles (LMNPs) was used as solid phase extract absorbent for screening of lipase inhibitors from this plant. Three flavonoids were found to bind to LMNPs and were identified as baicalin, wogonin, and oroxylin A by liquid chromatography-mass spectrometry (HPLC-MS). Their IC50 values were determined to be 229.22 ± 12.67, 153.71 ± 9.21, and 56.07 ± 4.90 μM, respectively. Fluorescence spectroscopy and molecular docking were used to probe the interactions between these flavonoids and lipase. All the flavonoids quenched the fluorescence of lipase statically by forming new complexes, implying their affinities with the enzyme. The thermodynamic analysis suggested that van der Waals force and hydrogen bond were the main forces between wogonin and lipase, while hydrophobic force was the main force for the other two flavonoids. The results from a molecular docking study further revealed that all of them could insert into the pocket of lipase binding to a couple of amino acid residues.

  8. Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors.

    PubMed

    Mateos-Diaz, E; Amara, S; Roussel, A; Longhi, S; Cambillau, C; Carrière, F

    2017-01-01

    Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases.

  9. Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

    PubMed

    Arnadottir, M; Nilsson-Ehle, P

    1994-01-01

    The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions.

  10. Developmental pattern of diacylglycerol lipase-α (DAGLα) immunoreactivity in brain regions important for song learning and control in the zebra finch (Taeniopygia guttata).

    PubMed

    Soderstrom, Ken; Wilson, Ashley R

    2013-11-01

    Zebra finch song is a learned behavior dependent upon successful progress through a sensitive period of late-postnatal development. This learning is associated with maturation of distinct brain nuclei and the fiber tract interconnections between them. We have previously found remarkably distinct and dense CB1 cannabinoid receptor expression within many of these song control brain regions, implying a normal role for endocannabinoid signaling in vocal learning. Activation of CB1 receptors via daily treatments with exogenous agonist during sensorimotor stages of song learning (but not in adulthood) results in persistent alteration of song patterns. Now we are working to understand physiological changes responsible for this cannabinoid-altered vocal learning. We have found that song-altering developmental treatments are associated with changes in expression of endocannabinoid signaling elements, including CB1 receptors and the principal CNS endogenous agonist, 2-AG. Within CNS, 2-AG is produced largely through activity of the α isoform of the enzyme diacylglycerol lipase (DAGLα). To better appreciate the role of 2-AG production in normal vocal development we have determined the spatial distribution of DAGLα expression within zebra finch CNS during vocal development. Early during vocal development at 25 days, DAGLα staining is typically light and of fibroid processes. Staining peaks late in the sensorimotor stage of song learning at 75 days and is characterized by fiber, neuropil and some staining of both small and large cell somata. Results provide insight to the normal role for endocannabinoid signaling in the maturation of brain regions responsible for song learning and vocal-motor output, and suggest mechanisms by which exogenous cannabinoid exposure alters acquisition of this form of vocal communication.

  11. Discovery of a novel series of benzimidazole derivatives as diacylglycerol acyltransferase inhibitors.

    PubMed

    Lee, Kyeong; Goo, Ja-Il; Jung, Hwa Young; Kim, Minkyoung; Boovanahalli, Shanthaveerappa K; Park, Hye Ran; Kim, Mun-Ock; Kim, Dong-Hyun; Lee, Hyun Sun; Choi, Yongseok

    2012-12-15

    A novel series of benzimidazole derivatives was prepared and evaluated for their diacylglycerol acyltransferase (DGAT) inhibitory activity using microsome from rat liver. Among the newly synthesized compounds, furfurylamine containing benzimidazole carboxamide 10j showed the most potent DGAT inhibitory effect (IC(50)=4.4 μM) and inhibited triglyceride formation in HepG2 cells. Furthermore, compound 10j reduced body weight gain of Institute of Cancer Research mice on a high-fat diet and decreased levels of total triglyceride, total cholesterol, and LDL-cholesterol in the blood accompanied with a significant increase in HDL-cholesterol level.

  12. A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics.

    PubMed

    Tschapalda, Kirsten; Zhang, Ya-Qin; Liu, Li; Golovnina, Kseniya; Schlemper, Thomas; Eichmann, Thomas O; Lal-Nag, Madhu; Sreenivasan, Urmila; McLenithan, John; Ziegler, Slava; Sztalryd, Carole; Lass, Achim; Auld, Douglas; Oliver, Brian; Waldmann, Herbert; Li, Zhuyin; Shen, Min; Boxer, Matthew B; Beller, Mathias

    2016-06-01

    Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

  13. Lipase

    MedlinePlus

    ... Lipase is used for indigestion, heartburn, allergy to gluten in wheat products (celiac disease), Crohn's disease, and ... that is associated with cystic fibrosis.Allergy to gluten in wheat products (celiac disease). Crohn's disease. Indigestion. ...

  14. Selective Monoacylglycerol Lipase Inhibitors: Antinociceptive versus Cannabimimetic Effects in Mice

    PubMed Central

    Wilkerson, Jenny L.; Mustafa, Mohammed; Abdullah, Rehab; Niphakis, Micah; Wiley, Jenny L.; Cravatt, Benjamin F.; Lichtman, Aron H.

    2015-01-01

    The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30–0.63]) > JZL184 (17.8 [11.6–27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69–1.02]) > JZL184 (24.9 [14.6–42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited

  15. Synthesis and multiparametric evaluation of thiadiazoles and oxadiazoles as diacylglycerol acyltransferase type 1 inhibitors.

    PubMed

    Mougenot, Patrick; Namane, Claudie; Fett, Eykmar; Goumy, Florence; Dadji-Faïhun, Rommel; Langot, Gwladys; Monseau, Catherine; Onofri, Bénédicte; Pacquet, François; Pascal, Cécile; Crespin, Olivier; Ben-Hassine, Majdi; Ragot, Jean-Luc; Van-Pham, Thao; Philippo, Christophe; Chatelain-Egger, Florence; Péron, Philippe; Le Bail, Jean-Christophe; Guillot, Etienne; Chamiot-Clerc, Philippe; Chabanaud, Marie-Aude; Pruniaux, Marie-Pierre; Ménegotto, Jérôme; Schmidt, Friedemann; Venier, Olivier; Viviani, Fabrice; Nicolai, Eric

    2016-01-01

    Chemical modulation of a formerly disclosed DGAT-1 inhibitor resulted in the identification of a compound with a suitable profile for preclinical development. Optimisation of solubility is discussed and a PK/PD study is presented.

  16. Rapid screening natural-origin lipase inhibitors from hypolipidemic decoctions by ultrafiltration combined with liquid chromatography-mass spectrometry.

    PubMed

    Xiao, Shun; Yu, Runru; Ai, Ni; Fan, Xiaohui

    2015-02-01

    Lipase inhibitors generate hypolipidemic effect that is helpful to control or treat some obesity diseases by inactivating catalytic activity of human pancreatic lipase, a key enzyme involved in triglyceride hydrolysis in vivo. Many traditional Chinese medicine (TCM) formulae have been effectively used to treat obesity and other fat related diseases for centuries and modern biological experiments demonstrate therapeutic effect of these formulae can be linked to their lipid-lowering capability in blood. These observations suggest that these hypolipidemic decoctions (HDs) could be a promising resource of natural-origin lipase inhibitors. This work described a rapid approach for screening lipase inhibitors from four widely used HDs, including Wu-Ling-San (WLS), Ze-Xie decoction (ZX), Xiao-Xian-Xiong decoction (XXX) and Xiao-Chai-Hu decoction (XCH), by ultrafiltration combing with high performance liquid chromatography-mass spectrometry (HPLC-MS). Our results showed sixteen natural-origin lipase inhibitors were discovered and identified by high resolution and multistage mass spectrometry. Inhibitory activities of two compounds were confirmed by a functional assay of lipase, which validated the reliability of our approach. Molecular docking simulation was then performed to investigate potential mechanism of action for these compounds. Together we present an efficient method for rapid screening lipase inhibitors from complex natural products, which can be easily accommodated to other important enzymatic system with therapeutic values.

  17. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    PubMed

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway.

  18. Monte Carlo method based QSAR modelling of natural lipase inhibitors using hybrid optimal descriptors.

    PubMed

    Kumar, A; Chauhan, S

    2017-03-08

    Obesity is one of the most provoking health burdens in the developed countries. One of the strategies to prevent obesity is the inhibition of pancreatic lipase enzyme. The aim of this study was to build QSAR models for natural lipase inhibitors by using the Monte Carlo method. The molecular structures were represented by the simplified molecular input line entry system (SMILES) notation and molecular graphs. Three sets - training, calibration and test set of three splits - were examined and validated. Statistical quality of all the described models was very good. The best QSAR model showed the following statistical parameters: r(2) = 0.864 and Q(2) = 0.836 for the test set and r(2) = 0.824 and Q(2) = 0.819 for the validation set. Structural attributes for increasing and decreasing the activity (expressed as pIC50) were also defined. Using defined structural attributes, the design of new potential lipase inhibitors is also presented. Additionally, a molecular docking study was performed for the determination of binding modes of designed molecules.

  19. Inhibitors of pancreatic lipase: state of the art and clinical perspectives

    PubMed Central

    Lunagariya, Nitin A.; Patel, Neeraj K.; Jagtap, Sneha C.; Bhutani, Kamlesh K.

    2014-01-01

    Obesity is a disorder of lipid metabolism and continues to be a global problem, ranking fifth for deaths worldwide. It also leads to diabetes, cardiovascular disorders, musculoskeletal disorders and some types of cancer. Obesity is regarded as the output of a long-term imbalance between energy intake and energy expenditure. Digestion and absorption of dietary lipids by pancreatic lipase, a major source of excess calorie intake, can be targeted for development of anti-obesity agents. Being the major factor of concern, food materials and edible plants are most widely studied for the anti-obesity activity, so that they can be incorporated in the routine diet. In this review, an attempt was made to present a current scenario of the bioactive compounds from plant and microbial origin that have been investigated for their pancreatic lipase inhibition. Compounds belonging to various classes of natural products such as alkaloids, carotenoids, glycosides, polyphenols, polysaccharides, saponins and terpenoids are well studied while lipophilic compounds from microbial sources are the most active against the pancreatic lipase. Few studies on the synthetic analogues, structurally similar to the triglycerides have been described in the review. Despite of tremendous research on the finding of potential pancreatic lipase inhibitor, very few compounds have entered the clinical studies and no new molecule after orlistat has been marketed. Along with HTS based screening, detailed structure-activity relationship studies on semi-synthetic and synthetic derivatives might also provide a direction for the development of potential lead(s) or pharmacophore for pancreatic lipase inhibition in order to treat and/or prevent obesity and related disorders. PMID:26417311

  20. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’ therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  1. Fast identification of lipase inhibitors in oolong tea by using lipase functionalised Fe3O4 magnetic nanoparticles coupled with UPLC-MS/MS.

    PubMed

    Zhu, Yuan-Ting; Ren, Xiao-Yun; Yuan, Li; Liu, Yi-Ming; Liang, Jian; Liao, Xun

    2015-04-15

    Oolong tea is an important member in tea family, which claims for various health benefits such as preventing obesity and improving lipid metabolism. In this work, using pancreatic lipase (PL) functionalised magnetic nanoparticles (PL-MNPs) as solid phase extraction absorbent in combination with ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), we developed a method for rapid screening and identification of lipase inhibitors from oolong tea. Three PL ligands were selectively extracted and identified as (-)-epigallocatechin-3-O-gallate (EGCG), (-)-gallocatechin-3-O-gallate (GCG) and (-)-epicatechin-3-O-gallate (ECG). Their lipase inhibitory activities were significantly higher than those non-ligands. Structure-activity analysis revealed that the presence of a galloyl moiety in the structure was required for binding to PL-MNPs, and therefore, exhibiting a strong inhibition on the enzyme. Taking advantages of the specificity in enzyme binding and the convenience of magnetic separation, this method has great potential for fast screening of lipase inhibitors from natural resources.

  2. Natural inhibitors of pancreatic lipase as new players in obesity treatment.

    PubMed

    de la Garza, Ana Laura; Milagro, Fermín I; Boque, Noemí; Campión, Javier; Martínez, J Alfredo

    2011-05-01

    Obesity is a multifactorial disease characterized by an excessive weight for height due to an enlarged fat deposition such as adipose tissue, which is attributed to a higher calorie intake than the energy expenditure. The key strategy to combat obesity is to prevent chronic positive impairments in the energy equation. However, it is often difficult to maintain energy balance, because many available foods are high-energy yielding, which is usually accompanied by low levels of physical activity. The pharmaceutical industry has invested many efforts in producing antiobesity drugs; but only a lipid digestion inhibitor obtained from an actinobacterium is currently approved and authorized in Europe for obesity treatment. This compound inhibits the activity of pancreatic lipase, which is one of the enzymes involved in fat digestion. In a similar way, hundreds of extracts are currently being isolated from plants, fungi, algae, or bacteria and screened for their potential inhibition of pancreatic lipase activity. Among them, extracts isolated from common foodstuffs such as tea, soybean, ginseng, yerba mate, peanut, apple, or grapevine have been reported. Some of them are polyphenols and saponins with an inhibitory effect on pancreatic lipase activity, which could be applied in the management of the obesity epidemic.

  3. Synthesis and kinetic evaluation of Cyclophostin and Cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases

    PubMed Central

    Point, Vanessa; Malla, Raj K.; Diomande, Sadia; Martin, Benjamin P.; Delorme, Vincent; Carriere, Frederic; Canaan, Stephane; Rath, Nigam P.; Spilling, Christopher D.; Cavalier, Jean-François

    2012-01-01

    New series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. Best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat towards same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these 7-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents. PMID:23095026

  4. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

    PubMed

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

  5. Piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Korhonen, Jani; Kuusisto, Anne; van Bruchem, John; Patel, Jayendra Z; Laitinen, Tuomo; Navia-Paldanius, Dina; Laitinen, Jarmo T; Savinainen, Juha R; Parkkari, Teija; Nevalainen, Tapio J

    2014-12-01

    The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.

  6. R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

    PubMed

    Shimomura, Tomoko; Nakano, Tomoyuki; Goto, Kaoru; Wakabayashi, Ichiro

    2017-02-01

    Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10 μM) inhibited IL-1β (10 ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1 μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

  7. Monoacylglycerol lipase inhibitors produce pro- or antidepressant responses via hippocampal CA1 GABAergic synapses

    PubMed Central

    Wang, Y; Gu, N; Duan, T; Kesner, P; Blaskovits, F; Liu, J; Lu, Y; Tong, L; Gao, F; Harris, C; Mackie, K; Li, J; Tan, Q; Hill, M N; Yuan, Z; Zhang, X

    2017-01-01

    The probability of suffering the mood disorder depression is up to 30% in women and 15% in men during their life span. Pharmacological options for depression are limited: conventional antidepressants have low efficacy and a delayed onset of action (several weeks). Here we investigate the antidepressant actions of inhibitors of monoacylglycerol lipase (MAGL), the major degradative enzyme of the endocannabinoid 2-arachidonoylglycerol. A low-dose of MAGL inhibitors produces antidepressant effects on acute stress-exposed mice, through glutamatergic synaptic long-term depression (LTD), without significant effects on chronic corticosterone-exposed mice. In contrast, a high-dose of MAGL inhibitors produces pro- or antidepressant effects on acute stress- or chronic corticosterone-exposed mice, respectively, through GABAergic synaptic disinhibition. In the hippocampus, in vivo inhibition of MAGL induces a CB1 cannabinoid receptor (CB1R)-dependent suppression of inhibitory GABAergic synapses and an in vivo LTD of excitatory glutamatergic synapses. LTD induction requires CB1R in astroglial cells (but not in GABAergic or glutamatergic neurons) and postsynaptic glutamate receptors. The conventional antidepressant fluoxetine produces rapid or delayed antidepressant effects in acute stress- or chronic corticosterone-exposed mice, respectively. We propose that depression-like behavior of animals in response to acute stress is the normal behavioral response, and thus, MAGL inhibitors, which produce antidepressant effects in chronic corticosterone-exposed animals through GABAergic synaptic disinhibition, represent a new class of rapidly-acting and long-lasting antidepressants. PMID:27001616

  8. Chitosan dosage regimen to trap fecal oil excretion after peroral lipase inhibitor administration in mice.

    PubMed

    Jang, Yura; Je, Young Tae; Yun, Cheol-Won; Chung, Hesson

    2017-01-01

    This study was designed to investigate the oil entrapment and systemic oil absorption-reducing activities of chitosan. High-molecular-weight chitosan formed gel aggregates with oil and bile salts in vitro. The oil/chitosan ratio and the molecular weight of chitosan were optimized for the in vivo study, and a molecular weight >100,000 was effective in reducing the oil contamination of mouse fur. The oil/chitosan weight ratio required for effective oil entrapment was less than 13 and 5 in the in vitro and in vivo experiments, respectively. Chitosan administration was most effective during meals, and high-molecular-weight chitosan could trap and facilitate the reduction of systemic absorption of oil droplets separated by orlistat. The activity of the lipase inhibitor was not altered by chitosan as evidenced by thin layer chromatography, and orlistat was not absorbed systemically by the co-administration of chitosan.

  9. Phorbol diesters inhibit enzymatic hydrolysis of diacylglycerols in vitro.

    PubMed Central

    Chabbott, H; Cabot, M C

    1986-01-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on diacylglycerol lipase activity was examined in rat serum, tissue, and cellular preparations by using di[14C]oleoylglycerol, [3H]palmitoylacetylglycerol, and membrane-resident phospholipase C-generated diacylglycerols as substrates. These experiments were conducted to address whether phorbol esters can mimic diacylglycerols in interacting with enzymes other than protein kinase C. Serum hydrolysis of palmitoylacetylglycerol, assayed by the formation of [3H]palmitic acid, was inhibited by PMA, 4-O-methyl-PMA, or phorbol 12,13-dibutyrate (in order of decreasing potency). The hydrolysis of palmitoylacetylglycerol was inhibited more than 40% by the addition of PMA at a 1:1 molar ratio with substrate. The inhibition resembled the competitive type, with a Ki of approximately 2.7 microM. PMA in the 10-60 microM range also inhibited hydrolysis of palmitoylacetylglycerol by lipases from rat brain microsomes and by homogenates of C3H/10T1/2 mouse fibroblasts. PMA was likewise inhibitory when assayed in an intramembrane enzyme-substrate milieu in which diacylglycerols were generated, in situ, by treatment of [3H]palmitate-labeled cell homogenates with phospholipase C. Collectively, these data demonstrate that PMA, which is now thought to act by mimicry of diacylglycerols, can inhibit the action of diacylglycerol lipase. It is possible that such a mechanism is linked to the multiplicity of responses elicited by phorbol diesters and that other agents may function by means of enzyme interactions (post-phospholipase C) to influence the levels of the cellular diacylglycerol mediators. PMID:3458169

  10. Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

    PubMed Central

    Bai, Tingli; Zhang, Daozhong; Lin, Shuangjun; Long, Qingshan; Wang, Yemin; Ou, Hongyu; Kang, Qianjin; Deng, Zixin; Liu, Wen

    2014-01-01

    Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-β-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the α-branched 3,5-dihydroxy fatty acid β-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two β-ketoacyl–acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the α-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the α-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3β-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the β-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique β-lactone ring. PMID:25239907

  11. Structure–activity studies in the development of a hydrazone based inhibitor of adipose-triglyceride lipase (ATGL)

    PubMed Central

    Mayer, Nicole; Schweiger, Martina; Melcher, Michaela-Christina; Fledelius, Christian; Zechner, Rudolf; Zimmermann, Robert; Breinbauer, Rolf

    2015-01-01

    Adipose triglyceride lipase (ATGL) catalyzes the degradation of cellular triacylglycerol stores and strongly determines the concentration of circulating fatty acids (FAs). High serum FA levels are causally linked to the development of insulin resistance and impaired glucose tolerance, which eventually progresses to overt type 2 diabetes. ATGL-specific inhibitors could be used to lower circulating FAs, which can counteract the development of insulin resistance. In this article, we report about structure–activity relationship (SAR) studies of small molecule inhibitors of ATGL based on a hydrazone chemotype. The SAR indicated that the binding pocket of ATGL requests rather linear compounds without bulky substituents. The best inhibitor showed an IC50 = 10 μM in an assay with COS7-cell lysate overexpressing murine ATGL. PMID:25778769

  12. Bioengineering recombinant diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 115 DGAT sequences are identified from 69 organisms in the GenBank databases. Only a few papers have been published in the last 28 years on the exp...

  13. Effects of RHC 80267, a diglyceride lipase inhibitor, on prolactin secretion and calcium uptake in GH/sub 3/ pituitary cells

    SciTech Connect

    Camoratto, A.M.; Grandison, L.

    1987-01-19

    The effect of the diglyceride lipase inhibitor RHC 80267 on the prolactin secretory process was examined in clonal anterior pituitary GH/sub 3/ cells. This compound reduced basal prolactin secretion as well as secretion induced by TRH and phospholipase C but not that induced by phorbol myristate acetate. Although exogenous phospholipase C increased diglyceride, no increase in the products of diglyceride lipase was detected. Moreover, low doses of RHC 80267 were observed to effectively block potassium-stimulated /sup 45/calcium influx. It is unlikely that RHC 80267 inhibits prolactin release solely by inhibiting diglyceride lipase. These data suggest blockage of plasma membrane calcium channels as an alternate mechanism for the inhibitory actions of RHC 80267 on intact GH/sub 3/ cells. These observations may have implications for RHC 80267 action in other cell types.

  14. (4-Phenoxyphenyl)tetrazolecarboxamides and related compounds as dual inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Holtfrerich, Angela; Hanekamp, Walburga; Lehr, Matthias

    2013-05-01

    Inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principle enzymes involved in the degradation of endogenous cannabinoids like anandamide and 2-arachidonoylglycerol, have potential utility in the treatment of several disorders including pain, inflammation and anxiety. In the present study, the effectivity and selectivity of eight known FAAH and MAGL inhibitors for inhibition of the appropriate enzyme were measured applying in vitro assays, which work under comparable conditions. Because many of the known FAAH and MAGL inhibitors simply consist of a lipophilic scaffold to which a heterocyclic system is bound, furthermore, different heterocyclic structures were evaluated for their contribution to enzyme inhibition by attaching them to the same lipophilic backbone, namely 4-phenoxybenzene. One of the most active compound synthesized during this investigation was N,N-dimethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-carboxamide (16) (IC50 FAAH: 0.012 μM; IC50 MAGL: 0.028 μM). This inhibitor was systematically modified in the lipophilic 4-phenoxyphenyl region. Structure-activity relationship studies revealed that the inhibitory potency against FAAH and MAGL, respectively, could still be increased by replacement of the phenoxy residue of 16 by 3-chlorophenoxy (45) or pyrrol-1-yl groups (49). Finally, the tetrazolecarboxamide 16 and some related compounds were tested for metabolic stability with rat liver S9 fractions showing that these kind of FAAH/MAGL inhibitors are readily inactivated by cleavage of the bond between the tetrazole ring and its carboxamide substituent.

  15. Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

    SciTech Connect

    Schalk-Hihi, Céline; Schubert, Carsten; Alexander, Richard; Bayoumy, Shariff; Clemente, Jose C.; Deckman, Ingrid; DesJarlais, Renee L.; Dzordzorme, Keli C.; Flores, Christopher M.; Grasberger, Bruce; Kranz, James K.; Lewandowski, Frank; Liu, Li; Ma, Hongchang; Maguire, Diane; Macielag, Mark J.; McDonnell, Mark E.; Haarlander, Tara Mezzasalma; Miller, Robyn; Milligan, Cindy; Reynolds, Charles

    2011-12-22

    A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

  16. Sequence analysis of diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the final step of triacylglycerol (TAG) biosynthesis in eukaryotes. DGATs esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA. Plants and animals deficient in DGATs accumulate less TAG and over-expression of DGATs increases TAG. DGAT knock...

  17. Brominated polyunsaturated lipids from the Chinese sponge Xestospongia testudinaria as a new class of pancreatic lipase inhibitors.

    PubMed

    Liang, Lin-Fu; Wang, Ting; Cai, You-Sheng; He, Wen-Fei; Sun, Peng; Li, Yu-Fen; Huang, Qi; Taglialatela-Scafati, Orazio; Wang, He-Yao; Guo, Yue-Wei

    2014-05-22

    Chemical analysis of the Chinese marine sponge Xestospongia testudinaria afforded a library of brominated polyunsaturated lipids including eight new compounds, named xestonarienes A-H (3-10) and thirteen known analogues (11-23). The structures of the new compounds were elucidated by detailed spectroscopic analysis and by comparison with literature data. The isolated lipids were evaluated for their inhibitory activity against pancreatic lipase (PL), an essential enzyme for efficient fat digestion and the major metabolite, 14, exhibited a marked inhibitory activity (IC50 = 3.11 μM), similar to that of the positive control Orlistat (IC50 = 0.78 μM). The preliminary structure-activity relationships on the series of compounds clearly evidenced that a terminal (E)-enyne functionality, a diyne within the chain, and methyl ester group are all key functional groups for the activity of this class of PL inhibitors. Further biological investigation on compound 14 revealed a significant decrease in the plasma triglyceride level following an oral lipid challenge in C57BLKS/J male mice. Acute toxicology study demonstrated that compound 14 was non-toxic up to 1600 mg/kg p.o in mice. This is the first report of the PL inhibitory activity for brominated polyunsaturated lipids and the obtained results qualify compound 14 as a potent and bioavailable drug candidate for a mild and safe treatment to prevent and reduce obesity.

  18. Optimal Extraction Conditions of Anti-obesity Lipase Inhibitor from Phellinus linteus and Nutritional Characteristics of the Extracts

    PubMed Central

    Lee, Jong-Kug; Song, Jung-Hwa

    2010-01-01

    In an effort to develop novel mushroom-derived anti-obesity nutraceuticals, water and ethanol extracts containing the lipaseinhibitory compound from Phellinus linteus were prepared, and their nutritional components were determined. The optimal conditions for the extraction of P. linteus lipase inhibitor involved the treatment of the fruiting bodies with distilled water at 80℃ for 72 hr and 80% ethanol at 100℃ for 60 hr, respectively. The distilled water extract and ethanol extract contained 10.9% and 6.11% of crude protein, and 0.96% and 15.86% of crude fat, respectively. Additionally, the distilled water extract contained a large quantity of minerals, including 239.5 mg of K, 39.3 mg of Mg, and 39.3 mg of Na. The free amino acid content of the distilled water extracts was also higher than that of the ethanol extracts, and in particular, the distilled water extracts contained 5,139 mg of asparagine, 3,891 mg of tryptophan, 2,598 mg of alanine, and 2,066 mg of serine in 100 g of the distilled water extracts. 100 g of the distilled water and ethanol extracts were found to contain 12.31 g and 8.16 g of malic acid, respectively. PMID:23956626

  19. Comparative biochemical characterization of the monoacylglycerol lipase inhibitor KML29 in brain, spinal cord, liver, spleen, fat and muscle tissue.

    PubMed

    Pasquarelli, Noemi; Porazik, Christoph; Hanselmann, Johannes; Weydt, Patrick; Ferger, Boris; Witting, Anke

    2015-04-01

    Monoacylglycerol lipase (MAGL) is part of the endocannabinoid and the prostaglandin signaling system. MAGL degrades the endocannabinoid 2-arachidonoylglycerol (2-AG) into glycerol and arachidonic acid. MAGL-induced arachidonic acid is the primary source for prostaglandin synthesis in the brain. 2-AG mainly induces neuroprotective and anti-inflammatory effects, whereas prostaglandins are related to pro-inflammatory effects inducing neurotoxicity. Therefore, inhibition of MAGL represents a promising target for neurological diseases characterized by inflammation. However, as 2-AG is an agonist for the cannabinoid receptor 1 (CB1), inhibition of MAGL might be associated with unwanted cannabimimetic effects. Here, we show that oral administration of KML29, a highly selective inhibitor of MAGL, induced large and dose-dependent changes in 2-AG levels in vivo in brain and spinal cord of mice. Of note, MAGL inhibition by KML29 induced a decrease in prostaglandin levels in brain and most peripheral tissues but not in the spinal cord. MAGL expression was highest in fat, liver and brain, whereas the cytosolic phospholipase A2 (cPLA2), a further enzyme responsible for arachidonic acid production, was highly expressed in spinal cord, muscle and spleen. In addition, high doses (10 mg/kg) of KML29 induced some cannabimimetic effects in vivo in the tetrad test, including hypothermia, analgesia and hypomotility without induction of cataleptic behavior. In summary, inhibition of MAGL by KML29 represents a promising strategy for targeting the cannabinoid and prostaglandin system of the brain with only a moderate induction of cannabimimetic effects.

  20. The Selective Monoacylglycerol Lipase Inhibitor MJN110 Produces Opioid-Sparing Effects in a Mouse Neuropathic Pain Model.

    PubMed

    Wilkerson, Jenny L; Niphakis, Micah J; Grim, Travis W; Mustafa, Mohammed A; Abdullah, Rehab A; Poklis, Justin L; Dewey, William L; Akbarali, Hamid; Banks, Matthew L; Wise, Laura E; Cravatt, Benjamin F; Lichtman, Aron H

    2016-04-01

    Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required μ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

  1. Lipase Test

    MedlinePlus

    ... known as: LPS Formal name: Lipase Related tests: Amylase , Trypsin , Trypsinogen At a Glance Test Sample The ... lipase is most often used, along with an amylase test , to help diagnose and monitor acute pancreatitis . ...

  2. Identification of 1H-indene-(1,3,5,6)-tetrol derivatives as potent pancreatic lipase inhibitors using molecular docking and molecular dynamics approach.

    PubMed

    Kalathiya, Umesh; Padariya, M; Baginski, M

    2016-11-01

    Pancreatic lipase is a potential therapeutic target to treat diet-induced obesity in humans, as obesity-related diseases continue to be a global problem. Despite intensive research on finding potential inhibitors, very few compounds have been introduced to clinical studies. In this work, new chemical scaffold 1H-indene-(1,3,5,6)-tetrol was proposed using knowledge-based approach, and 36 inhibitors were derived by modifying its functional groups at different positions in scaffold. To explore binding affinity and interactions of ligands with protein, CDOCKER and AutoDock programs were used for molecular docking studies. Analyzing results of rigid and flexible docking algorithms, inhibitors C_12, C_24, and C_36 were selected based on different properties and high predicted binding affinities for further analysis. These three inhibitors have different moieties placed at different functional groups in scaffold, and to characterize structural rationales for inhibitory activities of compounds, molecular dynamics simulations were performed (500 nSec). It has been shown through simulations that two structural fragments (indene and indole) in inhibitor can be treated as isosteric structures and their position at binding cleft can be replaced by each other. Taking into account these information, two lines of inhibitors can further be developed, each line based on a different core scaffold, that is, indene/indole.

  3. Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

    PubMed Central

    Woo, Dong Ho; Jung, Sung Jun; Zhu, Mei Hong; Park, Chul-Kyu; Kim, Yong Ho; Oh, Seog Bae; Lee, C Justin

    2008-01-01

    The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1), is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC). However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG) directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG) neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5)P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C. PMID:18826653

  4. Characterization of the Yeast DGK1-encoded CTP-dependent Diacylglycerol Kinase*♦

    PubMed Central

    Han, Gil-Soo; O'Hara, Laura; Siniossoglou, Symeon; Carman, George M.

    2008-01-01

    The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2+ or Mg2+ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 °C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent Km = 6.5 mol %) and saturation kinetics with respect to CTP (apparent Km = 0.3 mm). dCTP was both a substrate (apparent Km = 0.4 mm) and competitive inhibitor (apparent Ki = 0.4 mm) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases. PMID:18458076

  5. Diacylglycerol kinase θ: regulation and stability.

    PubMed

    Tu-Sekine, Becky; Goldschmidt, Hana; Petro, Elizabeth; Raben, Daniel M

    2013-01-01

    Given the well-established roles of diacylglycerol (DAG) and phosphatidic acid (PtdOH) in a variety of signaling cascades, it is not surprising that there is an increasing interest in understanding their physiological roles and mechanisms that regulate their cellular levels. One class of enzymes capable of coordinately regulating the levels of these two lipids is the diacylglycerol kinases (DGKs). These enzymes catalyze the transfer of the γ-phosphate of ATP to the hydroxyl group of DAG, which generates PtdOH while reducing DAG. As these enzymes reciprocally modulate the relative levels of these two signaling lipids, it is essential to understand the regulation and roles of these enzymes in various tissues. One system where these enzymes play important roles is the nervous system. Of the ten mammalian DGKs, eight of them are readily detected in the mammalian central nervous system (CNS): DGK-α, DGK-β, DGK-γ, DGK-η, DGK-ζ, DGK-ι, DGK-ε, and DGK-θ. Despite the increasing interest in DGKs, little is known about their regulation. We have focused some attention on understanding the enzymology and regulation of one of these DGK isoforms, DGK-θ. We recently showed that DGK-θ is regulated by an accessory protein containing polybasic regions. We now report that this accessory protein is required for the previously reported broadening of the pH profile observed in cell lysates in response to phosphatidylserine (PtdSer). Our data further reveal DGK-θ is regulated by magnesium and zinc, and sensitive to the known DGK inhibitor R599022. These data outline new parameters involved in regulating DGK-θ.

  6. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  7. Characterization and identification of a lipoprotein lipase from Manduca sexta flight muscle.

    PubMed

    Van Heusden, M C

    1993-10-01

    Lipoprotein lipase (LpL) activity in Manduca sexta flight muscle tissue was measured using in vivo radiolabeled lipophorin as a substrate. LpL hydrolyses diacylglycerol in the low density lipophorin (that occurs during flight) at a higher rate than diacylglycerol in the high density lipophorin (present in the resting insect). LpL has a pH-optimum of 7.5 and is less sensitive to NaCl than mammalian LpL. LpL is inhibited by bovine albumin and chicken ovalbumin. LpL is inhibited by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), which indicates the presence of an active site serine similar to mammalian LpL. Flight muscle LpL shows affinity for immobilized copper as well as for immobilized heparin. Using radiolabeled DFP, a protein of 37 kDa was identified (after SDS-PAGE) as the DFP-binding protein in a partially purified preparation of LpL. This 37 kDa protein is proposed to be the LpL or a subunit thereof.

  8. Does triacylglycerol biosynthesis require diacylglycerol acyltransferase (DAGAT)?

    PubMed

    Fraser, T; Waters, A; Chatrattanakunchai, S; Stobart, K

    2000-12-01

    Microsomal membrane preparations from the developing seeds of sunflower (Helianthus annuus L.) catalyse the conversion of sn-glycerol-3-phosphate and acyl-CoA to triacylglycerol via phosphatidic acid and diacylglycerol. The formation of diacylglycerol from phosphatidic acid was Mg2+ dependent and in the presence of EDTA phosphatidic acid accumulated. This property was used to generate large quantities of endogenous radioactive phosphatidic acid in the membranes. On addition of Mg2+ the phosphatidic acid was used in triacylglycerol formation. Acyl-CoA had little effect on the label which accumulated in triacylglycerol from phosphatidic acid. Diacylglycerol acyltransferase, therefore, may not play a major role in oil formation as originally envisaged and other enzymes, including diacylglycerol:diacylglycerol transacylase [Stobart, Mancha, Lenman, Dahlqvist and Stymne (1997) Planta 203, 58-66] may have important biosynthetic functions.

  9. Lipase test

    MedlinePlus

    ... cholecystitis Chronic pancreatitis Enzyme Familial lipoprotein lipase deficiency Pancreatic cancer Triglyceride level Review Date 2/4/2015 Updated ... team. Related MedlinePlus Health Topics Gastroenteritis Genetic Disorders Pancreatic Cancer Pancreatic Diseases Pancreatitis Browse the Encyclopedia A.D. ...

  10. Response of cardiac myocytes to a ramp increase of diacylglycerol generated by photolysis of a novel caged diacylglycerol.

    PubMed Central

    Huang, X P; Sreekumar, R; Patel, J R; Walker, J W

    1996-01-01

    To test the responsiveness of living cells to the intracellular messenger diacylglycerol, we developed a prototype caged diacylglycerol compound, 3-O-(alpha-carboxyl-2,4-dinitrobenzyl)-1 ,2-dioctanoyl-rac-glycerol (designated alpha-carboxyl caged diC(8)), that produces dioctanoylglycerol (diC(8)) on photolysis. Alpha-Carboxyl caged diC(8) is biologically inert toward diacylglycerol kinase and protein kinase C in vitro and is readily incorporated into cardiac myocyte membranes, where it has no effect before irradiation. Exposure to near-UV light releases biologically active diC8 in good yield (quantum efficiency = 0.2). Here we examine a cellular response to controlled elevation of diC8 within single cardiac myocytes. Twitch amplitude was monitored in electrically stimulated myocytes, and a ramp increase in the concentration of diC(8) was generated by continuous irradiation of cells loaded with the caged compound. The myocyte response was biphasic with a positive inotropic phase (39% increase in twitch amplitude), followed by a large negative inotropic phase (>80% decrease). The time to peak inotropy for both phases depended on the light intensity, decreasing from 376 +/- 51 S to 44 +/- 5 s (positive phase) and 422 +/- 118 S to 51 +/- 9 S (negative phase) as the light intensity was increased eightfold. Both phases were inhibited by the protein kinase C inhibitor chelethyrine chloride. An increase in extracellular K+ from 5 mM to 20 mM to partially depolarize the cell membrane eliminated the positive inotropic phase, but the negative inotropic response was largely unaltered. The results reveal new features in the response of cardiac muscle to diacylglycerol, including a positive inotropic phase and a complex responsiveness to a simple linear increase in diacylglycerol. The effects of photoreleased diC(8) were similar to the effects of opiate agonists selective for kappa receptors, consistent with a major role for diacylglycerol in these responses. Images FIGURE 2

  11. Regulation of adipose triglyceride lipase by rosiglitazone

    PubMed Central

    Liu, L.-F.; Purushotham, A.; Wendel, A. A.; Koba, K.; DeIuliis, J.; Lee, K.; Belury, M. A.

    2013-01-01

    Aim To elucidate the mechanism by which rosiglitazone regulates adipose triglyceride lipase (ATGL). Methods Male C57Bl/6 mice were treated with rosiglitazone daily (10 mg/kg body weight), and adipose tissues were weighed and preserved for mRNA and protein analysis of ATGL. In parallel, preadipocyte (3T3-L1) cells were differentiated with insulin/dexamethasone/3-isobutyl-1-methlxanthine cocktail or rosiglitazone, and ATGL levels were measured with real-time PCR, western blotting and immunohistochemistry. Results Rosiglitazone concomitantly promoted differentiation of pre-adipocytes to functional adipocytes and induced mRNA levels of ATGL. The peroxisome proliferator-activated receptor-γ (PPARγ) antagonist bisphenol A diglycidyl ether significantly abrogated the induction of mRNA, but not protein levels of ATGL by rosiglitazone in differentiated 3T3-L1 adipocytes. In the presence of epinephrine rosiglitazone stimulated free fatty acid release and increased diacylglycerol acyltransferase-1 (DGAT-1) mRNA suggest that ATGL and DGAT-1 may be cooperatively involved in rosiglitazone-stimulated triglyceride hydrolysis and fatty acid re-esterification in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with rosiglitazone or insulin did not appear to alter localization of ATGL staining surrounding lipid droplets. Finally, we found that rosiglitazone increased ATGL mRNA levels in 3T3-L1 adipocytes in the presence of cycloheximide, an inhibitor of protein synthesis, suggesting that rosiglitazone regulation of ATGL occurs at the transcriptional level. Conclusions Rosiglitazone directly regulates transcription of ATGL, likely through a PPARγ-mediated mechanism. PMID:18643838

  12. Diacylglycerol kinases in membrane trafficking

    PubMed Central

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Diacylglycerol kinases (DGKs) belong to a family of cytosolic kinases that regulate the phosphorylation of diacylglycerol (DAG), converting it into phosphatidic acid (PA). There are 10 known mammalian DGK isoforms, each with a different tissue distribution and substrate specificity. These differences allow regulation of cellular responses by fine-tuning the delicate balance of cellular DAG and PA. DGK isoforms are best characterized as mediators of signal transduction and immune function. However, since recent studies reveal that DAG and PA are also involved in the regulation of endocytic trafficking, it is therefore anticipated that DGKs also plays an important role in membrane trafficking. In this review, we summarize the literature discussing the role of DGK isoforms at different stages of endocytic trafficking, including endocytosis, exocytosis, endocytic recycling, and transport from/to the Golgi apparatus. Overall, these studies contribute to our understanding of the involvement of PA and DAG in endocytic trafficking, an area of research that is drawing increasing attention in recent years. PMID:27057419

  13. Inhibition of monoacylglycerol lipase by troglitazone, N-arachidonoyl dopamine and the irreversible inhibitor JZL184: comparison of two different assays

    PubMed Central

    Björklund, E; Norén, E; Nilsson, J; Fowler, CJ

    2010-01-01

    BACKGROUND AND PURPOSE Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays. EXPERIMENTAL APPROACH MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated. KEY RESULTS Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC50 values of 1.1, 4.9, 0.78 and 3.1 µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ12,14-prostaglandin J2≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC50 41 µM) than hydrolysis of NPA by the human MGL lysates. CONCLUSIONS AND IMPLICATIONS ‘Hits’ in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators. PMID:20735405

  14. Neutrophil chemotaxis by Propionibacterium acnes lipase and its inhibition.

    PubMed Central

    Lee, W L; Shalita, A R; Suntharalingam, K; Fikrig, S M

    1982-01-01

    The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10(-4) mM) completely inhibited lipase activity as well as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracycline hydrochloride and erythromycin base at concentrations of 10(-1) mM and 1 mM, respectively, caused 100% inhibition of PMN migration toward lipase or zymosan-activated serum. The inhibiting activity of the antibiotics was directed against cells independently of any effect on lipase. Chemotaxis by P. acnes lipase suggests a wider role for this enzyme in the inflammatory process and the pathogenesis of acne vulgaris. Images PMID:7054130

  15. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  16. Repeated Low-Dose Administration of the Monoacylglycerol Lipase Inhibitor JZL184 Retains Cannabinoid Receptor Type 1–Mediated Antinociceptive and Gastroprotective Effects

    PubMed Central

    Kinsey, Steven G.; Wise, Laura E.; Ramesh, Divya; Abdullah, Rehab; Selley, Dana E.; Cravatt, Benjamin F.

    2013-01-01

    The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug–induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [3H]SR141716A binding and CP55,940 [(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ9-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid

  17. Inhibition of hydroxycinnamoyl-CoA thioesterases in ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) by lipase inhibitors.

    PubMed

    Flores-Sanchez, Isvett Josefina; Gang, David Roger

    2013-11-01

    Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean β-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively.

  18. Coordinated regulation of endocannabinoid-mediated retrograde synaptic suppression in the cerebellum by neuronal and astrocytic monoacylglycerol lipase

    PubMed Central

    Liu, Xiaojie; Chen, Yao; Vickstrom, Casey R.; Li, Yan; Viader, Andreu; Cravatt, Benjamin F.; Liu, Qing-song

    2016-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic depression including depolarization-induced suppression of excitation (DSE) and inhibition (DSI). 2-AG is degraded primarily by monoacylglycerol lipase (MAGL), which is expressed in neurons and astrocytes. Using knockout mice in which MAGL is deleted globally or selectively in neurons or astrocytes, we investigated the relative contribution of neuronal and astrocytic MAGL to the termination of DSE and DSI in Purkinje cells (PCs) in cerebellar slices. We report that neuronal MAGL plays a predominant role in terminating DSE at climbing fiber (CF) to PC synapses, while both neuronal and astrocytic MAGL significantly contributes to the termination of DSE at parallel fiber (PF) to PC synapses and DSI at putative Stellate cell to PC synapses. Thus, DSE and DSI at different synapses is not uniformly affected by global and cell type-specific knockout of MAGL. Additionally, MAGL global knockout, but not cell type-specific knockout, caused tonic activation and partial desensitization of the CB1 receptor at PF-PC synapses. This tonic CB1 activation is mediated by 2-AG since it was blocked by the diacylglycerol lipase inhibitor DO34. Together, these results suggest that both neuronal and astrocytic MAGL contribute to 2-AG clearance and prevent CB1 receptor over-stimulation in the cerebellum. PMID:27775008

  19. Lid mobility in lipase SMG1 validated using a thiol/disulfide redox potential probe.

    PubMed

    Guo, Shaohua; Popowicz, Grzegorz Maria; Li, Daoming; Yuan, Dongjuan; Wang, Yonghua

    2016-05-01

    Most lipases possess a lid domain above the catalytic site that is responsible for their activation. Lipase SMG1 from Malassezia globose CBS 7966 (Malassezia globosa LIP1), is a mono- and diacylglycerol lipase with an atypical loop-like lid domain. Activation of SMG1 was proposed to be solely through a gating mechanism involving two residues (F278 and N102). However, through disulfide bond cross-linking of the lid, this study shows that full activation also requires mobility of the lid domain, contrary to a previous proposal. The newly introduced disulfide bond makes lipase SMG1 eligible as a ratiometric thiol/disulfide redox potential probe, when it is coupled with chromogenic substrates. This redox-switch lipase could also be of potential use in cascade biocatalysis.

  20. Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

    PubMed

    Albert, A P; Piper, A S; Large, W A

    2005-08-01

    Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel

  1. Expression and purification of diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG and over-expression of DGATs increases TAG. DGAT knockout mice are resistant to ...

  2. Bioengineering recombinant tung tree diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Plants and animals defici...

  3. The Immunomodulatory Functions of Diacylglycerol Kinase ζ

    PubMed Central

    Singh, Brenal K.; Kambayashi, Taku

    2016-01-01

    The generation of diacylglycerol (DAG) is critical for promoting immune cell activation, regulation, and function. Diacylglycerol kinase ζ (DGKζ) serves as an important negative regulator of DAG by enzymatically converting DAG into phosphatidic acid (PA) to shut down DAG-mediated signaling. Consequently, the loss of DGKζ increases DAG levels and the duration of DAG-mediated signaling. However, while the enhancement of DAG signaling is thought to augment immune cell function, the loss of DGKζ can result in both immunoactivation and immunomodulation depending on the cell type and function. In this review, we discuss how different immune cell functions can be selectively modulated by DGKζ. Furthermore, we consider how targeting DGKζ can be potentially beneficial for the resolution of human diseases by either promoting immune responses important for protection against infection or cancer or dampening immune responses in immunopathologic conditions such as allergy and septic shock. PMID:27656643

  4. Determination of the quantitative stereoselectivity fingerprint of lipases during hydrolysis of a prochiral triacylglycerol.

    PubMed

    Mitchell, David Alexander; Rodriguez, Jorge A; Carrière, Frederic; Krieger, Nadia

    2008-06-01

    We propose a method for characterizing quantitatively the stereoselectivity of lipases during hydrolysis of triacylglycerols. Although it is of general applicability, we demonstrate it specifically for sn-1,3-regiospecific lipases. In this case the method generates a "stereoselectivity fingerprint" that consists of ratios of the specificity constants for the various reactions that produce and consume the 1,2-sn- and 2,3-sn-diacylglycerols. We use the method to determine the stereoselectivity fingerprint of several lipases during the hydrolysis of the prochiral substrate triolein. Our method opens up the possibility of correlating quantitative fingerprints with structural information, in the quest to elucidate the mechanisms underlying the stereoselectivity of lipases.

  5. Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007.

    PubMed

    Yuan, Dongjuan; Lan, Dongming; Xin, Ruipu; Yang, Bo; Wang, Yonghua

    2016-01-01

    A screening method along with the combination of genome sequence of microorganism, pairwise alignment, and lipase classification was used to search the thermostable lipase. Then, a potential thermostable lipase (named MAS1) from marine Streptomyces sp. strain W007 was expressed in Pichia pastoris X-33, and the biochemical properties were characterized. Lipase MAS1 belongs to the subfamily I.7, and it has 38% identity to the well-characterized Bacillus subtilis thermostable lipases in the subfamily I.4. The purified enzyme was estimated to be 29 kDa. The enzyme showed optimal temperature at 40 °C, and retained more than 80% of initial activity after 1 H incubation at 60 °C, suggesting that MAS1 was a thermostable lipase. MAS1 was an alkaline enzyme with optimal pH value at 7.0 and had stable activity for 12 H of incubation at pH 6.0-9.0. It was stable and retained about 90% of initial activity in the presence of Cu(2+) , Ca(2+) , Ni(2+) , and Mg(2+) , whereas 89.05% of the initial activity was retained when ethylene diamine tetraacetic acid was added. MAS1 showed the tolerance to organic solvents, but was inhibited by various surfactants. MAS1 was verified to be a triglyceride lipase and could hydrolyze triacylglycerol and diacylglycerol. The result represents a good example for researchers to discover thermostable lipase for industrial application.

  6. Acid Lipase Disease

    MedlinePlus

    ... Page You are here Home » Disorders » All Disorders Acid Lipase Disease Information Page Acid Lipase Disease Information Page What research is being ... research to understand lipid storage diseases such as acid lipase deficiency. Additional research studies hope to identify ...

  7. Diacylglycerol kinase ε localizes to subsurface cisterns of cerebellar Purkinje cells.

    PubMed

    Hozumi, Yasukazu; Fujiwara, Hiroki; Kaneko, Kenya; Fujii, Satoshi; Topham, Matthew K; Watanabe, Masahiko; Goto, Kaoru

    2017-02-13

    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGKε is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGKε play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGKε antibody. DGKε was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGKε was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGKε was also distributed in close apposition to DG lipase-α, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGKε-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKε plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.

  8. Synthesis and evaluation of new bis-1,3,4,2-triazaphospholinoalkane derivatives as in vitro α-amylase and lipase inhibitors.

    PubMed

    Hamzaoui, Salwa; Ben Salah, Bochra; Hamden, Khaled; Rekik, Awatef; Kossentini, Mohamed

    2015-03-01

    A series of new 1,ω-bis-(5-alkyl-2-oxide-3-tosyl-1,3,4,2-triazaphospholino)alkanes 2 and 3 were prepared in good yields by the treatment of 1,ω-bis-(1-tosylamidrazone)alkanes 1 with two molar equivalents of phosphoryl trichloride and phenylphosphonic dichloride, respectively. All the newly synthesized compounds were characterized by IR, (1) H NMR, (13) C NMR, (31) P NMR, and elemental analysis. All the new compounds were screened for their inhibitory effect on the key enzymes related to diabetes and obesity, as α-amylase and lipase. The in vitro study revealed that these alkane derivatives exert an inhibitory action against these key enzymes, especially 2b with an IC50 of 16 μg/mL against α-amylase and lipase. Overall, the findings of the current study indicate that 2d exhibits attractive properties and can therefore be considered for future application in the development of antidiabetic and hypolipidemic drugs.

  9. Acyl migration kinetics of vegetable oil 1,2-diacylglycerols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The acyl migration kinetics of long-chain 1,2-diacylglycerol (1,2-DAG) to form 1,3-diacylglycerol (1,3-DAG) over the temperature range of 25 to 80 degrees Celsius were examined using proton NMR spectroscopy. The 1,2-DAG mole fraction of 0.32 at equilibrium was found to be insensitive to temperature...

  10. Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase

    SciTech Connect

    Dousset, N.; Negre, A.; Salvayre, R.; Rogalle, P.; Dang, Q.Q.; Douste-Blazy, L.

    1988-06-01

    A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate.

  11. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

    PubMed

    Ard, Ryan; Mulatz, Kirk; Pomoransky, Julia L; Parks, Robin J; Trinkle-Mulcahy, Laura; Bell, John C; Gee, Stephen H

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.

  12. Epigenetic silencing of diacylglycerol kinase gamma in colorectal cancer.

    PubMed

    Kai, Masahiro; Yamamoto, Eiichiro; Sato, Akiko; Yamano, Hiro-O; Niinuma, Takeshi; Kitajima, Hiroshi; Harada, Taku; Aoki, Hironori; Maruyama, Reo; Toyota, Mutsumi; Hatahira, Tomo; Nakase, Hiroshi; Sugai, Tamotsu; Yamashita, Toshiharu; Toyota, Minoru; Suzuki, Hiromu

    2017-02-20

    Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.

  13. Mesenchymal Stem Cells Respond to Hypoxia by Increasing Diacylglycerols.

    PubMed

    Lakatos, Kinga; Kalomoiris, Stefanos; Merkely, Béla; Nolta, Jan A; Fierro, Fernando A

    2016-02-01

    Mesenchymal stem cells (MSC) are currently being tested clinically for a plethora of conditions, with most approaches relying on the secretion of paracrine signals by MSC to modulate the immune system, promote wound healing, and induce angiogenesis. Hypoxia has been shown to affect MSC proliferation, differentiation, survival and secretory profile. Here, we investigate changes in the lipid composition of human bone marrow-derived MSC after exposure to hypoxia. Using mass spectrometry, we compared the lipid profiles of MSC derived from five different donors, cultured for two days in either normoxia (control) or hypoxia (1% oxygen). Hypoxia induced a significant increase of total triglycerides, fatty acids and diacylglycerols (DG). Remarkably, reduction of DG levels using the phosphatidylcholine-specific phospholipase C inhibitor D609 inhibited the secretion of VEGF and Angiopoietin-2, but increased the secretion of interleukin-8, without affecting significantly their respective mRNA levels. Functionally, incubation of MSC in hypoxia with D609 inhibited the potential of the cells to promote migration of human endothelial cells in a wound/scratch assay. Hence, we show that hypoxia induces in MSC an increase of DG that may affect the angiogenic potential of these cells.

  14. Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons.

    PubMed

    Tu, Peng; Kunert-Keil, Christiane; Lucke, Silke; Brinkmeier, Heinrich; Bouron, Alexandre

    2009-01-01

    The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca(2+) ([Ca(2+)]i) in nearly 35% of the KCl-responsive cells. These Ca(2+) responses disappeared in a Ca(2+)-free medium supplemented with EGTA. Mn(2+) quench experiments showed that OAG activated Ca(2+)-conducting channels that were also permeant to Ba(2+). The OAG-induced Ca(2+) responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[beta-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd(3+). Replacing Na(+) ions with N-methyl-D-glucamine diminished the amplitude of the OAG-induced Ca(2+) responses showing that the Ca(2+) entry was mediated via Na(+)-dependent and Na(+)-independent mechanisms. Experiments carried out with the fluorescent Na(+) indicator CoroNa Green showed that OAG elevated [Na(+)]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca(2+)]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca(2+) responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca(2+)]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca(2+)-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical

  15. Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca2+

    PubMed Central

    Chinopoulos, Christos; Starkov, Anatoly A.; Grigoriev, Sergey; Dejean, Laurent M.; Kinnally, Kathleen W.; Liu, Xibao; Ambudkar, Indu S.; Fiskum, Gary

    2008-01-01

    Mitochondria contribute to cytosolic Ca2+ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca2+ release from Ca2+-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na+/Ca2+ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca2+ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca2+ and relatively slowreuptake, a secondary progressive release of Ca2+ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca2+ efflux is abolished by La3+ (1mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca2+ efflux. Ca2+-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGsinduced Ca2+ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brainmitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La3+. The presence of a second messenger-sensitive Ca2+ release mechanism in mitochondria could have an important impact on intracellular Ca2+ homeostasis. PMID:16167179

  16. Alterations in endocannabinoid tone following chemotherapy-induced peripheral neuropathy: Effects of endocannabinoid deactivation inhibitors targeting fatty-acid amide hydrolase and monoacylglycerol lipase in comparison to reference analgesics following cisplatin treatment

    PubMed Central

    Guindon, Josée; Lai, Yvonne; Takacs, Sara M.; Bradshaw, Heather B.; Hohmann, Andrea G.

    2012-01-01

    SUMMARY Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and cold allodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid consists of endocannabinoids (e.g. anandamide (AEA) and 2-arachidonoylglycerol (2-AG)), cannabinoid receptors (e.g. CB1 and CB2) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB1 (AM251 3 mg/kg), CB2 (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB1, CB2, TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the neuropathy while amitriptyline, administered acutely, was ineffective. CB1 or CB2 antagonist

  17. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism.

    PubMed

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan; Maillet, Jean-Christian; Fottinger, Alexandra; Foley, Tanya; Byham, Michèle-Renée; Iqbal, Tasfia Ahmed; Yoneda, Atsuko; Couchman, John R; Parks, Robin J; Gee, Stephen H

    2012-10-01

    Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

  18. Triton WR1339, an inhibitor of lipoprotein lipase, decreases vitamin E concentration in some tissues of rats by inhibiting its transport to liver.

    PubMed

    Abe, Chisato; Ikeda, Saiko; Uchida, Tomono; Yamashita, Kanae; Ichikawa, Tomio

    2007-02-01

    The aim of this experiment was to clarify the contribution of the alpha-tocopherol transfer activity of lipoprotein lipase (LPL) to vitamin E transport to tissues in vivo. We studied the effect of Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoproteins by LPL on vitamin E distribution in rats. Vitamin E-deficient rats fed a vitamin E-free diet for 4 wk were injected with Triton WR1339 and administered by oral gavage an emulsion containing 10 mg of alpha-tocopherol, 10 mg of gamma-tocopherol, or 29.5 mg of a tocotrienol mixture with 200 mg of sodium taurocholate, 200 mg of triolein, and 50 mg of albumin. alpha-Tocopherol was detected in the serum and other tissues of the vitamin E-deficient rats, but gamma-tocopherol, alpha- and gamma-tocotrienol were not detected. Triton WR1339 injection elevated (P<0.05) the serum alpha-tocopherol concentration and inhibited (P<0.05) the elevation of alpha-tocopherol concentration in the liver, adrenal gland, and spleen due to the oral administration of alpha-tocopherol. Neither alpha-tocopherol administration nor Triton WR1339 injection affected (P>or=0.05) the alpha-tocopherol concentration in the perirenal adipose tissue, epididymal fat, and soleus muscle despite a high expression of LPL in the adipose tissue and muscle. These data show that alpha-tocopherol transfer activity of LPL in adipose tissue and muscle is not important for alpha-tocopherol transport to the tissue after alpha-tocopherol intake or that the amount transferred is small relative to the tissue concentration. Furthermore, Triton WR1339 injection tended to elevate the serum gamma-tocopherol (P=0.071) and alpha-tocotrienol (P=0.053) concentrations and lowered them (P<0.05) in the liver and adrenal gland of rats administered gamma-tocopherol or alpha-tocotrienol. These data suggest that lipolysis of triacylglycerol-rich chylomicron by LPL is necessary for postprandial vitamin E transport to the liver and subsequent transport to the

  19. Calcium inhibits diacylglycerol uptake by serum albumin.

    PubMed

    Ahyayauch, Hasna; Arana, Gorka; Sot, Jesús; Alonso, Alicia; Goñi, Félix M

    2009-03-01

    Serum albumin is an abundant protein in blood plasma, that is well-known for its ability to transport hydrophobic biomolecules and drugs. Recent hypotheses propose that serum albumin plays a role in the regulation of lipid metabolism in addition to its lipid transport properties. The present work explores the capacity of bovine serum albumin (BSA) to extract diacylglycerols (DAG) from phospholipid bilayers, and the inhibition of such interaction by divalent cations. Quantitative measurements using radioactive DAG and morphological evidence derived from giant unilamellar vesicles examined by confocal microscopy provide concurrent results. BSA extracts DAG from vesicles consisting of phosphatidylinositol/DAG. Long, saturated DAG species are incorporated more readily than the shorter-chain or unsaturated ones. Divalent cations hinder DAG uptake by BSA. For Ca(2+), the concentration causing half-maximal inhibition is approximately 10 muM; 90% inhibition is caused by 100 muM Ca(2+). Sr(2+) requires concentrations one order of magnitude higher, while Mg(2+) has virtually no effect. As an example on how DAG uptake by BSA, and its inhibition by Ca(2+), could play a regulating role in lipid metabolism, a PI-specific phospholipase C has been assayed in the presence of BSA and/or Ca(2+). BSA activates the enzyme by removing the end-product DAG, but the activation is reverted by Ca(2+) that inhibits DAG uptake.

  20. Diacylglycerol Kinases: Shaping Diacylglycerol and Phosphatidic Acid Gradients to Control Cell Polarity

    PubMed Central

    Baldanzi, Gianluca; Bettio, Valentina; Malacarne, Valeria; Graziani, Andrea

    2016-01-01

    Diacylglycerol kinases (DGKs) terminate diacylglycerol (DAG) signaling and promote phosphatidic acid (PA) production. Isoform specific regulation of DGKs activity and localization allows DGKs to shape the DAG and PA gradients. The capacity of DGKs to constrain the areas of DAG signaling is exemplified by their role in defining the contact interface between T cells and antigen presenting cells: the immune synapse. Upon T cell receptor engagement, both DGK α and ζ metabolize DAG at the immune synapse thus constraining DAG signaling. Interestingly, their activity and localization are not fully redundant because DGKζ activity metabolizes the bulk of DAG in the cell, whereas DGKα limits the DAG signaling area localizing specifically at the periphery of the immune synapse. When DGKs terminate DAG signaling, the local PA production defines a new signaling domain, where PA recruits and activates a second wave of effector proteins. The best-characterized example is the role of DGKs in protrusion elongation and cell migration. Indeed, upon growth factor stimulation, several DGK isoforms, such as α, ζ, and γ, are recruited and activated at the plasma membrane. Here, local PA production controls cell migration by finely modulating cytoskeletal remodeling and integrin recycling. Interestingly, DGK-produced PA also controls the localization and activity of key players in cell polarity such as aPKC, Par3, and integrin β1. Thus, T cell polarization and directional migration may be just two instances of the general contribution of DGKs to the definition of cell polarity by local specification of membrane identity signaling. PMID:27965956

  1. Solvent-Free Lipase-Catalyzed Synthesis of Diacylgycerols as Low-Calorie Food Ingredients

    PubMed Central

    Vázquez, Luis; González, Noemí; Reglero, Guillermo; Torres, Carlos

    2016-01-01

    Problems derived from obesity and overweight have recently promoted the development of fat substitutes and other low-calorie foods. On the one hand, fats with short- and medium-chain fatty acids are a source of quick energy, easily hydrolyzable and hardly stored as fat. Furthermore, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gastrointestinal tract, reducing the formation of chylomicron and lowers the serum level of triacylglycerols by decreasing its resynthesis in the enterocyte. In this work, these two effects were combined to synthesize short- and medium-chain 1,3-diacylglycerols, leading to a product with great potential as for their low-calorie properties. Lipase-catalyzed transesterification reactions were performed between short- and medium-chain fatty acid ethyl esters and glycerol. Different variables were investigated, such as the type of biocatalyst, the molar ratio FAEE:glycerol, the adsorption of glycerol on silica gel, or the addition of lecithin. Best reaction conditions were evaluated considering the percentage of 1,3-DAG produced and the reaction rate. Except Novozym 435 (Candida antarctica), other lipases required the adsorption of glycerol on silica gel to form acylglycerols. Lipases that gave the best results with adsorption were Novozym 435 and Lipozyme RM IM (Rhizomucor miehei) with 52 and 60.7% DAG at 32 h, respectively. Because of its specificity for sn-1 and sn-3 positions, lipases leading to a higher proportion of 1,3-DAG vs. 1,2-DAG were Lipozyme RM IM (39.8 and 20.9%, respectively) and Lipase PLG (Alcaligenes sp.) (35.9 and 19.3%, respectively). By adding 1% (w/w) of lecithin to the reaction with Novozym 435 and raw glycerol, the reaction rate was considerably increased from 41.7 to 52.8% DAG at 24 h. PMID:26904539

  2. Solvent-Free Lipase-Catalyzed Synthesis of Diacylgycerols as Low-Calorie Food Ingredients.

    PubMed

    Vázquez, Luis; González, Noemí; Reglero, Guillermo; Torres, Carlos

    2016-01-01

    Problems derived from obesity and overweight have recently promoted the development of fat substitutes and other low-calorie foods. On the one hand, fats with short- and medium-chain fatty acids are a source of quick energy, easily hydrolyzable and hardly stored as fat. Furthermore, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gastrointestinal tract, reducing the formation of chylomicron and lowers the serum level of triacylglycerols by decreasing its resynthesis in the enterocyte. In this work, these two effects were combined to synthesize short- and medium-chain 1,3-diacylglycerols, leading to a product with great potential as for their low-calorie properties. Lipase-catalyzed transesterification reactions were performed between short- and medium-chain fatty acid ethyl esters and glycerol. Different variables were investigated, such as the type of biocatalyst, the molar ratio FAEE:glycerol, the adsorption of glycerol on silica gel, or the addition of lecithin. Best reaction conditions were evaluated considering the percentage of 1,3-DAG produced and the reaction rate. Except Novozym 435 (Candida antarctica), other lipases required the adsorption of glycerol on silica gel to form acylglycerols. Lipases that gave the best results with adsorption were Novozym 435 and Lipozyme RM IM (Rhizomucor miehei) with 52 and 60.7% DAG at 32 h, respectively. Because of its specificity for sn-1 and sn-3 positions, lipases leading to a higher proportion of 1,3-DAG vs. 1,2-DAG were Lipozyme RM IM (39.8 and 20.9%, respectively) and Lipase PLG (Alcaligenes sp.) (35.9 and 19.3%, respectively). By adding 1% (w/w) of lecithin to the reaction with Novozym 435 and raw glycerol, the reaction rate was considerably increased from 41.7 to 52.8% DAG at 24 h.

  3. PRD125, a potent and selective inhibitor of sterol O-acyltransferase 2 markedly reduces hepatic cholesteryl ester accumulation and improves liver function in lysosomal acid lipase-deficient mice.

    PubMed

    Lopez, Adam M; Chuang, Jen-Chieh; Posey, Kenneth S; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L; Turley, Stephen D

    2015-11-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management.

  4. PRD125, a Potent and Selective Inhibitor of Sterol O-Acyltransferase 2 Markedly Reduces Hepatic Cholesteryl Ester Accumulation and Improves Liver Function in Lysosomal Acid Lipase-Deficient Mice

    PubMed Central

    Lopez, Adam M.; Chuang, Jen-Chieh; Posey, Kenneth S.; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L.

    2015-01-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal−/− mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal+/+ littermates (23 versus 1.8 mg, respectively). In Lal−/− males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal−/− mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management. PMID:26283692

  5. Monoacylglycerol Lipase Regulates Fever Response.

    PubMed

    Sanchez-Alavez, Manuel; Nguyen, William; Mori, Simone; Moroncini, Gianluca; Viader, Andreu; Nomura, Daniel K; Cravatt, Benjamin F; Conti, Bruno

    2015-01-01

    Cyclooxygenase inhibitors such as ibuprofen have been used for decades to control fever through reducing the levels of the pyrogenic lipid transmitter prostaglandin E2 (PGE2). Historically, phospholipases have been considered to be the primary generator of the arachidonic acid (AA) precursor pool for generating PGE2 and other eicosanoids. However, recent studies have demonstrated that monoacyglycerol lipase (MAGL), through hydrolysis of the endocannabinoid 2-arachidonoylglycerol, provides a major source of AA for PGE2 synthesis in the mammalian brain under basal and neuroinflammatory states. We show here that either genetic or pharmacological ablation of MAGL leads to significantly reduced fever responses in both centrally or peripherally-administered lipopolysaccharide or interleukin-1β-induced fever models in mice. We also show that a cannabinoid CB1 receptor antagonist does not attenuate these anti-pyrogenic effects of MAGL inhibitors. Thus, much like traditional nonsteroidal anti-inflammatory drugs, MAGL inhibitors can control fever, but appear to do so through restricted control over prostaglandin production in the nervous system.

  6. Lipase activity of Mucor pusillus.

    PubMed

    Somkuti, G A; Babel, F J

    1968-04-01

    Two strains of Mucor pusillus were examined for their ability to synthesize lipase in a complex medium used in the production of milk-clotting protease. Lipase activity of both strains reached maximal after 6 days of incubation under submerged conditions at 35 C. Lipase secreted into the medium hydrolyzed butterfat and vegetable lipids, as well as selected synthetic triglycerides. About 50% of lipase activity was destroyed after a 45-min heat treatment at 58 C.

  7. Diacylglycerol acyltransferase-1 inhibition enhances intestinal fatty acid oxidation and reduces energy intake in rats.

    PubMed

    Schober, Gudrun; Arnold, Myrtha; Birtles, Susan; Buckett, Linda K; Pacheco-López, Gustavo; Turnbull, Andrew V; Langhans, Wolfgang; Mansouri, Abdelhak

    2013-05-01

    Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50 = 0.3494) and HuTu80 (EC50 = 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect.

  8. Diacylglycerols induce both ion pumping in patch-clamped guard-cell protoplasts and opening of intact stomata.

    PubMed Central

    Lee, Y; Assmann, S M

    1991-01-01

    Stomatal guard cells in leaves regulate the apertures of microscopic pores through which photosynthetic gas exchange and water vapor loss occur. Environmental signals, including light, high humidity, and low CO2 concentrations, open stomata by increasing the volume of guard cells. Activation of a plasma membrane H+ pump initiates K+ and Cl- influx, accompanied by malate synthesis, resulting in osmotic water flow into the guard cells, a bowing apart of the guard-cell pair, and consequent stomatal opening. Physiological and electrophysiological techniques were employed to investigate the possibility that a second-messenger lipid, 1,2-diacylglycerol, is involved in the transduction of opening stimuli. The synthetic diacylglycerols 1,2-dihexanoylglycerol and 1,2-dioctanoylglycerol enhanced light-induced stomatal opening in Commelina communis and induced stomatal opening under darkness, whereas an isomer with no known second-messenger role, 1,3-dioctanoylglycerol, did not affect stomatal responses. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, the enzyme typically activated by 1,2-diacylglycerol in animal cells, inhibited light-stimulated stomatal opening and enhanced dark-induced stomatal closure. N-[(2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), which inhibits cyclic nucleotide-dependent protein kinases preferentially over lipid-dependent protein kinases such as protein kinase C, had little effect on stomatal apertures. Whole-cell patch clamping of guard-cell protoplasts of Vicia faba revealed that 1,2-dihexanoylglycerol and 1-oleoyl-2-acetylglycerol activated an ATP-dependent, voltage-independent current, suggesting activation of an electrogenic ion pump such as the H+ pump. Diacylglycerol or functionally similar lipids may act through protein phosphorylation to provide the intracellular signals that mediate H+-ATPase activation and stomatal opening in response to light or other opening stimuli. PMID:11607161

  9. Structural elements of ligand recognition site in secretory phospho-lipase A2 and structure-based design of specific inhibitors.

    PubMed

    Singh, Nagendra; Somvanshi, Rishi K; Sharma, Sujata; Dey, Sharmistha; Kaur, Punit; Singh, Tej P

    2007-01-01

    Phospholipases A2 (phosphotide 2-acylhydrolases, PLA2s, EC 3.1.1.4) are widely distributed enzymes in the animal world. They catalyze the hydrolysis of the sn-2 acyl ester linkage of phospholipids, producing fatty acids and lysophospholipids. The mammalian type II secreted phospholipase A2 (PLA2-II) is one of the most extensively studied member of low molecular weight (13-18 kDa) PLA2s. PLA2-II contains 120-125 amino acid residues and seven disulphide bridges. The important features of overall structure of PLA2-II contain an N-terminal helix, H1 (residues: 2-12), an external loop (residues: 14-23), a calcium binding loop (Ca2+-loop, residues: 25-35), a second alpha-helix, H2 (residues: 40-55), a short two stranded anti-parallel beta-sheet referred to as beta-wing (residues: 75-84), a third alpha-helix, H3 (residues: 90-108) which is antiparallel to H2 and two single helical turns, SH4 (residues: 114-117) and SH5 (residues: 121-125). The three-dimensional structure of PLA2-II has defined a conserved active site within a hydrophobic channel lined by invariant hydrophobic residues. The active site residues His48, Asp49, Tyr52 and Asp99 are directly connected to the channel. An important water molecule that bridges His48 and Asp49 through hydrogen bonds is a part of catalytic network. Based on the structures of various complexes of group II PLA2, the ligand-recognition site has been divided into six subsites consisting of residues 2-10 (subsite 1), residues 17-23 (subsite 2), residues 28-32 (subsite 3), residues 48-52 (subsite 4), residues 68-70 (subsite 5) and residues 98-106 (subsite 6). It is observed that most of the currently available ligands saturate only part of the ligand-recognition site leaving a wide scope to improve the ligand complementarity. Naturally, the ligands that interact with the largest number of subsites would also correspond to the maximum affinity. Therefore, for the design of potent inhibitors of PLA2, the stereochemical knowledge of the

  10. Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT isoforms have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii) which contains 80% high-value eleostearic acid in its seed oils. ...

  11. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) are responsible for the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Different forms of DGATs have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii), which contains approximately 80% high-valu...

  12. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG. Over-expression of DGATs increases TAG. DGAT knockout mice are resistant to diet-induced obesity and lack milk secr...

  13. Purification of recombinant tung tree diacylglycerol acyltransferases from E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Over-expression of DGATs ...

  14. Inhibition of diacylglycerol acyltransferase by alkamides isolated from the fruits of Piper longum and Piper nigrum.

    PubMed

    Lee, Seung Woong; Rho, Mun-Chual; Park, Hye Ran; Choi, Jung-Ho; Kang, Ji Yun; Lee, Jung Won; Kim, Koanhoi; Lee, Hyun Sun; Kim, Young Kook

    2006-12-27

    Pharmacological inhibition of acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) has emerged as a potential therapy for the treatment of obesity and type 2 diabetes. Bioassay-guided isolation of CHCl3 extracts of the fruits of Piper longum and Piper nigum (Piperaceae), using an in vitro DGAT inhibitory assay, lead to isolation of a new alkamide named (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (2), together with four known alkamides: retrofractamide C (1), pipernonaline (3), piperrolein B (4), and dehydropipernonaline (5). Compounds 2-5 inhibited DGAT with IC50 values of 29.8 (2), 37.2 (3), 20.1 (4), and 21.2 (5) microM, respectively, but the IC50 value for 1 was more than 900 microM. This finding indicates that compounds possessing piperidine groups (2-5) can be potential DGAT inhibitors.

  15. Diacylglycerol Kinases as Emerging Potential Drug Targets for a Variety of Diseases: An Update

    PubMed Central

    Sakane, Fumio; Mizuno, Satoru; Komenoi, Suguru

    2016-01-01

    Ten mammalian diacylglycerol kinase (DGK) isozymes (α–κ) have been identified to date. Our previous review noted that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes (Sakane et al., 2008). Since then, recent genome-wide association studies have implied several new possible relationships between DGK isozymes and diseases. For example, DGKθ and DGKκ have been suggested to be associated with susceptibility to Parkinson's disease and hypospadias, respectively. In addition, the DGKη gene has been repeatedly identified as a bipolar disorder (BPD) susceptibility gene. Intriguingly, we found that DGKη-knockout mice showed lithium (BPD remedy)-sensitive mania-like behaviors, suggesting that DGKη is one of key enzymes of the etiology of BPD. Because DGKs are potential drug targets for a wide variety of diseases, the development of DGK isozyme-specific inhibitors/activators has been eagerly awaited. Recently, we have identified DGKα-selective inhibitors. Because DGKα has both pro-tumoral and anti-immunogenic properties, the DGKα-selective inhibitors would simultaneously have anti-tumoral and pro-immunogenic (anti-tumor immunogenic) effects. Although the ten DGK isozymes are highly similar to each other, our current results have encouraged us to identify and develop specific inhibitors/activators against every DGK isozyme that can be effective regulators and drugs against a wide variety of physiological events and diseases. PMID:27583247

  16. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src

    PubMed Central

    Torres-Ayuso, Pedro; Daza-Martín, Manuel; Martín-Pérez, Jorge; Ávila-Flores, Antonia; Mérida, Isabel

    2014-01-01

    Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer. PMID:25339152

  17. Protein kinase C–independent inhibition of arterial smooth muscle K+ channels by a diacylglycerol analogue

    PubMed Central

    Rainbow, RD; Parker, AM; Davies, NW

    2011-01-01

    BACKGROUND AND PURPOSE Analogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K+ and Ca2+ channels. We investigated whether dioctanoyl-sn-glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K+ currents of rat mesenteric arterial smooth muscle in a PKC-independent manner. EXPERIMENTAL APPROACH Conventional whole-cell and inside-out patch clamp was used to measure the inhibition of K+ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide. KEY RESULTS Mesenteric artery smooth muscle Kv currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40-fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and KATP currents, and again a significant proportion of these blocks occurred independently of PKC activation. CONCLUSIONS AND IMPLICATIONS These results show that DiC8 has a direct effect on arterial smooth muscle K+ channels, and this precludes its use as a PKC activator when investigating PKC-mediated effects on vascular K+ channels. PMID:21323899

  18. A novel thermostable lipase from basidiomycete Bjerkandera adusta R59: characterisation and esterification studies.

    PubMed

    Bancerz, Renata; Ginalska, Grazyna

    2007-08-01

    Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50 degrees C, respectively. Both enzyme forms were highly thermostable up to 60 degrees C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.

  19. Role of lipase in Burkholderia cepacia complex (Bcc) invasion of lung epithelial cells.

    PubMed

    Mullen, T; Markey, K; Murphy, P; McClean, S; Callaghan, M

    2007-12-01

    The Burkholderia cepacia complex (Bcc) is a group of ten closely related species associated with life-threatening infection in cystic fibrosis (CF). These bacteria are highly antibiotic resistant, with some strains transmissible, and in a subgroup of patients, they can cause a rapid and fatal necrotising pneumonia. The Bcc organisms produce a range of exoproducts with virulence potential, including exopolysaccharide, proteases and lipases. Many members of the Bcc are also capable of epithelial cell invasion, although the mechanism(s) involved are poorly understood. This study investigates a role for Bcc lipase in epithelial cell invasion by Bcc strains. Lipase activity was measured in eight species of the Bcc. Strains that produced high levels of lipase were predominantly from the B. multivorans and B. cenocepacia species. Pre-treatment of two epithelial cell lines with Bcc lipase significantly increased invasion by two B. multivorans strains and one B. cenocepacia strain and did not affect either plasma membrane or tight junction integrity. Inhibition of Bcc lipase production by the lipase inhibitor Orlistat significantly decreased invasion by both B. multivorans and B. cenocepacia strains in a concentration-dependent manner. This study demonstrates the extent of lipase production across the Bcc and establishes a potential role for lipase in Bcc epithelial cell invasion.

  20. Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10.

    PubMed

    Sethi, Bijay Kumar; Nanda, Prativa Kumari; Sahoo, Santilata

    2016-01-01

    Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30°C for 96h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50°C, and substrate concentration of 1.5%. The enzyme was thermostable at 60°C for 1h, and the optimum enzyme-substrate reaction time was 30min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30°C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn(2+), followed by Mg(2+) and Fe(2+). Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

  1. Lingual lipase activity in the orosensory detection of fat by humans.

    PubMed

    Kulkarni, Bhushan V; Mattes, Richard D

    2014-06-15

    Lingual lipase generates nonesterified fatty acids (NEFA) from dietary fats during oral processing by lipolysis. Lingual lipase in rodents has strong lipolytic activity and plays a critical role in oral detection of fats. The functional activity of lingual lipase during oral processing of high-fat foods in humans remains poorly characterized. Five commonly consumed high-fat foods varying in physical states and fatty acid composition (almond, almond butter, olive oil, walnut, and coconut) were masticated by 15 healthy human subjects at the rate of one chew per second with and without lipase inhibitor orlistat. Salivary NEFA concentrations were measured. To determine the role of lingual lipase in oral fat detection, sensory ratings were obtained from the same 15 human subjects for almond butter with and without orlistat. Lingual lipase was active during oral processing of almond and coconut. No activity of lingual lipase was detected during processing of almond butter. There was only weak evidence lingual lipase is a determinant of oral fat detection. Lingual lipase may only contribute to NEFA generation and oral fat detection of fatty foods that require stronger oral processing effort.

  2. Lingual lipase activity in the orosensory detection of fat by humans

    PubMed Central

    Kulkarni, Bhushan V.

    2014-01-01

    Lingual lipase generates nonesterified fatty acids (NEFA) from dietary fats during oral processing by lipolysis. Lingual lipase in rodents has strong lipolytic activity and plays a critical role in oral detection of fats. The functional activity of lingual lipase during oral processing of high-fat foods in humans remains poorly characterized. Five commonly consumed high-fat foods varying in physical states and fatty acid composition (almond, almond butter, olive oil, walnut, and coconut) were masticated by 15 healthy human subjects at the rate of one chew per second with and without lipase inhibitor orlistat. Salivary NEFA concentrations were measured. To determine the role of lingual lipase in oral fat detection, sensory ratings were obtained from the same 15 human subjects for almond butter with and without orlistat. Lingual lipase was active during oral processing of almond and coconut. No activity of lingual lipase was detected during processing of almond butter. There was only weak evidence lingual lipase is a determinant of oral fat detection. Lingual lipase may only contribute to NEFA generation and oral fat detection of fatty foods that require stronger oral processing effort. PMID:24694384

  3. Genetics Home Reference: lysosomal acid lipase deficiency

    MedlinePlus

    ... Home Health Conditions lysosomal acid lipase deficiency lysosomal acid lipase deficiency Enable Javascript to view the expand/ ... Download PDF Open All Close All Description Lysosomal acid lipase deficiency is an inherited condition characterized by ...

  4. Plant lipases: partial purification of Carica papaya lipase.

    PubMed

    Rivera, Ivanna; Mateos-Díaz, Juan Carlos; Sandoval, Georgina

    2012-01-01

    Lipases from plants have very interesting features for application in different fields. This chapter provides an overview on some of the most important aspects of plant lipases, such as sources, applications, physiological functions, and specificities. Lipases from laticifers and particularly Carica papaya lipase (CPL) have emerged as a versatile autoimmobilized biocatalyst. However, to get a better understanding of CPL biocatalytic properties, the isolation and purification of individual C. papaya lipolytic enzymes become necessary. In this chapter, a practical protocol for partial purification of the latex-associated lipolytic activity from C. papaya is given.

  5. Rapid triacylglycerol turnover in Chlamydomonas reinhardtii requires a lipase with broad substrate specificity.

    PubMed

    Li, Xiaobo; Benning, Christoph; Kuo, Min-Hao

    2012-12-01

    When deprived of nitrogen (N), the photosynthetic microalga Chlamydomonas reinhardtii accumulates large quantities of triacylglycerols (TAGs), making it a promising source of biofuel. Prominent transcriptional changes associated with the conditions leading to TAG accumulation have been found, suggesting that the key enzymes for TAG metabolism might be among those that fluctuate in their expression during TAG synthesis and breakdown. Using a Saccharomyces cerevisiae lipase null mutant strain for functional complementation, we identified the CrLIP1 gene from Chlamydomonas based on its ability to suppress the lipase deficiency-related phenotypes of the yeast mutant. In Chlamydomonas, an inverse correlation was found between the CrLIP1 transcript level and TAG abundance when Chlamydomonas cultures were reversibly deprived of N. The CrLIP1 protein expressed and purified from Escherichia coli exhibited lipolytic activity against diacylglycerol (DAG) and polar lipids. The lipase domain of CrLIP1 is most similar to two human DAG lipases, DAGLα and DAGLβ. The involvement of CrLIP1 in Chlamydomonas TAG hydrolysis was corroborated by reducing the abundance of the CrLIP1 transcript with an artificial micro-RNA, which resulted in an apparent delay in TAG lipolysis when N was resupplied. Together, these data suggest that CrLIP1 facilitates TAG turnover in Chlamydomonas primarily by degrading the DAG presumably generated from TAG hydrolysis.

  6. Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase.

    PubMed

    Pyo, Young-Gil; Hong, Seung In; Kim, Yangha; Kim, Byung Hee; Kim, In-Hwan

    2012-01-01

    High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase-catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40 °C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20 °C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase-catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to -30 °C.

  7. Resveratrol regulates lipolysis via adipose triglyceride lipase.

    PubMed

    Lasa, Arrate; Schweiger, Martina; Kotzbeck, Petra; Churruca, Itziar; Simón, Edurne; Zechner, Rudolf; Portillo, María del Puy

    2012-04-01

    Resveratrol has been reported to increase adrenaline-induced lipolysis in 3T3-L1 adipocytes. The general aim of the present work was to gain more insight concerning the effects of trans-resveratrol on lipid mobilization. The specific purpose was to assess the involvement of the two main lipases: adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in the activation of lipolysis induced by this molecule. For lipolysis experiments, 3T3-L1 and human SGBS adipocytes as well as adipose tissue from wild-type, ATGL knockout and HSL knockout mice were used. Moreover, gene and protein expressions of these lipases were analyzed. Resveratrol-induced free fatty acids release but not glycerol release in 3T3-L1 under basal and isoproterenol-stimulating conditions and under isoproterenol-stimulating conditions in SGBS adipocytes. When HSL was blocked by compound 76-0079, free fatty acid release was still induced by resveratrol. By contrast, in the presence of the compound C, an inhibitor of adenosine monophosphate-activated protein kinase, resveratrol effect was totally blunted. Resveratrol increased ATGL gene and protein expressions, an effect that was not observed for HSL. Resveratrol increased fatty acids release in epididymal adipose tissue from wild-type and HSL knockout mice but not in that adipose tissue from ATGL knockout mice. Taking as a whole, the present results provide novel evidence that resveratrol regulates lipolytic activity in human and murine adipocytes, as well as in white adipose tissue from mice, acting mainly on ATGL at transcriptional and posttranscriptional levels. Enzyme activation seems to be induced via adenosine monophosphate-activated protein kinase.

  8. Residue Asn277 affects the stability and substrate specificity of the SMG1 lipase from Malassezia globosa.

    PubMed

    Lan, Dongming; Wang, Qian; Xu, Jinxin; Zhou, Pengfei; Yang, Bo; Wang, Yonghua

    2015-03-31

    Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273-278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.

  9. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    SciTech Connect

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-12-17

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. As a result, the active site architecture shows clear evidence of having arisen by convergent evolution.

  10. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    NASA Astrophysics Data System (ADS)

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-12-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

  11. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    PubMed Central

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-01-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. PMID:26673816

  12. The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi.

    PubMed Central

    Docampo, R; Pignataro, O P

    1991-01-01

    Using [32P]Pi and [3H]inositol as precursors, we have detected the presence of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and their derivatives inositol phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate respectively, in Trypanosoma cruzi epimastigotes. Using digitonin-permeabilized cells it was possible to detect a stimulation in the formation of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate as well as an increased generation of diacylglycerol in the presence of 1 mM-CaCl2. These results are consistent with the operation of a functional inositol phosphate/diacylglycerol pathway in T. cruzi, and constitute the first demonstration of the presence and activation of this pathway in a parasitic protozoan. These results also indicate that this pathway is conserved during evolution from lower to higher eukaryotic organisms. Images Fig. 1. PMID:2025225

  13. Diacylglycerol Kinases in T Cell Tolerance and Effector Function

    PubMed Central

    Chen, Shelley S.; Hu, Zhiming; Zhong, Xiao-Ping

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that regulate the relative levels of diacylglycerol (DAG) and phosphatidic acid (PA) in cells by phosphorylating DAG to produce PA. Both DAG and PA are important second messengers cascading T cell receptor (TCR) signal by recruiting multiple effector molecules, such as RasGRP1, PKCθ, and mTOR. Studies have revealed important physiological functions of DGKs in the regulation of receptor signaling and the development and activation of immune cells. In this review, we will focus on recent progresses in our understanding of two DGK isoforms, α and ζ, in CD8 T effector and memory cell differentiation, regulatory T cell development and function, and invariant NKT cell development and effector lineage differentiation. PMID:27891502

  14. Diacylglycerol acyltransferase: a key mediator of plant triacylglycerol synthesis.

    PubMed

    Lung, Shiu-Cheung; Weselake, Randall J

    2006-12-01

    Many plants deposit TAG in seeds and fruits as the major form of storage lipid. TAG production is of tremendous socioeconomic value in food, nutraceutical, and industrial applications, and thus numerous conventional and molecular genetic strategies have been explored in attempts to increase TAG content and modify the FA composition of plant seed oils. Much research has focused on the acyl-CoA-dependent reaction catalyzed by diacylglycerol acyltransferase (DGAT), which is an integral endoplasmic reticulum protein and has also been shown to be present in oil bodies and plastids. DGAT enzymes exhibit diverse biochemical properties among different plant species, many of which are summarized here. In addition to catalyzing a critical step in TAG biosynthesis, there is evidence that DGAT has roles in lipid metabolism associated with germination and leaf senescence. TAG can also be formed in plants via two different acyl-CoA-independent pathways, catalyzed by phospholipid: diacylglycerol acyltransferase and diacylglycerol transacylase. The current understanding of the terminal step in TAG formation in plants and the development of molecular genetic approaches aimed at altering TAG yield and FA composition of TAG are discussed.

  15. Psychrophilic Lipase from Arctic Bacterium

    PubMed Central

    Ramle, Zakiah; Rahim, Rashidah Abdul

    2016-01-01

    A lipase producer psychrophilic microorganism isolated from Arctic sample was studied. The genomic DNA of the isolate was extracted using modified CTAB method. Identification of the isolate by morphological and 16S rRNA sequence analysis revealed that the isolate is closely related to Arthrobacter gangotriensis (97% similarity). A. gangotriensis was determined as positive lipase producer based on the plate screening using specific and sensitive plate assay of Rhodamine B. The PCR result using Arthrobacter sp.’s full lipase gene sequence as the template primers emphasised a possible lipase gene at 900 bp band size. The gene is further cloned in a suitable vector system for expression of lipase. PMID:27965754

  16. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    PubMed

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.

  17. Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii[C][W

    PubMed Central

    Yoon, Kangsup; Han, Danxiang; Li, Yantao; Sommerfeld, Milton; Hu, Qiang

    2012-01-01

    Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production. PMID:23012436

  18. Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii.

    PubMed

    Yoon, Kangsup; Han, Danxiang; Li, Yantao; Sommerfeld, Milton; Hu, Qiang

    2012-09-01

    Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.

  19. Structure-function analysis of diacylglycerol acyltransferase sequences from tung tree and 82 other Organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferase family (DGATs) catalyzes the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGATs esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA. Understanding the roles of DGATs will help to create transgenic plants with v...

  20. Diacylglycerol kinase-ζ regulates mTORC1 and lipogenic metabolism in cancer cells through SREBP-1

    PubMed Central

    Torres-Ayuso, P; Tello-Lafoz, M; Mérida, I; Ávila-Flores, A

    2015-01-01

    Diacylglycerol kinases (DGKs) transform diacylglycerol (DAG) into phosphatidic acid (PA), balancing the levels of these key metabolic and signaling lipids. We previously showed that PA derived from the DGKζ isoform promotes mammalian target of rapamycin complex 1 (mTORC1) activation. This function might be crucial for the growth and survival of cancer cells, especially for those resistant to the allosteric mTOR inhibitor rapamycin. How this positive function of DGKζ coordinates with DAG metabolism and signaling is unknown. In this study, we used a rapamycin-resistant colon cancer cell line as a model to address the role of DGKζ in tumor cells. We found that DGKζ predominated over other PA sources such as DGKα or phospholipase D to activate mTORC1, and that its activity was a component of the rapamycin-induced feedback loops. We show that the DGKζ DAG-consuming function is central to cell homeostasis, as DAG negatively regulates levels of the lipogenic transcription factor SREBP-1. Our findings suggest a model in which simultaneous regulation of DAG and PA levels by DGKζ is integrated with mTOR function to maintain tumor cell homeostasis; we provide new evidence of the crosstalk between mTOR and lipid metabolism that will be advantageous in the design of drug therapies. PMID:26302180

  1. Diacylglycerol acyltransferase-1 inhibition enhances intestinal fatty acid oxidation and reduces energy intake in rats[S

    PubMed Central

    Schober, Gudrun; Arnold, Myrtha; Birtles, Susan; Buckett, Linda K.; Pacheco-López, Gustavo; Turnbull, Andrew V.; Langhans, Wolfgang; Mansouri, Abdelhak

    2013-01-01

    Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50 = 0.3494) and HuTu80 (EC50 = 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect. PMID:23449193

  2. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of...

  3. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of...

  4. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of...

  5. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of...

  6. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of...

  7. Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.

    PubMed Central

    Thomas, T P; Porcellati, F; Kato, K; Stevens, M J; Sherman, W R; Greene, D A

    1994-01-01

    Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells. Images PMID:8201009

  8. Biodiesel production with immobilized lipase: A review.

    PubMed

    Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

    2010-01-01

    Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored.

  9. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    DOE PAGES

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; ...

    2015-12-17

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternarymore » structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. As a result, the active site architecture shows clear evidence of having arisen by convergent evolution.« less

  10. Relative oxidative stability of diacylglycerol and triacylglycerol oils.

    PubMed

    Qi, Jin F; Wang, Xiang Y; Shin, Jung-Ah; Lee, Young-Hwa; Jang, Young-Seok; Lee, Jeung Hee; Hong, Soon-Taek; Lee, Ki-Teak

    2015-03-01

    To compare the oxidative stability between diacylglycerol (DAG) oil and conventional triacylglycerol (TAG) oil (that is, soybean oil), the prepared stripped diacylglycerol oil (SDO) and soybean oil (SSBO) were stored at 60 °C in the dark for 144 h. During storage peroxide values (POVs), contents of aldehydes, unsaturated fatty acids were measured to evaluate the oxidative stabilities of the 2 oils. The results showed the content of C18:2, C18:3, and total unsaturated fatty acid decreased faster in DAG oil than in soybean oil, whereas the decreased rate of C18:1 was similar in 2 oils. Also, both rate constants (K1 and K2) obtained from POV (K1 ) and total aldehydes (K2 ) indicated that DAG oil (K1 = 3.22 mmol/mol FA h(-1) , K2 = 0.023 h(-1)) was oxidized more rapidly than soybean oil (K1 = 2.56 mmol/mol FA h(-1) , K2 = 0.021 h(-1)), which was mainly due to the difference of acylglycerol composition of the 2 oils along with higher C18:3 (9.6%) in SDO than SSBO (5.7%). It is concluded that DAG was more easily oxidized than soybean oil at 60 °C in the dark for 144 h.

  11. Arachidonoyl-Specific Diacylglycerol Kinase ε and the Endoplasmic Reticulum

    PubMed Central

    Nakano, Tomoyuki; Matsui, Hirooki; Tanaka, Toshiaki; Hozumi, Yasukazu; Iseki, Ken; Kawamae, Kaneyuki; Goto, Kaoru

    2016-01-01

    The endoplasmic reticulum (ER) comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA) serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK) that phosphorylates diacylglycerol (DG) to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology, and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER. PMID:27917381

  12. Lipases in Medicine: An Overview.

    PubMed

    Loli, Heni; Narwal, Sunil Kumar; Saun, Nitin Kumar; Gupta, Reena

    2015-01-01

    Lipases are part of the family of hydrolases that act on carboxylic ester bonds. They are involved in catalyzing the hydrolysis of triglycerides (TG) into chylomicrons and very low density lipoprotein (VLDL) particles. Uses of lipases are evolving rapidly and currently they are reported to show high potential in medicine. Intensive study and investigations have led researchers to explore lipases for their use in substitution therapy, where in enzyme deficiency during diseased conditions is compensated by their external administration. In our body, they are used to break down fats present in food so that they can be absorbed in the intestine and deficiency of lipases leads to malabsorption of fats and fat-soluble vitamins. Lipases help a person who has cystic fibrosis, Alzheimer's disease, atherosclerosis and act as a candidate target for cancer prevention and therapy. They act as diagnostic tool and their presence or increasing levels can indicate certain infection or disease. Obesity causes metabolic disease and is a serious health problem around the world. Thus inhibiting digestive lipase to reduce fat absorption has become the main pharmacological approach to the treatment of obesity in recent years.

  13. The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

    PubMed Central

    Sánchez-Migallón, M P; Aranda, F J; Gómez-Fernández, J C

    1995-01-01

    We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed. PMID:7696508

  14. Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis.

    PubMed

    Farese, R V; Konda, T S; Davis, J S; Standaert, M L; Pollet, R J; Cooper, D R

    1987-05-01

    The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.

  15. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

  16. Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

    PubMed

    Cacas, Jean-Luc; Gerbeau-Pissot, Patricia; Fromentin, Jérôme; Cantrel, Catherine; Thomas, Dominique; Jeannette, Emmanuelle; Kalachova, Tetiana; Mongrand, Sébastien; Simon-Plas, Françoise; Ruelland, Eric

    2017-04-01

    Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [(33) P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

  17. Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

    PubMed Central

    Sethi, Bijay Kumar; Nanda, Prativa Kumari; Sahoo, Santilata

    2016-01-01

    Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation. PMID:26887237

  18. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity

    PubMed Central

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins. PMID:27630986

  19. Viscosity and compressibility of diacylglycerol under high pressure

    NASA Astrophysics Data System (ADS)

    Malanowski, Aleksander; Rostocki, A. J.; Kiełczyński, P.; Szalewski, M.; Balcerzak, A.; Kościesza, R.; Tarakowski, R.; Ptasznik, S.; Siegoczyński, R. M.

    2013-03-01

    The influence of high pressure on viscosity and compressibility of diacylglycerol (DAG) oil has been presented in this paper. The investigated DAG oil was composed of 82% of DAGs and 18% TAGs (triacylglycerols). The dynamic viscosity of DAG was investigated as a function of the pressure up to 400 MPa. The viscosity was measured by means of the surface acoustic wave method, where the acoustic waveguides were used as sensing elements. As the pressure was rising, the larger ultrasonic wave attenuation was observed, whereas amplitude decreased with the liquid viscosity augmentation. Measured changes of physical properties were most significant in the pressure range near the phase transition. Deeper understanding of DAG viscosity and compressibility changes versus pressure could shed more light on thermodynamic properties of edible oils.

  20. [The Integration and Regulation of Hormone-Sensitive Lipase in Reproductive System].

    PubMed

    Wang, Wei-yi; Xu, Guo-Heng

    2015-02-01

    Hormone-sensitive lipase (HSL) has long been considered as a classical rate-limiting enzyme during lipolysis since it was first described in 1960s. HSL is regulated mainly by catecholamine, including adrenalin. Studies in recent years indicated that the substrates for HSL are not only triglycerides, but also diacylglycerol with the catalytic activity is ten times that of triglycerides, glycerol esters and cholesterol esters, which overthrow the opinion that HSL is specific to triglyceride. The scientists have generated HSL gene knockout mice and confirmed HSL is widely located in the reproductive system, which indicates that HSL may play an important role in the regulation of physiological and pathophysiological process in the reproductive system. Here, we will focus on the features of the HSL gene, mRNA and its protein, and summarize the HSL functions in the reproductive system.

  1. Agonist-induced production of 1,2-diacylglycerol and phosphatidic acid in intact resistance arteries. Evidence that accumulation of diacylglycerol is not a prerequisite for contraction.

    PubMed

    Ohanian, J; Ollerenshaw, J; Collins, P; Heagerty, A

    1990-05-25

    The production of total amounts of 1,2-diacylglycerol as well as those specifically derived from inositol lipid hydrolysis was studied in intact rat resistance arteries stimulated with either noradrenaline, vasopressin, or angiotensin II at 20 s when the onset of contraction would be nearing its maximum, and at 5 min during the sustained phase of contraction. Total amounts of 1,2-diacylglycerol were not altered by any agonist at 20 s, or at 5 min. However, arachidonate-containing species of 1,2-diacylglycerol were differentially influenced being increased at 5 min by noradrenaline, and decreased at 20 s and 5 min by vasopressin. Only angiotensin II produced substantial increases in this class of 1,2-diacylglycerol at both time points. In order to investigate the fate of this second messenger total and inositol lipid derived phosphatidic acids were then measured at both 20 s and 5 min. Noradrenaline induced a rise in both total and arachidonate-containing phosphatidic acid at both times as did vasopressin. Only small increases were induced by angiotensin II at 20 s. These data demonstrate that the accumulation of 1,2-diacylglycerol generated from inositol lipid breakdown is only observed with activation by angiotensin II. Other agonists produced phosphatidic acids with time and the rate of generation of these lipids is agonist-specific. Thus phosphatidic acid may play a more prominent role during the sustained phase of contraction than previously anticipated.

  2. The SUGAR-DEPENDENT1 Lipase Limits Triacylglycerol Accumulation in Vegetative Tissues of Arabidopsis1[W

    PubMed Central

    Kelly, Amélie A.; van Erp, Harrie; Quettier, Anne-Laure; Shaw, Eve; Menard, Guillaume; Kurup, Smita; Eastmond, Peter J.

    2013-01-01

    There has been considerable interest recently in the prospect of engineering crops to produce triacylglycerol (TAG) in their vegetative tissues as a means to achieve a step change in oil yield. Here, we show that disruption of TAG hydrolysis in the Arabidopsis (Arabidopsis thaliana) lipase mutant sugar-dependent1 (sdp1) leads to a substantial accumulation of TAG in roots and stems but comparatively much lower TAG accumulation in leaves. TAG content in sdp1 roots increases with the age of the plant and can reach more than 1% of dry weight at maturity, a 50-fold increase over the wild type. TAG accumulation in sdp1 roots requires both ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL ACYLTRANSFERASE1 and can also be strongly stimulated by the provision of exogenous sugar. In transgenic plants constitutively coexpressing WRINKLED1 and DGAT1, sdp1 also doubles the accumulation of TAG in roots, stems, and leaves, with levels ranging from 5% to 8% of dry weight. Finally, provision of 3% (w/v) exogenous Suc can further boost root TAG content in these transgenic plants to 17% of dry weight. This level of TAG is similar to seed tissues in many plant species and establishes the efficacy of an engineering strategy to produce oil in vegetative tissues that involves simultaneous manipulation of carbohydrate supply, fatty acid synthesis, TAG synthesis, and also TAG breakdown. PMID:23686420

  3. Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

    PubMed Central

    Patel, Krutika; Trivedi, Ram N.; Durgampudi, Chandra; Noel, Pawan; Cline, Rachel A.; DeLany, James P.; Navina, Sarah; Singh, Vijay P.

    2016-01-01

    Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response. PMID:25579844

  4. Biochemical Characterization and Molecular Modeling of Pancreatic Lipase from a Cartilaginous Fish, the Common Stingray (Dasyatis pastinaca).

    PubMed

    Bouchaâla, Emna; BouAli, Madiha; Ben Ali, Yassine; Miled, Nabil; Gargouri, Youssef; Fendri, Ahmed

    2015-05-01

    In order to identify fish enzymes displaying novel biochemical properties, we have chosen the common stingray (Dasyatis pastinaca), one of the most primitive living jawed aquatic vertebrates as a starting biological material to purify a lipase. A stingray pancreatic lipase (SPL) was purified from delipidated pancreatic powder. The SPL molecular weight was around 55 kDa which is slightly higher than that of known classical pancreatic lipases (50 kDa). This increase in the molecular weight was due to glycosylation. Like classic pancreatic lipases, SPL was found to be much more active on short-chain triacylglycerols than on long-chain ones. Natural detergents act as inhibitors of the SPL activity. This inhibition can be reversed by the addition of stingray colipase. Starting from total pancreatic messenger RNAs (mRNAs), partial stingray pancreatic lipase complementary DNA (cDNA) was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into the PGEM-T vector. Partial amino acid sequence of the SPL was homologous to that of Japanese eel, porcine, and human pancreatic lipases. A 3D structure model of the sequenced part of SPL was built using the 3D structure of porcine pancreatic lipase as template, since both lipases shared an amino acid sequence identity of 60%.

  5. Safety assessment of heated diacylglycerol oil: subchronic toxicity study in rats.

    PubMed

    Morita, Osamu; Tamaki, Yasushi; Kirkpatrick, Jeannie B; Chengelis, Christopher P

    2008-08-01

    Diacylglycerol oil is an edible oil with similar taste and usability characteristics as conventional edible oil rich in triacylglycerol oil. The objective of the present study was to evaluate potential adverse effects of heated diacylglycerol and triacylglycerol oil in rats following subchronic administration. The heated diacylglycerol and triacylglycerol oils were prepared separately following deep frying potato slices at 180 degrees C for 8h per day for three days. Sprague Dawley rats were fed diets containing different ratios (concentrations) of heated to unheated diacylglycerol oil. The ratio of heated to unheated diacylglycerol was as follows: 0%/5.5% (control-1; Group 1), 1.0%/4.5% (Group 2), 2.75%/2.75% (Group 3), and 5.5%/0% (Group 4). Two additional groups received the feed containing 5.5% of unheated or 5.5% of heated triacylglycerol oil. Compared to the unheated oils, feeding of heated diacylglycerol or triacylglycerol oil did not reveal any toxicologically significant changes in clinical observation, body weights, body weight gains, feed consumption, ophthalmic examinations, functional observational battery and motor activity, clinical pathology evaluations and organ weights. Similarly, terminal necropsy did not reveal treatment-related gross or histopathology findings. Based on the results of this subchronic study, the no-observed-effect levels (NOELs) of heated diacylglycerol or triacylglycerol oil were 5.5%, the highest levels tested. The mean dietary exposure levels at the highest dose for the heated diacylglycerol and triacylglycerol oil for male and female rats ranged from 3,178 to 4,120 mg/kg/day.

  6. Antioxidant property and [Formula: see text]-glucosidase, [Formula: see text]-amylase and lipase inhibiting activities of Flacourtia inermis fruits: characterization of malic acid as an inhibitor of the enzymes.

    PubMed

    Alakolanga, A G A W; Kumar, N Savitri; Jayasinghe, Lalith; Fujimoto, Yoshinori

    2015-12-01

    Flacourtia inermis Roxb. (Flacourtiaceae), is a moderate sized tree cultivated in Sri Lanka for its fruits known as Lovi. The current study was undertaken to study the biological activity of extracts of the fruits in an attempt to increase the value of the under exploited fruit crops. Fruits of F. inermis were found to be rich in phenolics and anthocyanins. Polyphenol content of the fruits was determined to be 1.28 g gallic acid equivalents per 100 g of fresh fruit and anthocyanin content was estimated as 108 mg cyanidin-3-glucoside equivalents per 100 g of fresh fruits. The EtOAc extract showed moderate antioxidant activity in the DPPH radical scavenging assay with IC50 value of 66.2 ppm. The EtOAc and MeOH extracts of the fruits also exhibited inhibitory activities toward α-glucosidase, α-amylase and lipase enzymes with IC50values ranging from 549 to 710 ppm, 1021 to 1949 ppm and 1290 to 2096 ppm, respectively. The active principle for the enzyme inhibition was isolated through activity-guided fractionation and was characterized as (S)-malic acid. The results of this study indicate that F. inermis fruits have the potential to be used in health foods and in nutritional supplements.

  7. The role of diacylglycerol-carrying lipoprotein I in lipid transport during insect vitellogenesis.

    PubMed

    Chino, H; Downer, R G; Takahashi, K

    1977-06-22

    A diacylglycerol-carrying lipoprotein was isolated from mature eggs of the silkworm, Philosamia cynthia and compared, for physiochemical properties, with the major diacylglycerol-carrying lipoprotein I (LP-I) of hemolymph. The two molecules are identical an electrophoretic mobility, structural configuration as revealed by electron microscopy, and amino acid composition. In addition mannose was detected in the lipid-free protein moiety, thus enabling classification of the molecules as glycoproteins. The molecules differ in lipid content with egg-LP-I containing only 3.6% of the diacylglycerol content of hemolymph LP-I and the phospholipid and cholesterol components also showing a marked reduction. Analysis of the LP-I and vitellogenin (another diacylglycerol-carrying lipoprotein; LP-II) concentrations of mature eggs indicates that the amounts of the two glycolipoproteins in eggs are insufficient to account for the total acylglycerol content of the eggs. The results suggest that LP-I functions as a true carrier-protein serving to transport diacylglycerol from the fat body to the ovary. This proposal is supported by the observation that LP-I isolated from the egg retains the physiological capacity of hemolymph-LPi to take up diacylglycerol from fat body. Thus it is suggested that LP-I is the major source of lipid for vitellogenesis, whereas vitellogenin is the primary source of protein.

  8. Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

    2013-12-13

    Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

  9. Diacylglycerol Kinase-ε: Properties and Biological Roles

    PubMed Central

    Epand, Richard M.; So, Vincent; Jennings, William; Khadka, Bijendra; Gupta, Radhey S.; Lemaire, Mathieu

    2016-01-01

    In mammals there are at least 10 isoforms of diacylglycerol kinases (DGK). All catalyze the phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PA). Among DGK isoforms, DGKε has several unique features. It is the only DGK isoform with specificity for a particular species of DAG, i.e., 1-stearoyl-2-arachidonoyl glycerol. The smallest of all known DGK isoforms, DGKε, is also the only DGK devoid of a regulatory domain. DGKε is the only DGK isoform that has a hydrophobic segment that is predicted to form a transmembrane helix. As the only membrane-bound, constitutively active DGK isoform with exquisite specificity for particular molecular species of DAG, the functional overlap between DGKε and other DGKs is predicted to be minimal. DGKε exhibits specificity for DAG containing the same acyl chains as those found in the lipid intermediates of the phosphatidylinositol-cycle. It has also been shown that DGKε affects the acyl chain composition of phosphatidylinositol in whole cells. It is thus likely that DGKε is responsible for catalyzing one step in the phosphatidylinositol-cycle. Steps of this cycle take place in both the plasma membrane and the endoplasmic reticulum membrane. DGKε is likely present in both of these membranes. DGKε is the only DGK isoform that is associated with a human disease. Indeed, recessive loss-of-function mutations in DGKε cause atypical hemolytic-uremic syndrome (aHUS). This condition is characterized by thrombosis in the small vessels of the kidney. It causes acute renal insufficiency in infancy and most patients develop end-stage renal failure before adulthood. Disease pathophysiology is poorly understood and there is no therapy. There are also data suggesting that DGKε may play a role in epilepsy and Huntington disease. Thus, DGKε has many unique molecular and biochemical properties when compared to all other DGK isoforms. DGKε homologs also contain a number of conserved sequence features that are distinctive

  10. Differential effects of fenofibrate or simvastatin treatment of rats on hepatic microsomal overt and latent diacylglycerol acyltransferase activities.

    PubMed

    Waterman, Ian J; Zammit, Victor A

    2002-06-01

    Hepatic triacylglycerol secretion is elevated in insulin-resistant states. Microsomal diacylglycerol acyltransferase (DGAT) catalyzes the final reaction in the synthesis of triacylglycerol (TAG). We have previously described two DGAT activities in rat liver microsomes, one overt (cytosol-facing) and one latent (endoplasmic reticulum lumen-facing) (Owen MR, Corstorphine CG, Zammit VA: Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion. Biochem J 323:17-21, 1977). It was suggested that they are involved in the synthesis of TAG for the cytosolic droplet and VLDL lipidation, respectively. In the present study, we measured the overt and latent DGAT activities in rats fed diets containing one of two hypolipidemic drugs: fenofibrate (a peroxisome proliferator-activated receptor alpha [PPARalpha] agonist) and simvastatin (a 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitor). We found that the activities of the two DGATs could be varied independently by these treatments. Fenofibrate raised overt DGAT activity but lowered that of latent DGAT. In contrast, simvastatin markedly lowered overt DGAT activity without affecting that of latent DGAT. The increase in overt DGAT activity induced by fenofibrate could not be mimicked by feeding a diet enriched in n-3 polyunsaturated fatty acids (PUFA), which lowered overt DGAT activity but did not affect latent DGAT, suggesting that n-3 PUFA act through a mechanism independent of PPARalpha activation. The fibrate-induced increase in overt DGAT activity and the inhibition of latent DGAT may provide a mechanism through which acyl moieties are retained within the liver for oxidation through the pathways concomitantly upregulated by PPARalpha activation.

  11. A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin[S

    PubMed Central

    Camacho-Ruiz, María de los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A.

    2015-01-01

    A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. PMID:25748441

  12. Phosphatidic acid phosphatase and diacylglycerol acyltransferase: potential targets for metabolic engineering of microorganism oil.

    PubMed

    Jin, Hong-Hao; Jiang, Jian-Guo

    2015-04-01

    Oleaginous microorganism is becoming one of the most promising oil feedstocks for biodiesel production due to its great advantages in triglyceride (TAG) accumulation. Previous studies have shown that de novo TAG biosynthesis can be divided into two parts: the fatty acid biosynthesis pathway (the upstream part which generates acyl-CoAs) and the glycerol-3-phosphate acylation pathway (the downstream part in which three acyl groups are sequentially added onto a glycerol backbone). This review mainly focuses on two enzymes in the G3P pathway, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). The former catalyzes a dephosphorylation reaction, and the latter catalyzes a subsequent acylation reaction. Genes, functional motifs, transmembrane domains, action mechanism, and new studies of the two enzymes are discussed in detail. Furthermore, this review also covers diacylglycerol kinase, an enzyme that catalyzes the reverse reaction of diacylglycerol formation. In addition, PAP and DGAT are the conjunction points of the G3P pathway, the Kennedy pathway, and the CDP-diacylglycerol pathway (CDP-DAG pathway), and the mutual transformation between TAGs and phospholipids is discussed as well. Given that both the Kennedy and CDP-diacylglycerol pathways are in metabolic interlock (MI) with the G3P pathway, it is suggested that, via metabolic engineering, TAG accumulation can be improved by the two pathways based on the pivotal function of PAP and DGAT.

  13. Diacylglycerol isomers in extra virgin olive oil: Effect of different storage conditions.

    PubMed

    Caponio, Francesco; Paradiso, Vito M; Bilancia, Maria T; Summo, Carmine; Pasqualone, Antonella; Gomes, Tommaso

    2013-10-15

    An experimental investigation was carried out with the aim to investigate on the isomerisation of 1,2-diacylglycerols to 1,3-diacylglycerols as a function of the storage conditions, as well as to identify indices useful to evaluate the freshness of the oils. Two oils derived from two different cultivars (Coratina and Ogliarola barese) were stored for two years as follows: in bottles at dark; in clear glass bottles at light; in green glass bottles at light; in bottles at dark, the latter subjected to repeated opening and samplings to simulate domestic use. The obtained results evinced that during the storage period a significant increase in the 1,3-isomers was observed due to an isomerisation from the 1,2 to the 1,3 isomeric form, consequently the 1,3/1,2 ratio increased in both oils. The covariance analysis of the data showed that the isomerisation of diacylglycerols, taking place during time, was affected by the type of oil, probably due to the different initial hydrolysis level, but was not affected by the storage conditions. Among the parameters considered, the total diacylglycerols/1,3-diacylglycerols ratio could be used as freshness index of extra virgin olive oil, since it is not affected by either oil or storage conditions.

  14. Modeling species-specific diacylglycerol dynamics in the RAW 264.7 macrophage.

    PubMed

    Callender, Hannah L; Horn, Mary Ann; DeCamp, Dianne L; Sternweis, Paul C; Alex Brown, H

    2010-02-21

    A mathematical model of the G protein signaling pathway in RAW 264.7 macrophages downstream of P2Y(6) receptors activated by the ubiquitous signaling nucleotide uridine 5'-diphosphate is developed. The model, which is based on time-course measurements of inositol trisphosphate, cytosolic calcium, and diacylglycerol, focuses particularly on differential dynamics of multiple chemical species of diacylglycerol. When using the canonical pathway representation, the model predicted that key interactions were missing from the current network structure. Indeed, the model suggested that accurate depiction of experimental observations required an additional branch to the signaling pathway. An intracellular pool of diacylglycerol is immediately phosphorylated upon stimulation of an extracellular receptor for uridine 5'-diphosphate and subsequently used to aid replenishment of phosphatidylinositol. As a result of sensitivity analysis of the model parameters, key predictions can be made regarding which of these parameters are the most sensitive to perturbations and are therefore most responsible for output uncertainty.

  15. Selective immunostaining of Mehlis' gland of Opisthorchis viverrini by antibody against rat diacylglycerol kinase.

    PubMed

    Hipkaeo, Wiphawi; Chaisiwamongkol, Kowit; Kanla, Pipatphong; Maleewong, Wanchai; Intapan, Pewpan M; Sadaow, Lakkhana; Hozumi, Yasukazu; Goto, Kaoru; Kondo, Hisatake

    2014-09-01

    The Mehlis' gland of Opisthorchis viverrini was selectively and intensely immunopositive with an antibody against rat diacylglycerol kinase gamma, and its entire structure with associated radiating processes was clearly demonstrated by immuno-light microscopy. In immuno-electron microscopy, the immunopositive processes were revealed to contain many vesicles and vacuoles and the immunoreactive materials were deposited diffusely in the cytoplasm except for the vesicular interior. The present findings suggest that diacylglycerol kinase is present and plays roles in PKC (protein kinase C)-related signaling in the Mehlis' gland of O. viverrini. This further suggests the possibility of a new way to protect from the infection of O. viverrini in humans by using diacylglycerol kinase as a therapeutic target.

  16. Anti- and pro-lipase activity of selected medicinal, herbal and aquatic plants, and structure elucidation of an anti-lipase compound.

    PubMed

    Ado, Muhammad Abubakar; Abas, Faridah; Mohammed, Abdulkarim Sabo; Ghazali, Hasanah M

    2013-11-26

    Plants that help in slowing down the digestion of triacylglycerols (TAGs) in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98) medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL) activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents). Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4%) exhibited moderate inhibition (41%-80%) and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack) I.C Nielsen L. (jering), Cynometra cauliflora (nam-nam) and Aleurites moluccana (L.) Willd (candle nut/buah keras) had the highest (100%) anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis), activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.

  17. [Identification of catalytically active groups of wheat (Triticum aestivum L.) germ lipase].

    PubMed

    Korneeva, O S; Popova, T N; Kapranchikov, V S; Motina, E A

    2008-01-01

    The active site of wheat germ lipase was studied by the Dixon method and chemical modification. The profile of curve logV = f(pH), pK and ionization heat values, lipase photoinactivation, and lipase inactivation with diethylpyrocarbonate and dicyclohexylcarbodiimide led us to assume that the active site of the enzyme comprises the carboxylic group of aspartic or glutamic acid and the imidazole group of histidine. Apparently, the OH-group of serine plays a key role in catalysis: as a result of incubation for 1 h in the presence of phenylmethylsulfonyl fluoride, the enzyme activity decreased by more than 70%. It is shown that ethylenediamine tetraacetate is a noncompetitive inhibitor of lipase. Wheat germs are very healthful because they are rich in vitamins, essential amino acids, and proteins. For this reason, wheat germs are widely used in food, medical, and feed mill industries [1-3]. However, their use is limited by instability during storage, which is largely determined by the effect of hydrolytic and redox enzymes. Representative enzymes of this group are lipase (glycerol ester hydrolase, EC 3.1.1.3), which hydrolyzes triglycerides of higher fatty acids, and lipoxygenase (EC 1.13.11.13), which oxidizes polyunsaturated higher fatty acids.

  18. Organic Solvent Tolerant Lipases and Applications

    PubMed Central

    Kanwar, Shamsher S.

    2014-01-01

    Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents. The esterification reactions using lipase(s) could be performed in water-restricted organic media as organic solvent(s) not only improve(s) the solubility of substrate and reactant in reaction mixture but also permit(s) the reaction in the reverse direction, and often it is easy to recover the product in organic phase in two-phase equilibrium systems. The use of organic solvent tolerant lipase in organic media has exhibited many advantages: increased activity and stability, regiospecificity and stereoselectivity, higher solubility of substrate, ease of products recovery, and ability to shift the reaction equilibrium toward synthetic direction. Therefore the search for organic solvent tolerant enzymes has been an extensive area of research. A variety of fatty acid esters are now being produced commercially using immobilized lipase in nonaqueous solvents. This review describes the organic tolerance and industrial application of lipases. The main emphasis is to study the nature of organic solvent tolerant lipases. Also, the potential industrial applications that make lipases the biocatalysts of choice for the present and future have been presented. PMID:24672342

  19. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ....1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract. Its characterizing...

  20. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract. Its characterizing enzyme activity is that of a triacylglycerol hydrolase (EC 3.1.1.3). (b) The ingredient meets...

  1. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract. Its characterizing enzyme activity is that of a triacylglycerol hydrolase (EC 3.1.1.3). (b) The ingredient meets...

  2. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract. Its characterizing enzyme activity is that of a triacylglycerol hydrolase (EC 3.1.1.3). (b) The ingredient meets...

  3. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract. Its characterizing enzyme activity is that of a triacylglycerol hydrolase (EC 3.1.1.3). (b) The ingredient meets...

  4. Overexpression of Peanut Diacylglycerol Acyltransferase 2 in Escherichia coli

    PubMed Central

    Yang, Lianqun; Zhang, Bin; Chen, Gao; Bi, Yuping

    2013-01-01

    Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for

  5. Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds.

    PubMed

    Banaś, Walentyna; Sanchez Garcia, Alicia; Banaś, Antoni; Stymne, Sten

    2013-06-01

    The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.

  6. In vitro stability evaluation of coated lipase

    PubMed Central

    Liu, Lu Jie; Zhu, Jia; Wang, Bin; Cheng, Chu; Du, Yong Jie; Wang, Min Qi

    2017-01-01

    Objective The study was conducted to evaluate the stability of commercial coated lipase (CT-LIP) in vitro. Methods The capsules were tested under different conditions with a range of temperature, pH, dry heat treatment and steaming treatment, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in this work, respectively. Free lipase (uncoated lipase, UC-LIP) was the control group. Lipase relative activities measured in various treatments were used as a reference frame to characterize the stability. Results The lipase activities were decreased with increasing temperatures (p<0.05), and there was a markedly decline (p<0.01) in lipase comparative activities of UC-LIP at 80°C compared with CT-LIP group. Higher relative activities of lipase were observed in CT-LIP group compared with the free one under acidic ambient (pH 3 to 7) and an alkaline medium (pH 8 to 12). Residual lipase activities of CT-LIP group were increased (p<0.05) by 5.67% and 35.60% in dry heat and hydrothermal treatments, respectively. The lipase relative activity profile of CT-LIP was raised at first and dropped subsequently (p<0.05) compared with constantly reduced tendency of UC-LIP exposed to both SGF and SIF. Conclusion The results suggest that the CT-LIP possesses relatively higher stability in comparison with the UC-LIP in vitro. The CT-LIP could retain the potential property to provide sustained release of lipase and thus improved its bioavailability in the gastrointestinal tract. PMID:27507179

  7. Diacylglycerol production induced by growth hormone in Ob1771 preadipocytes arises from phosphatidylcholine breakdown

    SciTech Connect

    Catalioto, R.M.; Ailhaud, G.; Negrel, R. )

    1990-12-31

    Growth Hormone has recently been shown to stimulate the formation of diacylglycerol in Ob1771 mouse preadipocyte cells without increasing inositol lipid turnover. Addition of growth hormone to Ob1771 cells prelabelled with ({sup 3}H)glycerol or ({sup 3}H)choline led to a rapid, transient and stoechiometric formation of labelled diacylglycerol and phosphocholine, respectively. In contrast, no change was observed in the level of choline and phosphatidic acid whereas the release of water-soluble metabolites in ({sup 3}H)ethanolamine prelabelled cells exposed to growth hormone was hardly detectable. Stimulation by growth hormone of cells prelabelled with (2-palmitoyl 9, 10 ({sup 3}H))phosphatidylcholine also induced the production of labelled diacyglycerol. Pertussis toxin abolished both diacylglycerol and phosphocholine formation induced by growth hormone. It is concluded that growth hormone mediates diacylglycerol production in Ob1771 cells by means of phosphatidylcholine breakdown involving a phospholipase C which is likely coupled to the growth hormone receptor via a pertussis toxin-sensitive G-protein.

  8. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants with value-added properties and provide clues for therapeutic intervention for obes...

  9. Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis.

    PubMed

    Jerga, Agoston; Lu, Ying-Jie; Schujman, Gustavo E; de Mendoza, Diego; Rock, Charles O

    2007-07-27

    Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.

  10. Expression of tung seed diacylglycerol acyltransferases (DGAT) in E. coli and yeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG, resist obesity, and/or lack milk secretion. Over-expression of the DGATs increases TAG content in seeds and other t...

  11. Castor diacylglycerol acyltransferase type1(DGAT1)displays greater activity with diricinolein than Arabidopsis DGAT1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Castor oil contains the hydroxy fatty acid ricinoleate as a major (90%) component. The diacylglycerol acyltransferase (DGAT) carries out the final reaction step in the biosynthesis of triacylglycerol, the principal constituent of seed oil, and has been considered to be the step that controls the oil...

  12. Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGAT) are responsible for the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes, including DGAT1 and DGAT2 of tung tre...

  13. Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice.

    PubMed

    Nègre, A; Salvayre, R; Dousset, N; Rogalle, P; Dang, Q Q; Douste-Blazy, L

    1988-11-25

    Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols

  14. Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols

    PubMed Central

    Haïli, Nawel; Louap, Julien; Canonge, Michel; Jagic, Franjo; Louis-Mondésir, Christelle; Chardot, Thierry

    2016-01-01

    The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1pΔ19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85). Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1pΔ85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications. PMID:27780240

  15. Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice.

    PubMed

    Baumeier, Christian; Kaiser, Daniel; Heeren, Jörg; Scheja, Ludger; John, Clara; Weise, Christoph; Eravci, Murat; Lagerpusch, Merit; Schulze, Gunnar; Joost, Hans-Georg; Schwenk, Robert Wolfgang; Schürmann, Annette

    2015-05-01

    Caloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species.

  16. Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

    PubMed

    Haïli, Nawel; Louap, Julien; Canonge, Michel; Jagic, Franjo; Louis-Mondésir, Christelle; Chardot, Thierry; Briozzo, Pierre

    2016-01-01

    The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1pΔ19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85). Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1pΔ85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications.

  17. The uncoupling effect of diacylglycerol on gap junctional communication of mammalian heart cells is independent of protein kinase C.

    PubMed

    Bastide, B; Hervé, J C; Délèze, J

    1994-10-01

    Possible regulatory effects on cell-to-cell communication of a synthetic diacylglycerol, an activator of protein kinase C (PKC), were examined in pairs of synchronously beating ventricular myocytes of neonatal rats in primary culture. Junctional communication was estimated by measuring either the rate constant of dye diffusion, with the fluorescence recovery after photobleaching technique, or the cell-to-cell electrical conductance with a double whole-cell voltage clamp. The addition of a freshly prepared emulsion of 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 micrograms/ml), either in the bath or in the solution filling the patch pipet, was seen to interrupt intercellular communication within approximately 8 to 10 min. This effect is neither mimicked by stimulation of PKC by a phorbol ester, nor prevented by PKC inhibitors, making it unlikely that, in these cells, PKC activation could induce intercellular uncoupling. During OAG exposures, the intracellular calcium concentration was very modestly increased (by a factor 1.5 to 2), which does not suffice to account for uncoupling. OAG might trigger interruption of cell-to-cell communication by a mechanism analogous to that of other lipophilic molecules (such as aliphatic alcohols or long chain unsaturated fatty acids) which interfere with gap junctions.

  18. Advances in lipase-catalyzed esterification reactions.

    PubMed

    Stergiou, Panagiota-Yiolanda; Foukis, Athanasios; Filippou, Michalis; Koukouritaki, Maria; Parapouli, Maria; Theodorou, Leonidas G; Hatziloukas, Efstathios; Afendra, Amalia; Pandey, Ashok; Papamichael, Emmanuel M

    2013-12-01

    Lipase-catalyzed esterification reactions are among the most significant chemical and biochemical processes of industrial relevance. Lipases catalyze hydrolysis as well as esterification reactions. Enzyme-catalyzed esterification has acquired increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions attracted research interest during the past decade, due to an increased use of organic esters in biotechnology and the chemical industry. Lipases, as hydrolyzing agents are active in environments, which contain a minimum of two distinct phases, where all reactants are partitioned between these phases, although their distribution is not fixed and changes as the reaction proceeds. The kinetics of the lipase-catalyzed reactions is governed by a number of factors. This article presents a thorough and descriptive evaluation of the applied trends and perspectives concerning the enzymatic esterification, mainly for biofuel production; an emphasis is given on essential factors, which affect the lipase-catalyzed esterification reaction. Moreover, the art of using bacterial and/or fungal strains for whole cell biocatalysis purposes, as well as carrying out catalysis by various forms of purified lipases from bacterial and fungal sources is also reviewed.

  19. Purification of extracellular lipase from Pseudomonas aeruginosa.

    PubMed Central

    Stuer, W; Jaeger, K E; Winkler, U K

    1986-01-01

    Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s. Images PMID:3096967

  20. Organization of the human lipoprotein lipase gene and evolution of the lipase gene family.

    PubMed Central

    Kirchgessner, T G; Chuat, J C; Heinzmann, C; Etienne, J; Guilhot, S; Svenson, K; Ameis, D; Pilon, C; d'Auriol, L; Andalibi, A

    1989-01-01

    The human lipoprotein lipase gene was cloned and characterized. It is composed of 10 exons spanning approximately equal to 30 kilobases. The first exon encodes the 5'-untranslated region, the signal peptide plus the first two amino acids of the mature protein. The next eight exons encode the remaining 446 amino acids, and the tenth exon encodes the long 3'-untranslated region of 1948 nucleotides. The lipoprotein lipase transcription start site and the sequence of the 5'-flanking region were also determined. We compared the organization of genes for lipoprotein lipase, hepatic lipase, pancreatic lipase, and Drosophila yolk protein 1, which are members of a family of related genes. A model for the evolution of the lipase gene family is presented that involves multiple rounds of gene duplication plus exon-shuffling and intron-loss events. Images PMID:2602366

  1. Synthesis of hepatic lipase in liver and extrahepatic tissues

    SciTech Connect

    Doolittle, M.H.; Wong, H.; Davis, R.C.; Schotz, M.C.

    1987-11-01

    Immunoprecipitations of hepatic lipase from pulse-labeled rat liver have demonstrated that hepatic lipase is synthesized in two distinct molecular weight forms, HL-I (Mr = 51,000) and HL-II (Mr = 53,000). Both forms are immunologically related to purified hepatic lipase, but not to lipoprotein lipase. HL-I and HL-II are also kinetically related and represent different stages of intracellular processing. Glycosidase experiments suggest that HL-I is the high mannose microsomal form of the mature, sialylated HL-II enzyme. Hepatic lipase activity was detected in liver and adrenal gland but was absent in brain, heart, kidney, testes, small intestine, lung, and spleen. The adrenal and liver lipase activities were inhibited in a similar dose-dependent manner by hepatic lipase antiserum. Immunoblot analysis of partially purified adrenal lipase showed an immunoreactive band co-migrating with HL-II at 53,000 daltons which was absent in a control blot treated with preimmune serum. Adrenal lipase and authentic hepatic lipase yielded similar peptide maps, confirming the presence of the lipase in adrenal gland. However, incorporation of L-(/sup 35/S)methionine into immunoprecipitable hepatic lipase was not detected in this tissue. In addition, Northern blot analysis showed the presence of hepatic lipase mRNA in liver but not adrenal gland. The presence of hepatic lipase in adrenal gland in the absence of detectable synthesis or messenger suggests that hepatic lipase originates in liver and is transported to this extrahepatic site.

  2. Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo.

    PubMed

    Zhang, Jian; Kang, Min-Jung; Kim, Myung-Jin; Kim, Mi-Eun; Song, Ji-Hyun; Lee, Young-Min; Kim, Jung-In

    2008-01-01

    Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and 4 microg/ml. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with corn oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of 250 microg/ml, respectively. T. officinale extract showed dose-dependent inhibition with the IC(50) of 78.2 microg/ml. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AUC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary.

  3. Effects of the diacylglycerol complexing agent, cremophor, on nerve-conduction velocity and perfusion in diabetic rats.

    PubMed

    Jack, A M; Cameron, N E; Cotter, M A

    1999-01-01

    The contribution of diacylglycerol (DAG) and protein kinase C (PKC) to diabetic complications has been the subject of debate. In vascular tissues, diabetes increases DAG content, which activates PKC and causes abnormal tissue perfusion. Reduced nerve blood flow has been implicated in the development of neuropathy. However, nerve DAG/PKC activity is not increased and may even be reduced by diabetes, which has also been implicated in neuropathy. The aim was to test whether 2 weeks of treatment with cremophor, an agent that complexes DAG and prevents PKC activation, could correct nerve-conduction velocity (NCV) deficits in rats with 6 weeks of untreated diabetes, as predicted on a vascular hypothesis, or whether this worsened the deficits, as predicted for a direct effect on nerve fibers. Diabetes caused 17.9 +/- 0.9% (+/- SEM) and 15.5 +/- 1.6% reductions in sciatic motor and saphenous sensory NCV, respectively, that were largely (79.6 +/- 6.3% and 57.8 +/- 11.5%) corrected by 100 mg x kg(-1) x day(-1) cremophor treatment. The effects of cremophor on motor and sensory NCV were completely attenuated by co-treatment with the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine. In contrast, co-treatment with the cyclooxygenase inhibitor, flurbiprofen, had no effect on NCV. Sciatic nutritive and total endoneurial perfusion were 49.7 +/- 3.4% and 51.8 +/- 4.2% reduced by diabetes, respectively, and these deficits were 69.5 +/- 7.4% and 79.0 +/- 11.6% corrected by cremophor treatment. Thus the data suggest that an increased DAG/PKC vascular mechanism, perhaps linked to the nitric oxide system, contributes to the etiology of diabetic nerve dysfunction.

  4. Lipase catalyzed synthesis of silicone polyesters.

    PubMed

    Poojari, Yadagiri; Clarson, Stephen J

    2009-11-28

    Immobilized Candida antarctica lipase B (CALB) was successfully employed as a catalyst to synthesize silicone aromatic polyesters by the transesterification of dimethyl terephthalate with alpha,omega-bis(hydroxyalkyl)-terminated poly(dimethylsiloxane) in toluene under mild reaction conditions.

  5. Lipase and phospholipase biosensors: a review.

    PubMed

    Herrera-López, Enrique J

    2012-01-01

    Recent advances in the field of biology, electronics, and nanotechnology have improved the development of biosensors. A biosensor is a device composed of a biological recognition element and a sensor element. Biosensor applications are becoming increasingly important in areas such as biotechnology, pharmaceutics, food, and environment. Lipases and phospholipases are enzymes which have been used widely in food industry, oleochemical industry, biodegradable polymers, detergents, and other applications. In the medical industry, lipases and phospholipases are used as diagnostic tools to detect triglycerides, cholesterol, and phospholipids levels in blood samples. Therefore, the development of lipase and phospholipase biosensors is of paramount importance in the clinical area. This chapter introduces the reader into the preliminaries of biosensor and reviews recent developments of lipase and phospholipase biosensors.

  6. Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol.

    PubMed

    Díaz-Flores, Ernesto; Siliceo, María; Martínez-A, Carlos; Mérida, Isabel

    2003-08-01

    Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.

  7. The interconversion of diacylglycerol and phosphatidylcholine during triacylglycerol production in microsomal preparations of developing cotyledons of safflower (Carthamus tinctorius L.).

    PubMed

    Stobart, A K; Stymne, S

    1985-11-15

    Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalyse the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. Under these conditions the radioactive glycerol in sn-glycerol 3-phosphate accumulates in phosphatidic acid, phosphatidylcholine, diacyl- and tri-acylglycerol. The incorporation of glycerol into phosphatidylcholine is via diacylglycerol and probably involves a cholinephosphotransferase. The results show that the glycerol moiety and the acyl components in phosphatidylcholine exchange with the diacylglycerol during the biosynthesis of diacylglycerol from phosphatidic acid. The continuous reversible transfer of diacylglycerol with phosphatidylcholine, which operates during active triacylglycerol synthesis, will control in part the polyunsaturated-fatty-acid quality of the final seed oil.

  8. Daio-Orengedokuto inhibits HMG-CoA reductase and pancreatic lipase.

    PubMed

    Kim, Young-Suk; Jung, Eun-Ah; Shin, Ji-Eun; Chang, Jong-Chul; Yang, Hyung-Kil; Kim, Nam-Jae; Cho, Ki-Ho; Bae, Hyung-Sup; Moon, Sang-Kwan; Kim, Dong-Hyun

    2002-11-01

    To evaluate the antihyperlipidemic activities of Orengedokuto (OT) and Daio-Orengedokuto (DOT), the inhibitory effects of these polyprescriptions on HMG-CoA reductase and pancreatic lipase and on the rat hyperlipidemic model induced by Triton WR-1339 were measured. OT potently inhibited HMG-CoA reductase but did not inhibit lipase. Among their ingredients, Coptidis Rhizoma was the most potent inhibitor, followed by Rhei Rhizoma. The HMG-CoA reductase-inhibitory activity of 80% EtOH extract was superior to that of water extract. However, DOT potently inhibited HMG CoA-reductase as well as pancreatic lipase. In the rat hyperlipidemic model induced by Triton WR-1339, OT and DOT decreased serum total cholesterol and low-density lipoprotein cholesterol levels. DOT also decreased serum triglyceride levels, but OT did not decrease it. These results suggest that the antihyperlipidemic activity of DOT may originate from the inhibition of pancreatic lipase as well as HMG-CoA reductase.

  9. Medicinal Plants and Their Inhibitory Activities against Pancreatic Lipase: A Review

    PubMed Central

    Seyedan, Atefehalsadat; Alshawsh, Mohammed Abdullah; Alshagga, Mustafa Ahmed; Koosha, Sanaz; Mohamed, Zahurin

    2015-01-01

    Obesity is recognized as a major life style disorder especially in developing countries and it is prevailing at an alarming speed in new world countries due to fast food intake, industrialization, and reduction of physical activity. Furthermore, it is associated with a vast number of chronic diseases and disabilities. To date, relatively effective drugs, from either natural or synthetic sources, are generally associated with serious side effects, often leading to cessation of clinical trials or even withdrawal from the market. In order to find new compounds which are more effective or with less adverse effects compared to orlistat, the drug that has been approved for obesity, new compounds isolated from natural products are being identified and screened for antiobesity effects, in particular, for their pancreatic lipase inhibitory effect. Pancreatic lipase inhibitory activity has been extensively used for the determination of potential efficacy of natural products as antiobesity agents. In attempts to identify natural products for overcoming obesity, more researches have been focused on the identification of newer pancreatic lipase inhibitors with less unpleasant adverse effects. In this review, we consider the potential role of plants that have been investigated for their pancreatic lipase inhibitory activity. PMID:26640503

  10. Utilization of agroindustrial residues for lipase production by solid-state fermentation

    PubMed Central

    Damaso, Mônica Caramez Triches; Passianoto, Moisés Augusto; de Freitas, Sidinéa Cordeiro; Freire, Denise Maria Guimarães; Lago, Regina Celi Araujo; Couri, Sonia

    2008-01-01

    The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively. PMID:24031288

  11. Diacylglycerol Kinases (DGKs): Novel Targets for Improving T Cell Activity in Cancer

    PubMed Central

    Riese, Matthew J.; Moon, Edmund K.; Johnson, Bryon D.; Albelda, Steven M.

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the metabolism of diacylglycerol (DAG). Two isoforms of DGK, DGKα, and DGKζ, specifically regulate the pool of DAG that is generated as a second messenger after stimulation of the T cell receptor (TCR). Deletion of either isoform in mouse models results in T cells bearing a hyperresponsive phenotype and enhanced T cell activity against malignancy. Whereas, DGKζ appears to be the dominant isoform in T cells, rationale exists for targeting both isoforms individually or coordinately. Additional work is needed to rigorously identify the molecular changes that result from deletion of DGKs in order to understand how DAG contributes to T cell activation, the effect of DGK inhibition in human T cells, and to rationally develop combined immunotherapeutic strategies that target DGKs. PMID:27800476

  12. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

    PubMed Central

    Weigel, Christoph; Veldwijk, Marlon R.; Oakes, Christopher C.; Seibold, Petra; Slynko, Alla; Liesenfeld, David B.; Rabionet, Mariona; Hanke, Sabrina A.; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-01-01

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy. PMID:26964756

  13. Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis1

    PubMed Central

    Ståhl, Ulf; Carlsson, Anders S.; Lenman, Marit; Dahlqvist, Anders; Huang, Bangquan; Banaś, Walentyna; Banaś, Antoni; Stymne, Sten

    2004-01-01

    A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type. PMID:15247387

  14. Dramatic Differences in the Roles in Lipid Metabolism Of Two Isoforms of Diacylglycerol Kinase†

    PubMed Central

    Milne, Stephen B.; Ivanova, Pavlina T.; Armstrong, Michelle D.; Myers, David S.; Lubarda, Jovana; Shulga, Yulia V.; Topham, Matthew K.; Brown, H. Alex; Epand, Richard M.

    2009-01-01

    Lipid species change for SV40 transformed fibroblasts from wild-type or from diacylglycerol kinase-ε (DGKε) or diacylglycerol kinase-α (DGKα) knock out mice, were determined for glycerophospholipids, poly-phosphatidylinositides (GPInsPn), and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKε knock out cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKε knock out and wild-type cells, suggesting that DGKε catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsPn lipids and least for DAG. These findings indicate that DGKε plays a significant role in determining the enrichment of GPInsPn with 20:4 and that there is a pathway for the selective translocation of arachidonoyl-phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKα knock out mice. However, the cells from the DGKα knock out mice had a higher concentration of DAG, consistent with the lack of down regulation of the major fraction of DAG by DGKα, in contrast with DGKε that is primarily responsible for enrichment of GPInsPn with arachidonoyl acyl chains. PMID:18702510

  15. Modeling Species-Specific Diacylglycerol Dynamics in the RAW 264.7 Macrophage

    PubMed Central

    Callender, Hannah L.; Horn, Mary Ann; DeCamp, Dianne L.; Sternweis, Paul C.; Brown, H. Alex

    2009-01-01

    A mathematical model of the G protein signaling pathway in RAW 264.7 macrophages downstream of P2Y6 receptors activated by the ubiquitous signaling nucleotide uridine 5′-diphosphate is developed. The model, which is based on time-course measurements of inositol trisphosphate, cytosolic calcium, and diacylglycerol, focuses particularly on differential dynamics of multiple chemical species of diacylglycerol. When using the canonical pathway representation, the model predicted that key interactions were missing from the current network structure. Indeed, the model suggested that accurate depiction of experimental observations required an additional branch to the signaling pathway. An intracellular pool of diacylglycerol is immediately phosphorylated upon stimulation of an extracellular receptor for uridine 5′-diphosphate and subsequently used to aid replenishment of phosphatidylinositol. As a result of sensitivity analysis of the model parameters, key predictions can be made regarding which of these parameters are the most sensitive to perturbations and are therefore most responsible for output uncertainty. PMID:19883664

  16. Resolution of diacylglycerol moieties of natural glycerophospholipids by gas-liquid chromatography on polar capillary columns.

    PubMed

    Myher, J J; Kuksis, A

    1982-06-01

    A rapid and practical method has been developed for the gas-liquid chromatographic determination of the sn-1,2-diacylglycerol moieties of natural glycerophospholipids using polar wall-coated open tubular columns. The method gives complete resolution and quantitative estimates for all species according to molecular weight and degree of unsaturation, including stearoyl docosahexaenoylglycerol and related polyunsaturates. For this purpose the sn-1,2-diacylglycerols are obtained from the glycerophospholipids by hydrolysis with phospholipase C and are converted into the trimethylsilyl or tertiary-butyldimethylsilyl ethers. The silyl ethers are separated by gas-liquid chromatography on the capillary glass columns coated with a polar cyanopropylsiloxane polymer, in the temperature range 175-250 degrees C, using hydrogen as the carrier gas. Practical applications of the method are illustrated by analyses of the sn-1,2-diacylglycerol moieties of the phosphatidylcholines of soybean phosphatides, egg yolk, and rat liver. The method of analysis is applicable to other classes of glycerophospholipids and the total time requirements for the analysis of any one phospholipid class are comparable to those for a fatty acid analysis.

  17. Diacylglycerol-carrying lipoprotein of hemolymph of the locust and some insects.

    PubMed

    Chino, H; Kitazawa, K

    1981-09-01

    The diacylglycerol-carrying lipoprotein (DGLP) was purified from hemolymph of the locust, Locusta migratoria, by a rapid method which included a specific precipitation at low ionic concentration and DEAE-cellulose column chromatography. The final preparation was highly homogeneous as judged by gel electrophoresis, electron microscopy, and immunodiffusion. The locust DGLP molecule was almost spherical in shape with a diameter of about 130 A. The molecular weight, determined by a sedimentation equilibrium method, was approximately 580,000. The total lipid content amounted to about 40%. The lipids comprised diacylglycerol (33% of total lipid), hydrocarbon (21%), cholesterol (8%), and phospholipids (36%). The hydrocarbon fraction contained a number of n-alkanes and methylalkanes ranging from C25 to C38 in chain length. Mannose (3%) and glucosamine (0.5%) were associated with the apoprotein of DGLP. Apoprotein of locust DGLP consisted of two subunits, heavy chain (mol wt 250,000) and light chain (mol wt 85,000); carbohydrate (mannose) was associated only with the heavy chain. Tests of physiological function of DGLPs from locust, cockroach, and silkworm suggest that the insect DGLP serves multiple roles as a true carrier molecule in transporting diacylglycerol, cholesterol, and hydrocarbon from sites of storage, absorption, and synthesis to sites where these lipids are utilized as metabolic fuel, precursors for triacylglycerol and phospholipid synthesis, or structural components of cell membrane and cuticle. In addition, the insect DGLPs displayed no species-specificity in terms of the functions, whereas they were immunologically distinguishable.

  18. Marine invertebrate lipases: Comparative and functional genomic analysis.

    PubMed

    Rivera-Perez, Crisalejandra

    2015-09-01

    Lipases are key enzymes involved in lipid digestion, storage and mobilization of reserves during fasting or heightened metabolic demand. This is a highly conserved process, essential for survival. The genomes of five marine invertebrate species with distinctive digestive system were screened for the six major lipase families. The two most common families in marine invertebrates, the neutral an acid lipases, are also the main families in mammals and insects. The number of lipases varies two-fold across analyzed genomes. A high degree of orthology with mammalian lipases was observed. Interestingly, 19% of the marine invertebrate lipases have lost motifs required for catalysis. Analysis of the lid and loop regions of the neutral lipases suggests that many marine invertebrates have a functional triacylglycerol hydrolytic activity as well as some acid lipases. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these families of enzymes in marine invertebrates.

  19. Arabidopsis Lipins, PDAT1 Acyltransferase, and SDP1 Triacylglycerol Lipase Synergistically Direct Fatty Acids toward β-Oxidation, Thereby Maintaining Membrane Lipid Homeostasis[C][W

    PubMed Central

    Fan, Jilian; Yan, Chengshi; Roston, Rebecca; Shanklin, John

    2014-01-01

    Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal β-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves. PMID:25293755

  20. Diacylglycerol Is Required for the Formation of COPI Vesicles in the Golgi-to-ER Transport Pathway

    PubMed Central

    Fernández-Ulibarri, Inés; Vilella, Montserrat; Lázaro-Diéguez, Francisco; Sarri, Elisabet; Martínez, Susana E.; Jiménez, Nuria; Claro, Enrique; Mérida, Isabel; Burger, Koert N.J.

    2007-01-01

    Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation. PMID:17567948

  1. Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

    PubMed Central

    Klig, L S; Homann, M J; Kohlwein, S D; Kelley, M J; Henry, S A; Carman, G M

    1988-01-01

    A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant. Images PMID:2832385

  2. A double blind lipase for lipase comparison of a high lipase and standard pancreatic enzyme preparation in cystic fibrosis.

    PubMed Central

    Bowler, I M; Wolfe, S P; Owens, H M; Sheldon, T A; Littlewood, J M; Walters, M P

    1993-01-01

    A standard acid resistant microsphere pancreatic enzyme preparation was compared with identical capsules half filled with mini-tablets of a new high lipase preparation in a randomised double blind crossover study in children with cystic fibrosis. Each patient received his/her usual number of capsules and the same dose of lipase during each period of the study. Eighteen patients completed the study. There were fewer gastrointestinal symptoms when pancreatic enzyme was supplied as the high lipase preparation. There was also a significant improvement in fat absorption (17%, 95% confidence interval (CI) 6 to 27), reduction in faecal fat output (15.8 g/day, 95% CI 6.4 to 22.5), and faecal energy loss (789 kJ/day, 95% CI 211 to 1384). It is concluded that half filled capsules of the new high lipase preparation are more effective than the standard preparation and it is likely that filled capsules would allow patients to use fewer than half the number of pancreatic enzyme capsules. PMID:7683190

  3. Polyphenols extracted from black tea (Camellia sinensis) residue by hot-compressed water and their inhibitory effect on pancreatic lipase in vitro.

    PubMed

    Yuda, Naoki; Tanaka, Miyuki; Suzuki, Manabu; Asano, Yuzo; Ochi, Hiroshi; Iwatsuki, Keiji

    2012-12-01

    Polyphenols, retained in black tea wastes following the commercial production of tea beverages, represent an underutilized resource. The purpose of this study was to investigate the potential use of hot-compressed water (HCW) for the extraction of pancreatic lipase-inhibiting polyphenols from black tea residues. Black tea residues were treated with HCW at 10 °C intervals, from 100 to 200 °C. The resulting extracts were analyzed using high-performance liquid chromatography-mass spectrometry and assayed to determine their inhibitory effect on pancreatic lipase activity in vitro. Four theaflavins (TF), 5 catechins, 2 quercetin glycosides, quinic acid, gallic acid, and caffeine were identified. The total polyphenol content of extracts increased with increasing temperature but lipase inhibitors (TF, theaflavin 3-O-gallate, theaflavin 3'-O-gallate, theaflavin 3,3'-O-gallate, epigallocatechin gallate, and epicatechin gallate) decreased over 150 °C. All extracts inhibited pancreatic lipase but extracts obtained at 100 to 140 °C showed the greatest lipase inhibition (IC(50) s of 0.9 to 1.3 μg/mL), consistent with the optimal extraction of TFs and catechins except catechin by HCW between 130 and 150 °C. HCW can be used to extract pancreatic lipase-inhibiting polyphenols from black tea waste. These extracts have potential uses, as dietary supplements and medications, for the prevention and treatment of obesity.

  4. Interfacial Partitioning of a Loop Hinge Residue Contributes to Diacylglycerol Affinity of Conserved Region 1 Domains*

    PubMed Central

    Stewart, Mikaela D.; Cole, Taylor R.; Igumenova, Tatyana I.

    2014-01-01

    Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826–830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities. PMID:25124034

  5. Diet quality determines lipase gene expression and lipase/esterase activity in Daphnia pulex

    PubMed Central

    Schwarzenberger, Anke; Wacker, Alexander

    2017-01-01

    ABSTRACT We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments. PMID:28069588

  6. New Extremophilic Lipases and Esterases from Metagenomics

    PubMed Central

    López-López, Olalla; Cerdán, Maria E; González Siso, Maria I

    2014-01-01

    Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications. PMID:24588890

  7. Gastric lipase secretion in children with gastritis.

    PubMed

    Tomasik, Przemyslaw J; Wędrychowicz, Andrzej; Rogatko, Iwona; Zając, Andrzej; Fyderek, Krzysztof; Sztefko, Krystyna

    2013-07-29

    Gastric lipase is one of the prepancreatic lipases found in some mammalian species and in humans. Our knowledge of the hormonal regulation of gastric lipase secretion in children and adolescents is still very limited. The aim of this study was to compare the activity of human gastric lipase (HGL) in gastric juice in healthy adolescents and in patients with gastritis. The adolescents were allocated to three groups: the first including patients with Helicobacter pylori gastritis (HPG; n = 10), the second including patients with superficial gastritis caused by pathogens other than H. pylori (non-HPG; n = 14) and the control group including healthy adolescents (n = 14). Activity of HGL was measured in gastric juice collected during endoscopy. Plasma concentrations of cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) were measured in all adolescents. Activity of HGL in the non-HPG group was significantly lower than in the HPG group (p < 0.005) and the control group (p < 0.005). Mean plasma GIP levels in the control group were lower than in the non-HPG group (p < 0.003) and the HPG group (p < 0.01). We conclude that the regulation of HGL secretion by GLP-1 and CCK is altered in patients with gastritis. Moreover, GIP is a potent controller of HGL activity, both in healthy subjects and in patients with gastritis.

  8. Structural characterization of MAPLE deposited lipase biofilm

    NASA Astrophysics Data System (ADS)

    Aronne, Antonio; Ausanio, Giovanni; Bloisi, Francesco; Calabria, Raffaela; Califano, Valeria; Fanelli, Esther; Massoli, Patrizio; Vicari, Luciano R. M.

    2014-11-01

    Lipases (triacylglycerol ester hydrolases) are enzymes used in several industrial applications. Enzymes immobilization can be used to address key issues limiting widespread application at industrial level. Immobilization efficiency is related to the ability to preserve the native conformation of the enzyme. MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique, a laser deposition procedure for treating organic/polymeric/biomaterials, was applied for the deposition of lipase enzyme in an ice matrix, using near infrared laser radiation. Microscopy analysis showed that the deposition occurred in micrometric and submicrometric clusters with a wide size distribution. AFM imaging showed that inter-cluster regions are uniformly covered with smaller aggregates of nanometric size. Fourier transform infrared spectroscopy was used for both recognizing the deposited material and analyzing its secondary structure. Results showed that the protein underwent reversible self-association during the deposition process. Actually, preliminary tests of MAPLE deposited lipase used for soybean oil transesterification with isopropyl alcohol followed by gas chromatography-mass spectrometry gave results consistent with undamaged deposition of lipase.

  9. Rat Hormone Sensitive Lipase Inhibition by Cyclipostins and Their Analogs

    PubMed Central

    Vasilieva, Elena; Dutta, Supratik; Malla, Raj K.; Martin, Benjamin P.; Spilling, Christopher D.; Dupureur, Cynthia M.

    2015-01-01

    Cyclipostins are bicyclic lipophilic phosphate natural products. We report here that synthesized individual diastereomers of cyclipostins P and R have nanomolar IC50s toward hormone sensitive lipase (HSL). The less potent diastereomers of these compounds have 10-fold weaker IC50s. The monocyclic phosphate analog of cyclipostin P is nearly as potent as the bicyclic natural product. Bicyclic phosphonate analogs of both cyclipostins exhibit IC50s similar to those of the weaker diastereomer phosphates (about 400 nM). The monocyclic phosphonate analog of cyclipostin P has similar potency. A series of monocyclic phosphonate analogs in which a hydrophobic tail extends from the lactone side of the ring are considerably poorer inhibitors, with IC50s around 50 μM. Finally cyclophostin, a related natural product inhibitor of acetylcholinesterase (AChE) that lacks the hydrocarbon tail of cyclipostins, is not active against HSL. These results indicate a critical SAR for these compounds, the hydrophobic tail. The smaller lactone ring is not critical to activity, a similarity shared with cyclophostin and AChE. The HSL kinetics of inhibition for the cyclipostin P trans diastereomer were examined in detail. The reaction is irreversible with a KI of 40 nM and a rate constant for inactivation of 0.2 min−1. These results are similar to those observed for cyclophostin and AChE. PMID:25678014

  10. Regulation of hepatic lipase activity by sphingomyelin in plasma lipoproteins.

    PubMed

    Yang, Peng; Subbaiah, Papasani V

    2015-10-01

    Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content.

  11. Regulation of hepatic lipase activity by sphingomyelin in plasma lipoproteins

    PubMed Central

    Yang, Peng; Subbaiah, Papasani V.

    2015-01-01

    Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content. PMID:26193433

  12. Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding.

    PubMed Central

    Jetten, A M; Ganong, B R; Vandenbark, G R; Shirley, J E; Bell, R M

    1985-01-01

    Tumor-promoting phorbol esters induce ornithine decarboxylase (ODCase) activity and reduce epidermal growth factor (EGF) binding in rat tracheal epithelial 2C5 cells. Phorbol esters activate protein kinase C by interacting at the same site as sn-1,2-diacylglycerols, the presumed physiological regulators. The effects of added sn-1,2-diacylglycerols and those generated by phospholipase C treatment of 2C5 cells on ODCase induction and EGF binding were investigated to establish a role for protein kinase C in these cellular responses. Treatment of 2C5 cells with phospholipase C induced ODCase activity and reduced EGF binding, whereas phospholipases A2 and D were inactive. When sn-1,2-diacylglycerols containing fatty acids 3-10 carbons in length were added to 2C5 cells, those diacylglycerols containing fatty acids 5-10 carbons in length caused ODCase induction and reduction in EGF binding. sn-1,2-Dioctanoylglycerol was one of the most active compounds tested. It induced ODCase in a dose- (50-500 microM) and time-dependent manner. The reduction of binding of 125I-labeled EGF by sn-1,2-dioctanoylglycerol was also time and dose dependent and appeared to result from a change in EGF affinity and not the number of receptor sites. This series of sn-1,2-diacylglycerols showed similar structure-function relationships in their ability to induce ODCase activity, to decrease EGF binding, to stimulate protein kinase C, and to inhibit [3H]phorbol dibutyrate binding to the phorbol ester receptor. These data demonstrate biological activities for a number of diacylglycerols and indicate that protein kinase C activation is implicated in ODCase induction and decreased EGF binding. PMID:3157191

  13. Efficient biocatalyst by encapsulating lipase into nanoporous gold

    NASA Astrophysics Data System (ADS)

    Du, Xiaoyu; Liu, Xueying; Li, Yufei; Wu, Chao; Wang, Xia; Xu, Ping

    2013-04-01

    Lipases are one of the most important biocatalysts for biotechnological applications. Immobilization is an efficient method to increase the stability and reusability of lipases. In this study, nanoporous gold (NPG), a new kind of nanoporous material with tunable porosity and excellent biocompatibility, was employed as an effective support for lipase immobilization. The pore size of NPG and adsorption time played key roles in the construction of lipase-NPG biocomposites. The morphology and composition of NPG before and after lipase loading are verified using a scanning electron microscope, equipped with an energy-dispersive X-ray spectrometer. The resulting lipase-NPG biocomposites exhibited excellent catalytic activity and remarkable reusability. The catalytic activity of the lipase-NPG biocomposite with a pore size of 35 nm had no decrease after ten recycles. Besides, the lipase-NPG biocomposite exhibited high catalytic activity in a broader pH range and higher temperature than that of free lipase. In addition, the leaching of lipase from NPG could be prevented by matching the protein's diameter and pore size. Thus, the encapsulation of enzymes within NPG is quite useful for establishing new functions and will have wide applications for different chemical processes.

  14. Prediction and evaluation of the lipase inhibitory activities of tea polyphenols with 3D-QSAR models

    PubMed Central

    Li, Yi-Fang; Chang, Yi-Qun; Deng, Jie; Li, Wei-Xi; Jian, Jie; Gao, Jia-Suo; Wan, Xin; Gao, Hao; Kurihara, Hiroshi; Sun, Ping-Hua; He, Rong-Rong

    2016-01-01

    The extraordinary hypolipidemic effects of polyphenolic compounds from tea have been confirmed in our previous study. To gain compounds with more potent activities, using the conformations of the most active compound revealed by molecular docking, a 3D-QSAR pancreatic lipase inhibitor model with good predictive ability was established and validated by CoMFA and CoMISA methods. With good statistical significance in CoMFA (r2cv = 0.622, r2 = 0.956, F = 261.463, SEE = 0.096) and CoMISA (r2cv = 0.631, r2 = 0.932, F = 75.408, SEE = 0.212) model, we summarized the structure-activity relationship between polyphenolic compounds and pancreatic lipase inhibitory activities and find the bulky substituents in R2, R4 and R5, hydrophilic substituents in R1 and electron withdrawing groups in R2 are the key factors to enhance the lipase inhibitory activities. Under the guidance of the 3D-QSAR results, (2R,3R,2′R,3′R)-desgalloyloolongtheanin-3,3′-O-digallate (DOTD), a potent lipase inhibitor with an IC50 of 0.08 μg/ml, was obtained from EGCG oxidative polymerization catalyzed by crude polyphenol oxidase. Furthermore, DOTD was found to inhibit lipid absorption in olive oil-loaded rats, which was related with inhibiting the activities of lipase in the intestinal mucosa and contents. PMID:27694956

  15. Postnatal ethanol exposure alters levels of 2-arachidonylglycerol-metabolizing enzymes and pharmacological inhibition of monoacylglycerol lipase does not cause neurodegeneration in neonatal mice.

    PubMed

    Subbanna, Shivakumar; Psychoyos, Delphine; Xie, Shan; Basavarajappa, Balapal S

    2015-07-01

    The consumption of ethanol by pregnant women may cause neurological abnormalities, affecting learning and memory processes in children, and are collectively described as fetal alcohol spectrum disorders. However, the molecular mechanisms underlying these changes are still poorly understood. In our previous studies, we found that ethanol treatment of postnatal day 7 (P7) mice significantly enhances the anandamide levels but not the 2-arachidonylglycerol (2-AG) levels and induces widespread neurodegeneration, but the reason for the lack of significant effects of ethanol on the 2-AG level is unknown. In this study, we examined developmental changes in diacylglycerol lipase-α, β (DAGL-α and β) and monoacylglycerol lipase (MAGL). We found that the levels of these proteins were significantly higher in adult brains compared to those detected early in brain development. Next, we examined the influence of P7 ethanol treatment on these enzymes, finding that it differentially altered the DAGL-α protein and mRNA levels but consistently enhanced those of the DAGL-β. Interestingly, the ethanol treatment enhanced MAGL protein and mRNA levels. Inhibition of MAGL with KML29 failed to induce neurodegeneration in P7 mice. Collectively, these findings suggest that ethanol significantly activates DAGL-β and MAGL in the neonatal brain, resulting in no net change in 2-AG levels.

  16. Diacylglycerol kinase α regulates tubular recycling endosome biogenesis and major histocompatibility complex class I recycling.

    PubMed

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2014-11-14

    Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. While a number of diacylglycerol kinase isoforms have been implicated in clathrin-dependent internalization, MHC I lacks the typical motifs known to mediate clathrin-dependent endocytosis. Here we show that depletion of diacylglycerol kinase α (DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of MHC I internalization. We demonstrate that DGKα knock-down causes accumulation of intracellular and surface MHC I, resulting from decreased degradation. Furthermore, we provide evidence that DGKα is required for the generation of phosphatidic acid required for tubular recycling endosome (TRE) biogenesis. Moreover, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with phosphatidic acid, we propose a positive feedback loop in which DGKα generates phosphatidic acid to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.

  17. Enhanced Effector Responses in Activated CD8+ T Cells Deficient in Diacylglycerol Kinases

    PubMed Central

    Riese, Matthew J.; Wang, Liang-Chuan S.; Moon, Edmund K.; Joshi, Rohan P.; Ranganathan, Anjana; June, Carl H.; Koretzky, Gary A.; Albelda, Steven M.

    2013-01-01

    Recent clinical trials have shown promise in the use of chimeric antigen receptor(CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical utility and improve their efficacy. We hypothesized that, since CAR action requires proteins essential for TCR signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin demonstrated greatly enhanced cytokine production and cytotoxicity when co-cultured with a murine mesothelioma line that stably expresses mesothelin. Additionally, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs. PMID:23576561

  18. Distinct Properties of the Two Isoforms of CDP-Diacylglycerol Synthase

    PubMed Central

    2015-01-01

    CDP-diacylglycerol synthases (CDS) are critical enzymes that catalyze the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid (PA). Here we show in vitro that the two isoforms of human CDS, CDS1 and CDS2, show different acyl chain specificities for its lipid substrate. CDS2 is selective for the acyl chains at the sn-1 and sn-2 positions, the most preferred species being 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid. CDS1, conversely, shows no particular substrate specificity, displaying similar activities for almost all substrates tested. Additionally, we show that inhibition of CDS2 by phosphatidylinositol is also acyl chain-dependent, with the strongest inhibition seen with the 1-stearoyl-2-arachidonoyl species. CDS1 shows no acyl chain-dependent inhibition. Both CDS1 and CDS2 are inhibited by their anionic phospholipid end products, with phosphatidylinositol-(4,5)-bisphosphate showing the strongest inhibition. Our results indicate that CDS1 and CDS2 could create different CDP-DAG pools that may serve to enrich different phospholipid species with specific acyl chains. PMID:25375833

  19. Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration.

    PubMed

    Rainero, Elena; Caswell, Patrick T; Muller, Patricia A J; Grindlay, Joan; McCaffrey, Mary W; Zhang, Qifeng; Wakelam, Michael J O; Vousden, Karen H; Graziani, Andrea; Norman, Jim C

    2012-01-23

    Inhibition of αvβ3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of α5β1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase α (DGK-α), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent α5β1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-α drove RCP-dependent invasion in the absence of mutant p53 expression or αvβ3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-α is essential to connect expression of mutant p53s or inhibition of αvβ3 to RCP and for this Rab11 effector to drive the trafficking of α5β1 that is required for tumor cell invasion through three-dimensional matrices.

  20. Acute Manipulation of Diacylglycerol Reveals Roles in Nuclear Envelope Assembly & Endoplasmic Reticulum Morphology

    PubMed Central

    Peddie, Christopher J.; Chung, Gary H. C.; Wang, Alan; Yeh, Karen; Jethwa, Nirmal; Zhang, Qifeng; Wakelam, Michael J. O.; Woscholski, Rudiger; Byrne, Richard D.; Collinson, Lucy M.; Poccia, Dominic L.; Larijani, Banafshé

    2012-01-01

    The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum. PMID:23227247

  1. Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase.

    PubMed

    Greer, Michael S; Truksa, Martin; Deng, Wei; Lung, Shiu-Cheung; Chen, Guanqun; Weselake, Randall J

    2015-03-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus. Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs' native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications.

  2. A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity.

    PubMed

    McFie, Pamela J; Stone, Scot J

    2011-09-01

    Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.

  3. Membrane anchoring of diacylglycerol lactones substituted with rigid hydrophobic acyl domains correlates with biological activities.

    PubMed

    Raifman, Or; Kolusheva, Sofiya; Comin, Maria J; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Marquez, Victor E; Jelinek, Raz

    2010-01-01

    Synthetic diacylglycerol lactones (DAG lactones) are effective modulators of critical cellular signaling pathways downstream of the lipophilic second messenger diacylglycerol that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG lactones in which the hydrophobic moiety is a 'molecular rod', namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Use of assays employing chromatic biomimetic vesicles and biophysical techniques revealed that the mode of membrane anchoring of the DAG lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of the PKC translocation from the cytosol to membranes that is induced by the molecular-rod DAG lactones. This investigation emphasizes that the side residues of DAG lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions, and thus may further contribute to the diversity of biological actions of these synthetic biomimetic ligands.

  4. Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms.

    PubMed Central

    De La Roche, Marc A; Smith, Janet L; Rico, Maribel; Carrasco, Silvia; Merida, Isabel; Licate, Lucila; Côté, Graham P; Egelhoff, Thomas T

    2002-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system. PMID:12296770

  5. Gastric lipase: localization of the enzyme in the stomach

    SciTech Connect

    DeNigris, S.J.; Hamosh, M.; Hamosh, P.; Kasbekar, D.K.

    1986-03-05

    Isolated gastric glands prepared from human and rabbit stomach secrete lipase in response to secretagogues. They have investigated the localization of this enzyme in three species (rabbit, baboon, guinea pig). Gastric mucosa was sampled from the cardia (C), fundus-smooth (FS), fundus-ruggae (FR) and the antral area (A). Lipase activity was measured in mucosal homogenates using /sup 3/H-triolein as substrate and is expressed in units (U) = nmols free fatty acid released/min/mg wet weight. The localization of lipase is compared with that of pepsin (measured by hydrolysis of 2% hemoglobin at pH 1.8 and expressed in I.U.). Lipase is localized in a well defined area in the rabbit and is diffusely distributed in both guinea pig and baboon. The distribution of lipase and pepsin containing cells differs in all three species. The cellular origin of gastric lipase remains to be determined.

  6. A spectrophotometric assay for lipase activity utilizing immobilized triacylglycerols.

    PubMed

    Safarík, I

    1991-01-01

    New substrates for the determination of lipase activity have been developed. Triacylglycerols were immobilized by adsorption on an appropriate carrier or adsorbent yielding a lipase substrate in a powder form. The adsorbed triacylglycerols were easily hydrolyzed by lipases present in a reaction mixture. The released fatty acids were extracted with benzene and converted to the corresponding Cu (II) salts (copper soaps) which were measured spectrophotometrically.

  7. Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11.

    PubMed

    Lailaja, V P; Chandrasekaran, M

    2013-08-01

    Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 °C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30-80 °C) and pH (7.0-10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries.

  8. Quantification of the molecular species of diacylglycerols,triacylglycerols and tetraacylglycerols in lesquerella (Physaria fendleri) oil by HPLC and MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ten diacylglycerols (DAG), 74 triacylglycerols (TAG) and 13 tetraacylglycerols in the seed oil of Physaria fendleri were recently identified by HPLC and MS. These acylglycerols (AG) were quantified by HPLC with evaporative light scattering detector and electrospray ionization mass spectrometry of th...

  9. Mass spectrometry of the lithium adducts of diacylglycerols containing hydroxy FA in castor oil and two normal FA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass ...

  10. Beta2-chimaerin provides a diacylglycerol-dependent mechanism for regulation of adhesion and chemotaxis of T cells.

    PubMed

    Siliceo, María; García-Bernal, David; Carrasco, Silvia; Díaz-Flores, Ernesto; Coluccio Leskow, Federico; Leskow, Federico C; Teixidó, Joaquín; Kazanietz, Marcelo G; Mérida, Isabel

    2006-01-01

    The small GTPase Rac contributes to regulation of cytoskeletal rearrangement during chemokine-induced lymphocyte adhesion and migration in a multi-step process that is very precisely coordinated. Chimaerins are Rac1-specific GTPase-activating proteins of unknown biological function, which have a canonical diacylglycerol C1-binding domain. Here we demonstrate endogenous expression of beta2-chimaerin in T lymphocytes and study the functional role of this protein in phorbol ester and chemokine (CXCL12)-regulated T-cell responses. We used green fluorescent protein-tagged beta2-chimaerin and phorbol ester stimulation to investigate changes in protein localization in living lymphocytes. Our results demonstrate that active Rac cooperates with C1-dependent phorbol ester binding to induce sustained GFP-beta2-chimaerin localization to the membrane. Subcellular distribution of GFP beta2-chimaerin in living cells showed no major changes following CXCL12 stimulation. Nonetheless Rac1-GTP levels were severely inhibited in GFP-beta2-chimaerin-expressing cells, which displayed reduced CXCL12-induced integrin-dependent adhesion and spreading. This effect was dependent on chimaerin GTPase-activating protein function and required diacylglycerol generation. Whereas beta2-chimaerin overexpression decreased static adhesion, it enhanced CXCL12-dependent migration via receptor-dependent diacylglycerol production. These studies demonstrate that beta2-chimaerin provides a novel, diacylglycerol-dependent mechanism for Rac regulation in T cells and suggest a functional role for this protein in Rac-mediated cytoskeletal remodeling.

  11. New tools for exploring "old friends-microbial lipases".

    PubMed

    Nagarajan, Saisubramanian

    2012-11-01

    Fat-splitting enzymes (lipases), due to their natural, industrial, and medical relevance, attract enough attention as fats do in our lives. Starting from the paper that we write, cheese and oil that we consume, detergent that we use to remove oil stains, biodiesel that we use as transportation fuel, to the enantiopure drugs that we use in therapeutics, all these applications are facilitated directly or indirectly by lipases. Due to their uniqueness, versatility, and dexterity, decades of research work have been carried out on microbial lipases. The hunt for novel lipases and strategies to improve them continues unabated as evidenced by new families of microbial lipases that are still being discovered mostly by metagenomic approaches. A separate database for true lipases termed LIPABASE has been created recently which provides taxonomic, structural, biochemical information about true lipases from various species. The present review attempts to summarize new approaches that are employed in various aspects of microbial lipase research, viz., screening, isolation, production, purification, improvement by protein engineering, and surface display. Finally, novel applications facilitated by microbial lipases are also presented.

  12. Endothelial lipase is a major determinant of HDL level

    SciTech Connect

    Ishida, Tatsuro; Choi, Sungshin; Kundu, Ramendra K.; Hirata, Ken-Ichi; Rubin, Edward M.; Cooper, Allen D.; Quertermous, Thomas

    2003-01-30

    For the past three decades, epidemiologic studies have consistently demonstrated an inverse relationship between plasma HDL cholesterol (HDL-C) concentrations and coronary heart disease (CHD). Population-based studies have provided compelling evidence that low HDL-C levels are a risk factor for CHD, and several clinical interventions that increased plasma levels of HDL-C were associated with a reduction in CHD risk. These findings have stimulated extensive investigation into the determinants of plasma HDL-C levels. Turnover studies using radiolabeled apolipoprotein A-I, the major protein component of HDL, suggest that plasma HDL-C concentrations are highly correlated with the rate of clearance of apolipoprotein AI. However, the metabolic mechanisms by which HDL are catabolized have not been fully defined. Previous studies in humans with genetic deficiency of cholesteryl ester transfer protein, and in mice lacking the scavenger receptor BI (SR-BI), have demonstrated that these proteins participate in the removal of cholesterol from HDL, while observations in individuals with mutations in hepatic lipase indicate that this enzyme hydrolyzes HDL triglycerides. In this issue of the JCI, reports from laboratories of Tom Quertermous and Dan Rader now indicate that endothelial lipase (LIPG), a newly identified member of the lipase family, catalyzes the hydrolysis of HDL phospholipids and facilitates the clearance of HDL from the circulation. Endothelial lipase was initially cloned by both of these laboratories using entirely different strategies. Quertermous and his colleagues identified endothelial lipase as a transcript that was upregulated in cultured human umbilical vein endothelial cells undergoing tube formation, whereas the Rader group cloned endothelial lipase as a transcript that was upregulated in the human macrophage-like cell line THP-1 exposed to oxidized LDL. Database searches revealed that endothelial lipase shows strong sequence similarity to lipoprotein

  13. Comparative and functional genomics of lipases in holometabolous insects.

    PubMed

    Horne, Irene; Haritos, Victoria S; Oakeshott, John G

    2009-08-01

    Lipases have key roles in insect lipid acquisition, storage and mobilisation and are also fundamental to many physiological processes underpinning insect reproduction, development, defence from pathogens and oxidative stress, and pheromone signalling. We have screened the recently sequenced genomes of five species from four orders of holometabolous insects, the dipterans Drosophila melanogaster and Anopheles gambiae, the hymenopteran Apis mellifera, the moth Bombyx mori and the beetle Tribolium castaneum, for the six major lipase families that are also found in other organisms. The two most numerous families in the insects, the neutral and acid lipases, are also the main families in mammals, albeit not in Caenorhabditis elegans, plants or microbes. Total numbers of the lipases vary two-fold across the five insect species, from numbers similar to those in mammals up to numbers comparable to those seen in C. elegans. Whilst there is a high degree of orthology with mammalian lipases in the other four families, the great majority of the insect neutral and acid lipases have arisen since the insect orders themselves diverged. Intriguingly, about 10% of the insect neutral and acid lipases have lost motifs critical for catalytic function. Examination of the length of lid and loop regions of the neutral lipase sequences suggest that most of the insect lipases lack triacylglycerol (TAG) hydrolysis activity, although the acid lipases all have intact cap domains required for TAG hydrolysis. We have also reviewed the sequence databases and scientific literature for insights into the expression profiles and functions of the insect neutral and acid lipases and the orthologues of the mammalian adipose triglyceride lipase which has a pivotal role in lipid mobilisation. These data suggest that some of the acid and neutral lipase diversity may be due to a requirement for rapid accumulation of dietary lipids. The different roles required of lipases at the four discrete life stages of

  14. Biodiesel production by transesterification using immobilized lipase.

    PubMed

    Narwal, Sunil Kumar; Gupta, Reena

    2013-04-01

    Biodiesel can be produced by transesterification of vegetable or waste oil catalysed by lipases. Biodiesel is an alternative energy source to conventional fuel. It combines environmental friendliness with biodegradability, low toxicity and renewability. Biodiesel transesterification reactions can be broadly classified into two categories: chemical and enzymatic. The production of biodiesel using the enzymatic route eliminates the reactions catalysed under acid or alkali conditions by yielding product of very high purity. The modification of lipases can improve their stability, activity and tolerance to alcohol. The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel. However, this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability. The main factors affecting biodiesel production are composition of fatty acids, catalyst, solvents, molar ratio of alcohol and oil, temperature, water content, type of alcohol and reactor configuration. Optimization of these parameters is necessary to reduce the cost of biodiesel production.

  15. Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G) by Different Strategies

    PubMed Central

    Mendes, Adriano A.; Freitas, Larissa; de Carvalho, Ana Karine F.; de Oliveira, Pedro C.; de Castro, Heizir F.

    2011-01-01

    The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g−1 of support) was achieved when the lipase was immobilized on epoxy-SiO2-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g−1 of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g−1 of gel, and the highest activity (68.8 ± 2.70 IU·g−1 of support) was obtained when 20 mg of protein·g−1 was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO2-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase. PMID:21811674

  16. Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects on α-glucosidase and pancreatic lipase.

    PubMed

    Zhang, Bing; Deng, Zeyuan; Ramdath, D Dan; Tang, Yao; Chen, Peter X; Liu, Ronghua; Liu, Qiang; Tsao, Rong

    2015-04-01

    Phenolic extracts from 20 Canadian lentil cultivars (Lens culinaris) were evaluated for total phenolic contents and composition, antioxidant activities (DPPH, FRAP, ORAC), and inhibitory properties against α-glucosidase and pancreatic lipase. Twenty one phenolic compounds were identified in the present study, with the majority being flavonoids, including kaempeferol glycosides, catechin/epicatechin glucosides and procyanidins. These phenolic compounds not only contributed significantly to the antioxidant activities, but they were also good inhibitors of α-glucosidase and lipase, two enzymes, respectively, associated with glucose and lipid digestion in the human intestine, thus contributing significantly to the control of blood glucose levels and obesity. More interestingly, it was the flavonols, not the flavanols, which showed the inhibitory activities against α-glucosidase and pancreatic lipase. Our result provides supporting information for developing lentil cultivars and functional foods with improved health benefits and suggests a potential role of lentil consumption in managing weight and control of blood glucose.

  17. Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase.

    PubMed

    Arroyo, R; Sánchez-Muniz, F J; Cuesta, C; Burguillo, F J; Sánchez-Montero, J M

    1996-11-01

    The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3 +/- 0.1 mg/100 mg oil to 26.4 +/- 0.3 mg/100 mg oil after 90 fryings, and from 4.0 +/- 0.1 mg/100 mg oil to 27.7 +/- 0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (KMapp) and apparent maximum velocity of hydrolysis (Vmaxapp) were different in unused palm olein (5.1 +/- 0.7 and 166 +/- 7.6, respectively) than in sunflower oil (2.2 +/- 0.3 and 62 +/- 2.2, respectively). However, changes in KMapp and Vmaxapp were not related to the degree of alteration of the oils.

  18. Enantioselective nano liquid chromatographic separation of racemic pharmaceuticals: a facile one-pot in situ preparation of lipase-based polymer monoliths in capillary format.

    PubMed

    Ahmed, Marwa; Ghanem, Ashraf

    2014-11-01

    New affinity monolithic capillary columns of 150 µm internal diameter were prepared in situ fused glass capillary via either immobilization or encapsulation of Candida antarctica lipase B (CALB) on or within polymer monoliths, respectively. The immobilized lipase-based monoliths were prepared via copolymerization of 19.1% monomers (8.9% MMA and 10.2% GMA), 19.8% EDMA, and 61.1% porogens (54.2% formamide and 6.9% 1-propanol) w/w or 20% GMA, 20% EDMA, and 60% porogens (51.6% cyclohexanol and 8.4% 1-dodecanol) in the presence of AIBN (1%) as a radical initiator. This was followed by pumping a solution of lipase through the capillaries and rinsing with potassium phosphate buffer. On the other hand, the encapsulated lipase-based monoliths were prepared via copolymerization of 20% monomers (GMA), 20% EDMA, and 60% porogens (48% 1-propanol, 6% 1,4-butanediol) or 16.4% monomers (16% BuMA, 0.4% SPMA), 23.6% EDMA, and 60% porogens (36% 1-propanol, 18% 1,4-butanediol along with 6% lipase aqueous solution in potassium phosphate buffer. The prepared capillary columns were investigated for the enantioselective nano liquid chromatographic separation of a set of different classes of racemic pharmaceuticals, namely, α- and β-blockers, antiinflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs, and antiarrhythmic drugs. Run-to-run repeatability was quite satisfactory. The encapsulated lipase-based capillary monolith showed better enantioselective separations of most of the investigated compounds. Baseline separation was achieved for alprenolol, atenolol, bromoglutithimide, carbuterol, chloropheneramine, cizolertine carbinol, 4-hydroxy-3-methoxymandelic acid, desmethylcizolertine, nomifensine, normetanephrine, and sulconazole under reversed phase chromatographic conditions. A speculation about the understanding of the chiral recognition mechanism of

  19. Synthesis and Biological Evaluation of Several Bryostatin Analogues Bearing a Diacylglycerol Lactone C-Ring.

    PubMed

    Baumann, David O; McGowan, Kevin M; Kedei, Noemi; Peach, Megan L; Blumberg, Peter M; Keck, Gary E

    2016-09-02

    As an initial step in designing a simplified bryostatin hybrid molecule, three bryostatin analogues bearing a diacylglycerol lactone-based C-ring, which possessed the requisite pharmacophores for binding to protein kinase C (PKC) together with a modified bryostatin-like A- and B-ring region, were synthesized and evaluated. Merle 46 and Merle 47 exhibited binding affinity to PKC alpha with Ki values of 7000 ± 990 and 4940 ± 470 nM, respectively. Reinstallation of the trans-olefin and gem-dimethyl group present in bryostatin 1 in Merle 48 resulted in improved binding affinity, 363 ± 42 nM. While Merle 46 and 47 were only marginally active biologically, Merle 48 showed sufficient activity on the U937 cells to confirm that it was PMA-like for growth and attachment, as predicted by the substitution pattern of its A- and B-rings.

  20. CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis

    PubMed Central

    Pan, Weijun; Pham, Van N.; Stratman, Amber N.; Castranova, Daniel; Kamei, Makoto; Kidd, Kameha R.; Lo, Brigid D.; Shaw, Kenna M.; Torres-Vazquez, Jesus; Mikelis, Constantinos M.; Gutkind, J. Silvio; Davis, George E.

    2012-01-01

    Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis. PMID:22649102

  1. DAG tales: the multiple faces of diacylglycerol--stereochemistry, metabolism, and signaling.

    PubMed

    Eichmann, Thomas Oliver; Lass, Achim

    2015-10-01

    The neutral lipids diacylglycerols (DAGs) are involved in a plethora of metabolic pathways. They function as components of cellular membranes, as building blocks for glycero(phospho)lipids, and as lipid second messengers. Considering their central role in multiple metabolic processes and signaling pathways, cellular DAG levels require a tight regulation to ensure a constant and controlled availability. Interestingly, DAG species are versatile in their chemical structure. Besides the different fatty acid species esterified to the glycerol backbone, DAGs can occur in three different stereo/regioisoforms, each with unique biological properties. Recent scientific advances have revealed that DAG metabolizing enzymes generate and distinguish different DAG isoforms, and that only one DAG isoform holds signaling properties. Herein, we review the current knowledge of DAG stereochemistry and their impact on cellular metabolism and signaling. Further, we describe intracellular DAG turnover and its stereochemistry in a 3-pool model to illustrate the spatial and stereochemical separation and hereby the diversity of cellular DAG metabolism.

  2. Isolation and biochemical characterization of Bacillus pumilus lipases from the Antarctic.

    PubMed

    Arifin, Arild Ranlym; Kim, Soon-Ja; Yim, Joung Han; Suwanto, Antonius; Kim, Hyung Kwoun

    2013-05-01

    Lipase-producing bacterial strains were isolated from Antarctic soil samples using the tricaprylin agar plate method. Seven strains with relatively strong lipase activities were selected. All of them turned out to be Bacillus pumilus strains by the 16S rRNA gene sequence analysis. Their corresponding lipase genes were cloned, sequenced, and compared. Finally, three different Bacillus pumilus lipases (BPL1, BPL2, and BPL3) were chosen. Their amino acid sequence identities were in the range of 92-98% with the previous Bacillus pumilus lipases. Their optimum temperatures and pHs were measured to be 40 degrees C and pH 9. Lipase BPL1 and lipase BPL2 were stable up to 30 degrees C, whereas lipase BPL3 was stable up to 20 degrees C. Lipase BPL2 was stable within a pH range of 6-10, whereas lipase BPL1 and lipase BPL3 were stable within a pH range of 5-11, showing strong alkaline tolerance. All these lipases exhibited high hydrolytic activity toward pnitrophenyl caprylate (C8). In addition, lipase BPL1 showed high hydrolytic activity toward tributyrin, whereas lipase BPL2 and lipase BPL3 hydrolyzed tricaprylin and castor oil preferentially. These results demonstrated that the three Antarctic Bacillus lipases were alkaliphilic and had a substrate preference toward short- and mediumchain triglycerides. These Antarctic Bacillus lipases might be used in detergent and food industries.

  3. Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons

    PubMed Central

    Tabet, Ricardos; Moutin, Enora; Becker, Jérôme A. J.; Heintz, Dimitri; Fouillen, Laetitia; Flatter, Eric; Krężel, Wojciech; Alunni, Violaine; Koebel, Pascale; Dembélé, Doulaye; Tassone, Flora; Bardoni, Barbara; Mandel, Jean-Louis; Vitale, Nicolas; Muller, Dominique; Le Merrer, Julie; Moine, Hervé

    2016-01-01

    Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine. PMID:27233938

  4. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

  5. Plasma lipidomic profile signature of hypertension in Mexican American families: specific role of diacylglycerols.

    PubMed

    Kulkarni, Hemant; Meikle, Peter J; Mamtani, Manju; Weir, Jacquelyn M; Barlow, Christopher K; Jowett, Jeremy B; Bellis, Claire; Dyer, Thomas D; Johnson, Matthew P; Rainwater, David L; Almasy, Laura; Mahaney, Michael C; Comuzzie, Anthony G; Blangero, John; Curran, Joanne E

    2013-09-01

    Both as a component of metabolic syndrome and as an independent entity, hypertension poses a continued challenge with regard to its diagnosis, pathogenesis, and treatment. Previous studies have documented connections between hypertension and indicators of lipid metabolism. Novel technologies, such as plasma lipidomic profiling, promise a better understanding of disorders in which there is a derangement of the lipid metabolism. However, association of plasma lipidomic profiles with hypertension in a high-risk population, such as Mexican Americans, has not been evaluated before. Using the rich data and sample resource from the ongoing San Antonio Family Heart Study, we conducted plasma lipidomic profiling by combining high-performance liquid chromatography with tandem mass spectroscopy to characterize 319 lipid species in 1192 individuals from 42 large and extended Mexican American families. Robust statistical analyses using polygenic regression models, liability threshold models, and bivariate trait analyses implemented in the SOLAR software were conducted after accounting for obesity, insulin resistance, and relative abundance of various lipoprotein fractions. Diacylglycerols, in general, and the DG 16:0/22:5 and DG 16:0/22:6 lipid species, in particular, were significantly associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP), as well as liability of incident hypertension measured during 7140.17 person-years of follow-up. Four lipid species, including the DG 16:0/22:5 and DG 16:0/22:6 species, showed significant genetic correlations with the liability of hypertension in bivariate trait analyses. Our results demonstrate the value of plasma lipidomic profiling in the context of hypertension and identify disturbance of diacylglycerol metabolism as an independent biomarker of hypertension.

  6. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    SciTech Connect

    Martinson, E.A.; Goldstein, D.; Brown, J.H. )

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  7. Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons.

    PubMed

    Tabet, Ricardos; Moutin, Enora; Becker, Jérôme A J; Heintz, Dimitri; Fouillen, Laetitia; Flatter, Eric; Krężel, Wojciech; Alunni, Violaine; Koebel, Pascale; Dembélé, Doulaye; Tassone, Flora; Bardoni, Barbara; Mandel, Jean-Louis; Vitale, Nicolas; Muller, Dominique; Le Merrer, Julie; Moine, Hervé

    2016-06-28

    Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

  8. The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation.

    PubMed

    Elamin, Ayssar A; Stehr, Matthias; Spallek, Ralf; Rohde, Manfred; Singh, Mahavir

    2011-09-01

    Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 µM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.

  9. Activation of a bacterial lipase by its chaperone.

    PubMed Central

    Hobson, A H; Buckley, C M; Aamand, J L; Jørgensen, S T; Diderichsen, B; McConnell, D J

    1993-01-01

    The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase. Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans. Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA. Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA. However, no lipase activity is observed in the absence of limA. limA has been overexpressed and encodes a 33-kDa protein, Lim. If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation. Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody. The Lim protein has therefore the properties of a chaperone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685908

  10. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance

    PubMed Central

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  11. Importance of the lid and cap domains for the catalytic activity of gastric lipases.

    PubMed

    Miled, N; Bussetta, C; De caro, A; Rivière, M; Berti, L; Canaan, S

    2003-09-01

    Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

  12. Structural Elucidation of Diglycosyl Diacylglycerol and Monoglycosyl Diacylglycerol from Streptococcus pneumoniae by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

    PubMed Central

    Tatituri, Raju Venkata Veera; Brenner, Michael B.; Turk, John; Hsu, Fong-Fu

    2013-01-01

    The cell wall of the pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) contains glucopyranosyl diacylglycerol (GlcDAG) and galactoglucopyranosyldiacylglycerol (GalGlcDAG). The specific GlcDAG consisting of vaccenic acid substituent at sn-2 was recently identified as another glycolipid antigen family recognized by invariant natural killer T cells (iNKT cells). Here, we describe a linear ion-trap (LIT) multiple-stage (MSn) mass spectrometric approach towards structural analysis of GalGlcDAG and GlcDAG. Structural information derived from MSn (n = 2,3) on the [M + Li]+ adduct ions desorbed by electrospray ionization (ESI) affords identification of the fatty acid substituents, assignment of the fatty acyl groups on the glycerol backbone, as well as the location of double bond along the fatty acyl chain. The identification of the fatty acyl groups and determination of their regio-specificity were confirmed by MSn (n = 2,3) on the [M + NH4]+ ions. We establish the structures of GalGlcDAG and GlcDAG isolated from S. pneumoniae, in which the major species consists of a 16:1- or 18:1-fatty acid substituent mainly at sn-2, and the double bond of the fatty acid is located at ω-7 (n-7). More than one isomers were found for each mass in the family. This mass spectrometric approach provides a simple method to achieve structure identification of this important lipid family that would be very difficult to define using the traditional method. PMID:22282097

  13. Lipase-catalyzed synthesis of ascorbyl oleate in acetone: optimization of reaction conditions and lipase reusability.

    PubMed

    Stojanović, Marija; Velićković, Dušan; Dimitrijević, Aleksandra; Milosavić, Nenad; Knežević-Jugović, Zorica; Bezbradica, Dejan

    2013-01-01

    Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60°C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4(th) hour of reaction enabled increase of yield from 16.7 mmol g⁻¹ to 19.3 mmol g⁻¹. Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation.

  14. Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras.

    PubMed

    Griffon, Nathalie; Budreck, Elaine C; Long, Christopher J; Broedl, Uli C; Marchadier, Dawn H L; Glick, Jane M; Rader, Daniel J

    2006-08-01

    The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.

  15. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries.

  16. Inhibitory activity of benzophenones from Anemarrhena asphodeloides on pancreatic lipase.

    PubMed

    Jo, Yang Hee; Kim, Seon Beom; Ahn, Jong Hoon; Liu, Qing; Hwang, Bang Yeon; Lee, Mi Kyeong

    2013-04-01

    Pancreatic lipase is a key enzyme for lipid absorption by hydrolysis of total dietary fats. Therefore, inhibition of pancreatic lipase is suggested to be an effective therapy in the regulation of obesity. The EtOAc-soluble fraction of Anemarrhena asphodeloides rhizomes significantly inhibited pancreatic lipase activity as assessed using porcine pancreatic lipase as an in vitro assay system. Further fractionation of the EtOAc-soluble fraction of A. asphodeloides led to the isolation of a new benzophenone glycoside, zimoside A (1), together with the eleven known compounds iriflophenone (2), 2,4',6-trihydroxy-4-methoxybenzophenone (3), foliamangiferoside A (4), (2,3-dihydroxy-4-methoxyphenyl)(4-hydroxyphenyl)-methanone (5), 1,4,5,6,-tetrahydroxyxanthone (6), isosakuranetin (7), 4-hydroxybenzoic acid (8), 4-hydroxyacetophenone (9), vanillic acid (10), tyrosol (11) and 5-hydroxymethyl-2-furaldehyde (12). Among the isolated compounds, 3, 5 and 10 showed significant inhibition of pancreatic lipase activity.

  17. Effect of clindamycin, erythromycin, lincomycin, and tetracycline on growth and extracellular lipase production by propionibacteria in vitro.

    PubMed Central

    Unkles, S E; Gemmell, C G

    1982-01-01

    Two propionibacteria identified as Propionibacterium acnes and Propionibacterium granulosum were grown anaerobically in the presence of growth subinhibitory concentrations (0.25 and 0.5 minimal inhibitory concentrations) of clindamycin, erythromycin, lincomycin, and tetracycline. Viable counts and assays of extracellular lipase were performed on samples taken at 24-h intervals over a 96-h period. The results showed that lincomycin and clindamycin could inhibit the production of the enzyme by both strains with little effect on their growth rates. Tetracycline caused inhibition of lipase production by P. granulosum only. Although production of the enzyme by P. acnes was delayed in the presence of tetracycline, the final titer was the same as the control. Erythromycin had little effect on growth and enzyme production of either strain. It is possible, therefore, that certain antibiotics used in acne therapy may act not only as bactericidal agents but also as inhibitors of enzyme production under non-growth-limiting conditions. PMID:7081974

  18. Mechanism of acetaldehyde-induced deactivation of microbial lipases

    PubMed Central

    2011-01-01

    Background Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. Results Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. Conclusions Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of

  19. 21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg....

  20. 21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg....

  1. 21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg....

  2. 21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg....

  3. 21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Lipase enzyme preparation derived from Rhizopus... Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No. 9001-62-1), which is obtained from...

  4. A rare entity in ED: Normal lipase level in acute pancreatitis.

    PubMed

    Limon, Onder; Sahin, Erkan; Kantar, Funda Ugur; Oray, Deniz; Ugurhan, Asli Aydinoglu

    2016-03-01

    Acute pancreatitis can have a variable presentation and diagnosis is based on clinical presentation, serum amylase and lipase levels and computed tomography. Negative predictive value of serum lipase in diagnosing acute pancreatitis is approximately to 100 percent and a normal blood lipase level in acute pancreatitis is an extremely rare condition. Here we reported two cases with normal serum amylase and lipase levels.

  5. Molecular characterization of a proteolysis-resistant lipase from Bacillus pumilus SG2.

    PubMed

    Sangeetha, R; Arulpandi, I; Geetha, A

    2014-01-01

    Proteolysis-resistant lipases can be well exploited by industrial processes which employ both lipase and protease as biocatalysts. A proteolysis resistant lipase from Bacillus pumilus SG2 was isolated, purified and characterized earlier. The lipase was resistant to native and commercial proteases. In the present work, we have characterized the lip gene which encodes the proteolysis-resistant lipase from Bacillus pumilus SG2. The parameters and structural details of lipase were analysed. The lip gene consisted of 650 bp. The experimental molecular weight of SG2 lipase was nearly double that of its theoretical molecular weight, thus suggesting the existence of the functional lipase as a covalent dimer. The proteolytic cleavage sites of the lipase would have been made inaccessible by dimerisation, thus rendering the lipase resistant to protease.

  6. Analysis of the Staphylococcus aureus DgkB Structure Reveals a Common Catalytic Mechanism for the Soluble Diacylglycerol Kinases

    SciTech Connect

    Miller, Darcie J.; Jerga, Agoston; Rock, Charles O.; White, Stephen W.

    2008-08-11

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg{sup 2+} site and an associated Asp{center_dot}water{center_dot}Mg{sup 2+} network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp{center_dot}water{center_dot}Mg{sup 2+} network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  7. Analysis of the Staphylococcus aureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerol kinases.

    PubMed

    Miller, Darcie J; Jerga, Agoston; Rock, Charles O; White, Stephen W

    2008-07-01

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp*water*Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp*water*Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  8. Production of lipases by four anoxygenic purple non-sulphur phototrophic bacteria.

    PubMed

    Munjam, Srinivas; Girisham, S; Reddy, S M

    Production of lipases by Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodocyclus gelatinosus and Rhodocyclus tenuis in different synthetic media was investigated. Rc. gelatinosus followed by Rb. sphaeroides were good producers of lipases, while Rps. palustris and Rc. tenuis were poor in lipase secretion. Lipase secretion by Rc. gelatinosus was adaptive in nature, while other three bacterial behavior was inconsistent. No positive correlation could be observed between growth and lipase production.

  9. The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis1[W][OA

    PubMed Central

    Hu, Zhaohui; Ren, Zhonghai; Lu, Chaofu

    2012-01-01

    We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds. PMID:22371508

  10. Inhibition of diacylglycerol kinase alpha restores restimulation-induced cell death and reduces immunopathology in XLP-1

    PubMed Central

    Ruffo, Elisa; Malacarne, Valeria; Larsen, Sasha E.; Das, Rupali; Patrussi, Laura; Wülfing, Christoph; Biskup, Christoph; Kapnick, Senta M.; Verbist, Katherine; Tedrick, Paige; Schwartzberg, Pamela L.; Baldari, Cosima T.; Rubio, Ignacio; Nichols, Kim E.; Snow, Andrew L.; Baldanzi, Gianluca; Graziani, Andrea

    2016-01-01

    X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8+ T cells following Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in SAP, an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase alpha (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ. Here, we show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the pro-apoptotic proteins NUR77 and NOR1. Importantly, pharmacological inhibition of DGKα prevents the excessive CD8+ T cell expansion and IFNγ production that occur in Sap-deficient mice following Lymphocytic Choriomeningitis Virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients. PMID:26764158

  11. Inhibition of diacylglycerol kinase α restores restimulation-induced cell death and reduces immunopathology in XLP-1.

    PubMed

    Ruffo, Elisa; Malacarne, Valeria; Larsen, Sasha E; Das, Rupali; Patrussi, Laura; Wülfing, Christoph; Biskup, Christoph; Kapnick, Senta M; Verbist, Katherine; Tedrick, Paige; Schwartzberg, Pamela L; Baldari, Cosima T; Rubio, Ignacio; Nichols, Kim E; Snow, Andrew L; Baldanzi, Gianluca; Graziani, Andrea

    2016-01-13

    X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.

  12. Harmaline and hispidin from Peganum harmala and Inonotus hispidus with binding affinity to Candida rugosa lipase: In silico and in vitro studies.

    PubMed

    Benarous, Khedidja; Bombarda, Isabelle; Iriepa, Isabel; Moraleda, Ignacio; Gaetan, Herbette; Linani, Abderrahmane; Tahri, Djillali; Sebaa, Mohamed; Yousfi, Mohamed

    2015-10-01

    The inhibitory effect of phenolic compounds and alkaloids of Inonotus hispidus and Peganum harmala on Candida rugosa lipase was investigated, also, their antioxidant activities using DPPH, ABTS and phosphomolybdenum were studied in this paper. The phenolic extracts have shown a stronger antiradical activity than the alkaloids extracts. The enzymatic inhibition produced by these extracts is described here for the first time. The results have shown that the phenolic and the alkaloid extracts are good inhibitors of C. rugosa lipase. Thus, the inhibitor molecules (harmaline and hispidin) have been isolated from P. harmala and I. hispidus. Their structures were elucidated by (1)H NMR analysis. Molecular docking has been achieved using AutoDock Vina program to discuss the nature of interactions and the mechanism of inhibition. Therefore, these isolated molecules could be used in the treatment of candidiasis.

  13. Lipase-catalyzed aza-Michael reaction on acrylate derivatives.

    PubMed

    Steunenberg, Peter; Sijm, Maarten; Zuilhof, Han; Sanders, Johan P M; Scott, Elinor L; Franssen, Maurice C R

    2013-04-19

    A methodology has been developed for an efficient and selective lipase-catalyzed aza-Michael reaction of various amines (primary and secondary) with a series of acrylates and alkylacrylates. Reaction parameters were tuned, and under the optimal conditions it was found that Pseudomonas stutzeri lipase and Chromobacterium viscosum lipase showed the highest selectivity for the aza-Michael addition to substituted alkyl acrylates. For the first time also, some CLEAs were examined that showed a comparable or higher selectivity and yield than the free enzymes and other formulations.

  14. Obtaining lipases from byproducts of orange juice processing.

    PubMed

    Okino-Delgado, Clarissa Hamaio; Fleuri, Luciana Francisco

    2014-11-15

    The presence of lipases was observed in three byproducts of orange juice processing: peel, core and frit. The enzymes were characterised biochemically over a wide pH range from neutral (6-7) to alkaline (8-9). The optimal temperature for the activity of these byproducts showed wide range at 20°C to 70°C, indicating fairly high thermostability. The activities were monitored on p-NP-butyrate, p-NP-laurate and p-NP-palmitate. For the first time, lipase activity was detected in these residues, reaching 68.5 lipase U/g for the crude extract from fractions called frit.

  15. Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function.

    PubMed

    Skjold-Jørgensen, Jakob; Vind, Jesper; Moroz, Olga V; Blagova, Elena; Bhatia, Vikram K; Svendsen, Allan; Wilson, Keith S; Bjerrum, Morten J

    2017-01-01

    Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.

  16. Angiopoietin-like protein 3 inhibits lipoprotein lipase activity through enhancing its cleavage by proprotein convertases.

    PubMed

    Liu, Jun; Afroza, Huq; Rader, Daniel J; Jin, Weijun

    2010-09-03

    Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.

  17. Function and Localization of the Arabidopsis thaliana Diacylglycerol Acyltransferase DGAT2 Expressed in Yeast

    PubMed Central

    Aymé, Laure; Baud, Sébastien; Dubreucq, Bertrand; Joffre, Florent; Chardot, Thierry

    2014-01-01

    Diacylglycerol acyltransferases (DGATs) catalyze the final and only committed step of triacylglycerol synthesis. DGAT activity is rate limiting for triacylglycerol accumulation in mammals, plants and microbes. DGATs belong to three different evolutionary classes. In Arabidopsis thaliana, DGAT1, encoded by At2g19450, is the major DGAT enzyme involved in triacylglycerol accumulation in seeds. Until recently, the function of DGAT2 (At3g51520) has remained elusive. Previous attempts to characterize its enzymatic function by heterologous expression in yeast were unsuccessful. In the present report we demonstrate that expression of a codon-optimized version of the DGAT2 gene is able to restore neutral lipid accumulation in the Saccharomyces cerevisiae mutant strain (H1246), which is defective in triacylglycerol biosynthesis. Heterologous expression of codon-optimized DGAT2 and DGAT1 induced the biogenesis of subcellular lipid droplets containing triacylglycerols and squalene. Both DGAT proteins were found to be associated with these lipid droplets. The fatty acid composition was affected by the nature of the acyltransferase expressed. DGAT2 preferentially incorporated C16:1 fatty acids whereas DGAT1 displayed preference for C16:0, strongly suggesting that these enzymes have contrasting substrate specificities. PMID:24663078

  18. Cloning and Functional Analysis of Three Diacylglycerol Acyltransferase Genes from Peanut (Arachis hypogaea L.)

    PubMed Central

    Zhang, Xiaowen; Chen, Mingna; Chen, Na; Pan, Lijuan; Wang, Tong; Wang, Mian; Yang, Zhen; Wang, Quanfu; Yu, Shanlin

    2014-01-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final and only committed acylation step in the synthesis of triacylglycerols. In this study, three novel AhDGATs genes were identified and isolated from peanut. Quantitative real-time RT-PCR analysis indicated that the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons, whereas the transcript abundances of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined. During seed development, transcript levels of AhDGAT1-1 remained relatively low during the initial developmental stage but increased gradually during later stages, peaking at 50 days after pegging (DAP). Levels of AhDGAT1-2 transcripts were higher at 10 and 60 DAPs and much lower during other stages, whereas AhDGAT3-3 showed higher expression levels at 20 and 50 DAPs. In addition, AhDGAT transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. The activity of the three AhDGAT genes was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant. The recombinant yeasts restored lipid body formation and TAG biosynthesis, and preferentially incorporated unsaturated C18 fatty acids into lipids. The present study provides significant information useful in modifying the oil deposition of peanut through molecular breeding. PMID:25181516

  19. Studies on CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity in rat arterial wall.

    PubMed

    Sasaki, N; Matsuoka, N; Shirai, K; Saito, Y; Kumagai, A

    1982-06-01

    The properties of CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT) (EC 2.7.8.2.), which catalyzes de novo synthesis of phosphatidylcholine, were studied in rat arterial wall. The optimal pH of CPT of the arterial wall was about 8.5. On subcellular fractionation of the arterial wall, the highest activity was found in the microsome-rich fraction; the cytosolic fraction showed only a trace of activity. The Michaelis constant (KM) for CDP-choline was 0.019 mM. The CPT activity of a homogenate of arterial wall increased linearly with increase in concentration of diolein up to 3.2 mM. 20 mM magnesium and 0.2 mM manganese ions caused marked activation respectively and essential for the activity. Calcium, barium, cobalt, copper, and ferrous ions were inhibitory. 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM glycoletherdiamine-N,N,N'N'-tetraacetic acid (GEDTA) increased the activity in the presence of 10 mM magnesium ion. Sonication of the enzyme solution and addition of high concentration of detergent, such as Triton X-100 and Tween 20, markedly decreased the activity. Porcine liver phosphatidylcholine, phosphatidylethanolamine, and especially polyenephosphatidylcholine increased CPT activity of the arterial wall, while lysophosphatidylcholine was strongly inhibitory. The properties of arterial CPT activity under various conditions are discussed.

  20. Expression pattern of diacylglycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana.

    PubMed

    Lu, Chaofu Lu; de Noyer, Shen Bayon; Hobbs, Douglas H; Kang, Jinling; Wen, Yancheng; Krachtus, Dieter; Hills, Matthew J

    2003-05-01

    Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter gene beta-glucuronidase (GUS) fused with DNA sequences flanking the DGAT1 coding region. GUS activity was not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinating seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot analyses, precursor feeding and DGAT assay. In addition, DGAT1 gene expression is up-regulated by glucose and associated with glucose-induced changes in seedling development.

  1. tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum.

    PubMed

    Smith, Angela M; Harrison, Jesse S; Grube, Christopher D; Sheppe, Austin E F; Sahara, Nahoko; Ishii, Ryohei; Nureki, Osamu; Roy, Hervé

    2015-11-01

    Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Alanyl-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.

  2. Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L.).

    PubMed

    Chi, Xiaoyuan; Hu, Ruibo; Zhang, Xiaowen; Chen, Mingna; Chen, Na; Pan, Lijuan; Wang, Tong; Wang, Mian; Yang, Zhen; Wang, Quanfu; Yu, Shanlin

    2014-01-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final and only committed acylation step in the synthesis of triacylglycerols. In this study, three novel AhDGATs genes were identified and isolated from peanut. Quantitative real-time RT-PCR analysis indicated that the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons, whereas the transcript abundances of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined. During seed development, transcript levels of AhDGAT1-1 remained relatively low during the initial developmental stage but increased gradually during later stages, peaking at 50 days after pegging (DAP). Levels of AhDGAT1-2 transcripts were higher at 10 and 60 DAPs and much lower during other stages, whereas AhDGAT3-3 showed higher expression levels at 20 and 50 DAPs. In addition, AhDGAT transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. The activity of the three AhDGAT genes was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant. The recombinant yeasts restored lipid body formation and TAG biosynthesis, and preferentially incorporated unsaturated C18 fatty acids into lipids. The present study provides significant information useful in modifying the oil deposition of peanut through molecular breeding.

  3. A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica.

    PubMed

    Li, Da-Wei; Cen, Shi-Ying; Liu, Yu-Hong; Balamurugan, Srinivasan; Zheng, Xin-Yan; Alimujiang, Adili; Yang, Wei-Dong; Liu, Jie-Sheng; Li, Hong-Ye

    2016-07-10

    Oleaginous microalgae have received a considerable attention as potential biofuel feedstock. However, lack of industry-suitable strain with lipid rich biomass limits its commercial applications. Targeted engineering of lipogenic pathways represents a promising strategy to enhance the efficacy of microalgal oil production. In this study, a type 2 diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme in triacylglycerol (TAG) biosynthesis, was identified and overexpressed in heterokont oleaginous microalga Nannochloropsis oceanica for the first time. Overexpression of DGAT2 in Nannochloropsis increased the relative transcript abundance by 3.48-fold in engineered microalgae cells. TAG biosynthesis was subsequently accelerated by DGAT2 overexpression and neutral lipid content was significantly elevated by 69% in engineered microalgae. The fatty acid profile determined by GC-MS revealed that fatty acid composition was altered in engineered microalgae. Saturated fatty acids and polyunsaturated fatty acids were found to be increased whereas monounsaturated fatty acids content decreased. Furthermore, DGAT2 overexpression did not show negative impact on algal growth parameters. The present investigation showed that the identified DGAT2 would be a potential candidate for enhancing TAG biosynthesis and might facilitate the development of promising oleaginous strains with industrial potential.

  4. Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol

    PubMed Central

    Storch, Ursula; Forst, Anna-Lena; Pardatscher, Franziska; Erdogmus, Serap; Philipp, Maximilian; Gregoritza, Manuel; Mederos y Schnitzler, Michael; Gudermann, Thomas

    2017-01-01

    The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the Gq/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+/H+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels. PMID:27994151

  5. Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium

    PubMed Central

    Kaylor, Joanna J.; Radu, Roxana A.; Bischoff, Nicholas; Makshanoff, Jacob; Hu, Jane; Lloyd, Marcia; Eddington, Shannan; Bianconi, Tran; Bok, Dean; Travis, Gabriel H.

    2015-01-01

    Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1 -/- mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1 -/- mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE’s) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1 -/- mice. Further, total retinyl-ester synthase activity was reduced in both dgat1 -/- retina and RPE. PMID:25974161

  6. Occurrence of sulfoquinovosyl diacylglycerol in some members of the family Rhizobiaceae.

    PubMed

    Cedergren, R A; Hollingsworth, R I

    1994-08-01

    A radiolabeled component of a membrane extract of Rhizobium meliloti 2011 cells grown in the presence of 35S-labeled sulfate was isolated by silica flash chromatography and purified by high performance liquid chromatography (HPLC). Based on 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analyses, the structure of the compound was determined to be sulfoquinovosyl diacylglycerol (SQDG). NMR analyses indicated substantial heterogeneity in the fatty acid composition and that an important group was the cyclopropyl fatty acids. This first report of the occurrence of SQDG outside of the plant kingdom, photosynthetic bacteria or diatoms deserves special attention as, in this case, the bacterium is one that can fix nitrogen in symbiosis with plants. The origins of the bacterium's ability to synthesize this class of membrane lipids is an important question. Membrane extracts of other strains of the family Rhizobiaceae were screened for the presence of SQDG. The occurrence of SQDG in the symbiotic organisms was confirmed, while no SQDG was detected in either the Agrobacterium tumefaciens or the Escherichia coli strains tested. The current function of these lipids in symbiosis and the commonality of the ability of bacteria that function as plant symbionts to synthesize such molecules are all germane to studies of the Rhizobium/legume symbiosis.

  7. Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4

    PubMed Central

    Murakami, Eri; Shionoya, Takao; Komenoi, Suguru; Suzuki, Yuji; Sakane, Fumio

    2016-01-01

    Diacylglycerol kinase (DGK) phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line), DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner. PMID:27643686

  8. Diversity and evolution of plant diacylglycerol acyltransferase (DGATs) unveiled by phylogenetic, gene structure and expression analyses

    PubMed Central

    Turchetto-Zolet, Andreia Carina; Christoff, Ana Paula; Kulcheski, Franceli Rodrigues; Loss-Morais, Guilherme; Margis, Rogerio; Margis-Pinheiro, Marcia

    2016-01-01

    Abstract Since the first diacylglycerol acyltransferase (DGAT) gene was characterized in plants, a number of studies have focused on understanding the role of DGAT activity in plant triacylglycerol (TAG) biosynthesis. DGAT enzyme is essential in controlling TAGs synthesis and is encoded by different genes. DGAT1 and DGAT2 are the two major types of DGATs and have been well characterized in many plants. On the other hand, the DGAT3 and WS/DGAT have received less attention. In this study, we present the first general view of the presence of putative DGAT3 and WS/DGAT in several plant species and report on the diversity and evolution of these genes and its relationships with the two main DGAT genes (DGAT1 and DGAT2). According to our analyses DGAT1, DGAT2, DGAT3 and WS/DGAT are very divergent genes and may have distinct origin in plants. They also present divergent expression patterns in different organs and tissues. The maintenance of several types of genes encoding DGAT enzymes in plants demonstrates the importance of DGAT activity for TAG biosynthesis. Evolutionary history studies of DGATs coupled with their expression patterns help us to decipher their functional role in plants, helping to drive future biotechnological studies. PMID:27706370

  9. Diacylglycerol kinase ϵ deficiency preserves glucose tolerance and modulates lipid metabolism in obese mice.

    PubMed

    Mannerås-Holm, Louise; Schönke, Milena; Brozinick, Joseph T; Vetterli, Laurène; Bui, Hai-Hoang; Sanders, Philip; Nascimento, Emmani B M; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R

    2017-02-28

    Diacylglycerol kinases (DGKs) catalyze the phosphorylation and conversion of DAG into phosphatidic acid. DGK isozymes have unique primary structures, expression patterns, subcellular localizations, regulatory mechanisms and DAG preferences. DGKε has a hydrophobic segment that promotes its attachment to membranes and shows substrate specificity for DAG with an arachidonoyl acyl chain in the sn-2 position of the substrate. We determined the role of DGKε in the regulation of energy and glucose homeostasis in relation to diet-induced insulin resistance and obesity using DGKε deficient (KO) and wild-type mice. Lipidomic analysis revealed elevated unsaturated and saturated DAG species in skeletal muscle of DGKε KO mice, which was paradoxically associated with increased glucose tolerance. While skeletal muscle insulin sensitivity was unaltered, whole body respiratory exchange ratio was reduced, and abundance of mitochondrial markers was increased, indicating a greater reliance on fat oxidation and intracellular lipid metabolism in DGKε KO mice. Thus, the increased intracellular lipids in skeletal muscle from DGKε KO mice may undergo rapid turnover due to increased mitochondrial function and lipid oxidation, rather than storage, which in turn may preserve insulin sensitivity. In conclusion, DGKε plays a role in glucose and energy homeostasis by modulating lipid metabolism in skeletal muscle.

  10. Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology.

    PubMed

    Liu, Qin; Siloto, Rodrigo M P; Lehner, Richard; Stone, Scot J; Weselake, Randall J

    2012-10-01

    Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identified in numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGAT-related acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to the hydrophobic properties of theses enzymes and difficulties associated with purification. This review examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2, using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.

  11. tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum

    PubMed Central

    Smith, Angela M.; Harrison, Jesse S.; Grube, Christopher D.; Sheppe, Austin E.F.; Sahara, Nahoko; Ishii, Ryohei; Nureki, Osamu; Roy, Hervé

    2015-01-01

    Summary Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane, and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine, and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Ala-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, while formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells. PMID:26235234

  12. An Unconventional Diacylglycerol Kinase That Regulates Phospholipid Synthesis and Nuclear Membrane Growth*♦

    PubMed Central

    Han, Gil-Soo; O'Hara, Laura; Carman, George M.; Siniossoglou, Symeon

    2008-01-01

    Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Δ cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis. PMID:18458075

  13. Determination of physicochemical properties of diacylglycerol oil at high pressure by means of ultrasonic methods.

    PubMed

    Kiełczyński, Piotr; Szalewski, Marek; Balcerzak, Andrzej; Wieja, Krzysztof; Malanowski, Aleksander; Kościesza, Rafał; Tarakowski, Rafał; Rostocki, Aleksander J; Siegoczyński, Ryszard M

    2014-12-01

    The purpose of the paper is to address, using ultrasonic methods, the impact of temperature and pressure on the physicochemical properties of liquids on the example of diacylglycerol (DAG) oil. The paper presents measurements of sound velocity, density and volume of DAG oil sample in the pressure range from atmospheric pressure up to 0.6GPa and at temperatures ranging from 20 to 50°C. Sound speed measurements were performed in an ultrasonic setup with a DAG oil sample located in the high-pressure chamber. An ultrasonic method that uses cross-correlation method to determine the time-of-flight of the ultrasonic pulses through the liquid was employed to measure the sound velocity in DAG oil. This method is fast and reliable tool for measuring sound velocity. The DAG oil density at high pressure was determined from the monitoring of sample volume change. The adiabatic compressibility and isothermal compressibility have been calculated on the basis of experimental data. Discontinuities in isotherms of the sound speed versus pressure point to the existence of phase transitions in DAG oil. The ultrasonic method presented in this study can be applied to investigate the physicochemical parameters of other liquids not only edible oils.

  14. Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction

    PubMed Central

    Sun, Jingjing; Chen, Yiling; Sheng, Jun; Sun, Mi

    2015-01-01

    To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition. PMID:26240816

  15. Tetracycline Inhibition of a Lipase from Corynebacterium acnes

    PubMed Central

    Weaber, K.; Freedman, R.; Eudy, W. W.

    1971-01-01

    A lipase which hydrolyzes triglycerides (tricaprylin and trilaurin) and naphthyl laurate was obtained from the broth of Corynebacterium acnes cultures by ammonium sulfate fractionation. Ca2+ and sodium taurocholate stimulated activity of the enzyme. Ethylenediaminetetraacetic acid (EDTA) did not inhibit activity of the Ca2+-activated enzyme, but lipolytic activity was inhibited by EDTA in the absence of Ca2+. Tetracycline (10−4m) produced a slight inhibition of the lipase activity with 5 × 10−5m or less showing no effect on the lipase activity. However, complete inhibition by tetracycline at 10−4m was observed for Ca2+-activated enzyme. Tetracycline inhibition of the C. acnes lipase could be demonstrated at concentrations as low as 10−6m. PMID:4252558

  16. Lipase Activity among Bacteria Isolated from Amazonian Soils

    PubMed Central

    Willerding, André Luis; de Oliveira, Luiz Antonio; Moreira, Francisco Wesen; Germano, Mariana Gomes; Chagas, Aloísio Freitas

    2011-01-01

    The objective of this study was to select lipase-producing bacteria collected from different counties of the Amazon region. Of the 440 bacteria strains, 181 were selected for the lipase assay in qualitative tests at Petri dishes, being 75 (41%) lipase positive. The enzymatic index was determined during fifteen days at different temperatures (30°, 35°, 40°, and 45°C). The highest lipase activity was observed within 72 hours at 30°C. Twelve bacteria strains presented an index equal to or greater than the standard used like reference, demonstrating the potential of microbial resource. After the bioassay in Petri dishes, the selected bacteria strains were analyzed in quantitative tests on p-nitrophenyl palmitate (p-NPP). A group of the strains was selected for other phases of study with the use in oleaginous substrates of the Amazonian flora, aiming for the application in processes like oil biotransformation. PMID:22007294

  17. Lipase-catalyzed ethanolysis of borage oil: a kinetic study.

    PubMed

    Torres, Carlos F; Hill, Charles G; Otero, Cristina

    2004-01-01

    Ethanolysis of borage oil catalyzed by two commercial lipases (from Pseudomonas cepacia and Candida antarctica) was studied using two different methodologies. Multiresponse models derived from a generalized Michaelis-Menten mechanism were utilized to describe the rates of formation of ethyl esters of the primary fatty acids present in the precursor oil. The relative rate constants determined for each of the fatty acid residues indicated that both lipases discriminate against release of gamma-linolenic acid residues under the reaction conditions studied. However, both lipases also released some of the residues located at the sn-2 position, indicating that for the experimental conditions studied, both lipases are nonspecific. Moreover, inactivation of Novozym 435 was rapid. Because the half-life of this enzyme (ca. 2.2 h) is comparable to the half-life of the reaction, the intrinsic reaction rate and enzyme deactivation must both be considered in modeling the kinetics.

  18. Discrimination of thermostable and thermophilic lipases using support vector machines.

    PubMed

    Zhao, Wei; Wang, Xunzhang; Deng, Riqiang; Wang, Jinwen; Zhou, Hongbo

    2011-07-01

    Discriminating thermophilic lipases from their similar thermostable counterparts is a challenging task and it would help to design stable proteins. In this study, the distributions of N (N=2, 3) neighboring amino acids and the non-adjacent di-residue coupling patterns in the sequences of 65 thermostable and 77 thermophilic lipases had been systematically analyzed. It was found that the hydrophobic residues Leu, Pro, Met, Phe, Trp, as well as the polar residue Tyr had higher occurrence in thermophilic lipases than thermostable ones. The occurrence frequencies of KC EE KE RE, VE, YI, EK, VK, EV, YV, EY, KY, VY and YY in thermophilic proteins were significantly higher, while the occurrence frequencies of QC, QH, QN, HQ, MQ, NQ, QQ, TQ, QS and QT were significantly lower. CXP or CPX showed significantly positive to lipase thermostability, while XXQ or QXX showed significantly negative to lipase thermostability. Non-adjacent di-residue coupling patterns of PR14, RY32, YR47, LE53, LE64, PP64, RP70 and PP101 were significantly different in thermophilic lipases and their thermostable counterparts. The composition of dipeptide, tripeptide and non-adjacent di-residue patterns contained more information than amino acid composition. A statistical method based on support vector machines (SVMs) was developed for discriminating thermophilic and thermostable lipases. The accuracy of this method for the training dataset was 97.17?. Furthermore, the highest accuracy of the method for testing datasets was 98.41?. The influence of some specific patterns on lipase thermostability was also discussed.

  19. Lipases at interfaces: unique interfacial properties as globular proteins.

    PubMed

    Reis, P; Miller, R; Krägel, J; Leser, M; Fainerman, V B; Watzke, H; Holmberg, K

    2008-06-01

    The adsorption behavior of two globular proteins, lipase from Rhizomucor miehei and beta-lactoglobulin, at inert oil/water and air/water interfaces was studied by the pendant drop technique. The kinetics and adsorption isotherms were interpreted for both proteins in different environments. It was found that the adopted mathematical models well describe the adsorption behavior of the proteins at the studied interfaces. One of the main findings is that unique interfacial properties were observed for lipase as compared to the reference beta-lactoglobulin. A folded drop with a "skinlike" film was formed for the two proteins after aging followed by compression. This behavior is normally associated with protein unfolding and covalent cross-linking at the interface. Despite this, the lipase activity was not suppressed. By highlighting the unique interfacial properties of lipases, we believe that the presented work contributes to a better understanding of lipase interfacial activation and the mechanisms regulating lipolysis. The results indicate that the understanding of the physical properties of lipases can lead to novel approaches to regulate their activity.

  20. Role of the lid hydrophobicity pattern in pancreatic lipase activity.

    PubMed

    Thomas, Annick; Allouche, Maya; Basyn, Frédéric; Brasseur, Robert; Kerfelec, Brigitte

    2005-12-02

    Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240-252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile salt-coated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240-252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.

  1. Effects of diacylglycerols and Ca2+ on structure of phosphatidylcholine/phosphatidylserine bilayers.

    PubMed Central

    Goldberg, E M; Lester, D S; Borchardt, D B; Zidovetzki, R

    1994-01-01

    The combined effects of the diacylglycerols (DAGs) with the various acyl chains and Ca2+ on the structure of phosphatidylcholine/phosphatidylserine (4:1 mole/mole) bilayers were studied using 2H- and 31P NMR. The following DAG- and Ca(2+)-induced bilayer perturbations were identified. 1) Increased tendency to form nonbilayer lipid phases was induced by diolein or stearoylarachidonoylglycerol, and was synergistically enhanced by the addition of Ca2+. 2) "Transverse" bilayer perturbation was induced by dioctanoylglycerol. The addition of this DAG caused increased ordering of the phospholipid acyl side chains in the region adjacent to the headgroup, with the concomitant decrease of the order toward the bilayer interior. 3) Separation of the phosphatidylcholine and phosphatidylserine bilayer components was induced by combinations of relatively high (1:5 mole/mole to phosphatidylserine) Ca2+ and 25 mol% (to the phospholipids) of diolein, stearoylarachidonoylglycerol, or oleoylacetylglycerol. 4) Lateral phase separation of the bilayers on the regions of different fluidities was induced by dipalmitin. These physicochemical effects were correlated with the effects of these DAGs and Ca2+ on the activity of protein kinase C. The increased tendency to form nonbilayer lipid phases and the transverse bilayer perturbations correlated with the increased protein kinase C activity, whereas the actual presence of the nonbilayer lipid phases, as well as the separation of the phosphatidylcholine and phosphatidylserine components, was associated with the decrease in the protein kinase C activity. The lateral phase separation of the bilayer on gel-like and liquid crystalline regions did not have an effect on the activity of the enzyme. These results demonstrate the importance of the physicochemical properties of the membranes in the process of activation of protein kinase C. PMID:8161692

  2. Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

    PubMed Central

    Cao, Heping; Shockey, Jay M.; Klasson, K. Thomas; Chapital, Dorselyn C.; Mason, Catherine B.; Scheffler, Brian E.

    2013-01-01

    Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms. PMID:24146944

  3. Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol

    PubMed Central

    Aznar-Moreno, Jose; Denolf, Peter; Van Audenhove, Katrien; De Bodt, Stefanie; Engelen, Steven; Fahy, Deirdre; Wallis, James G.; Browse, John

    2015-01-01

    DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world’s most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop. PMID:26195728

  4. Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol.

    PubMed

    Essemine, Jemâa; Govindachary, Sridharan; Ammar, Saïda; Bouzid, Sadok; Carpentier, Robert

    2011-09-01

    Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5min) mild (40°C) or strong (44°C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.

  5. Substrate selectivity of diacylglycerol kinase in PDGF-stimulated 3T3 cells

    SciTech Connect

    MacDonald, M.L.; Mack, K.F.; Glomset, J.A.

    1987-05-01

    The authors investigated the properties of Diacylglycerol (DAG) Kinase in 3T3 cells. PDGF treatment caused an increase in DAG mass, an increase in incorporation of /sup 32/P into phosphatidic acid (PA) and phosphatidylinositol (PI), and an increase in the rate of phosphorylation of membrane DAG in vitro. The mechanism of enhanced phosphorylation of DAG was studied with dicaprylin (diC/sub 10/) as a probe. Cells were prelabeled with /sup 32/P and treated with PDGF or carrier. DiC/sub 10/ was added to the cell medium before harvesting. With PDGF treatment, the radioactivity in endogenous PA increased fourfold, whereas the radioactivity in PA/sub 10/ and PI/sub 10/ was consistently decreased. To verify that the PDGF effect on PA/sub 10/ formation in intact cells was due to reduced phosphorylation of diC/sub 10/ by DAG kinase, cells were treated with PDGF and/or diC/sub 10/, freeze-thawed, and then incubated with Mg(/sup 32/P)ATP. The rate of phosphorylation of cell-associated diC/sub 10/ was decreased 50% by PDGF treatment. This effect could not be explained by decreased intracellular levels of diC/sub 10/, or by saturation of DAG kinase with endogenous DAGs. Therefore, it seemed that endogenous DAGs, derived from PI, might be better substrates for DAG kinase than is diC/sub 10/. In studies of the properties of DAG kinase with pure DAGs in mixed detergent micelles, they found that the enzyme phosphorylated arachidonoyl-DAG more readily than diC/sub 10/. The selectivity of DAG kinase may play a key role in the formation of arachidonoyl species of PI.

  6. Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

    PubMed Central

    Goldberg, E M; Lester, D S; Borchardt, D B; Zidovetzki, R

    1995-01-01

    The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C. PMID:8519996

  7. PTH (parathyroid hormone) elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line

    SciTech Connect

    Civitelli, R.; Reid, I.R.; Westbrook, S.; Avioli, L.V.; Hruska, K.A. )

    1988-11-01

    Parathyroid hormone (PTH)-stimulated signal transduction through mechanisms alternate to adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) production were studied in UMR 106-01 cells, a cell line with an osteoblastic phenotype. PTH produced transient, dose-related increases in cytosolic calcium ((Ca{sup 2+}){sub i}), inositol trisphosphates, and diacylglycerol (DAG). Both inositol 1,4,5-trisphosphate (Ins-1,4,5P{sub 3}) and inositol 1,3,4-trisphosphate (Ins-1,3,4P{sub 3}) production were rapidly stimulated by PTH. Consistent with the production of Ins-1,3,4P{sub 3}, rapid stimulation of late eluting inositol tetrakisphosphate was observed. The effects on the inositol phosphates were induced rapidly, consistent with roles as signals for changes in (Ca{sup 2+}){sub i}. In saponin-permeabilized UMR 106-01 cells, Ins-1,4,5P{sub 3} stimulated {sup 45}Ca release from a nonmitochondrial intracellular pool. Thus the hypothesis that PTH-stimulated Ins-1,4,5P{sub 3} production initiates Ca{sup 2+} release and contributes to transient elevations of (Ca{sup 2+}){sub i} is supported. These data suggest that stimulation of cAMP production during PTH stimulation may negatively affect production of rises in (Ca{sup 2+}){sub i} during PTH stimulation. The inactivation of the inhibitory G protein of adenylate cyclase by pertussis toxin could explain its action similar to cAMP analogues. Cyclci nucleotides diminish the effects of PTH on (Ca{sup 2+}){sub i}, probably interacting on a biochemical step subsequent to or independent of Ins-1,4,5P{sub 3} release.

  8. Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol.

    PubMed

    Aznar-Moreno, Jose; Denolf, Peter; Van Audenhove, Katrien; De Bodt, Stefanie; Engelen, Steven; Fahy, Deirdre; Wallis, James G; Browse, John

    2015-10-01

    DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop.

  9. Oxalic acid and diacylglycerol 36:3 are cross-species markers of sleep debt.

    PubMed

    Weljie, Aalim M; Meerlo, Peter; Goel, Namni; Sengupta, Arjun; Kayser, Matthew S; Abel, Ted; Birnbaum, Morris J; Dinges, David F; Sehgal, Amita

    2015-02-24

    Sleep is an essential biological process that is thought to have a critical role in metabolic regulation. In humans, reduced sleep duration has been associated with risk for metabolic disorders, including weight gain, diabetes, obesity, and cardiovascular disease. However, our understanding of the molecular mechanisms underlying effects of sleep loss is only in its nascent stages. In this study we used rat and human models to simulate modern-day conditions of restricted sleep and addressed cross-species consequences via comprehensive metabolite profiling. Serum from sleep-restricted rats was analyzed using polar and nonpolar methods in two independent datasets (n = 10 per study, 3,380 measured features, 407 identified). A total of 38 features were changed across independent experiments, with the majority classified as lipids (18 from 28 identified). In a parallel human study, 92 metabolites were identified as potentially significant, with the majority also classified as lipids (32 of 37 identified). Intriguingly, two metabolites, oxalic acid and diacylglycerol 36:3, were robustly and quantitatively reduced in both species following sleep restriction, and recovered to near baseline levels after sleep restriction (P < 0.05, false-discovery rate < 0.2). Elevated phospholipids were also noted after sleep restriction in both species, as well as metabolites associated with an oxidizing environment. In addition, polar metabolites reflective of neurotransmitters, vitamin B3, and gut metabolism were elevated in sleep-restricted humans. These results are consistent with induction of peroxisome proliferator-activated receptors and disruptions of the circadian clock. The findings provide a potential link between known pathologies of reduced sleep duration and metabolic dysfunction, and potential biomarkers for sleep loss.

  10. Lipase-catalyzed synthesis of (S)-naproxen ester prodrug by transesterification in organic solvents.

    PubMed

    Tsai, S W; Tsai, C S; Chang, C S

    1999-06-01

    A lipase-catalyzed enantioselective transesterification process was developed for the synthesis of (S)-naproxen 2-N-morpholinoethyl ester prodrug from racemic 2,2,2-trifluoroethyl naproxen ester in organic solvents. By selecting isooctane and 37 degrees C as the best solvent and temperature, the apparent fits of the initial conversion rates for transesterification and hydrolysis side reaction suggest a ping-pong Bi-Bi enzymatic mechanism with the alcohol as a competitive enzyme inhibitor. Improvements in the initial conversion rate and the productivity for the desired (S)-ester product were obtained after comparing with the result of an enantioselective esterification process. Studies of water content in isooctane and alcohol containing various N,N-dialkylamino groups on the enzyme activity and enantioselectivity, as well as the recovery of (S)-ester product by using extraction, were also reported.

  11. Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system

    PubMed Central

    Schlosburg, Joel E.; Blankman, Jacqueline L.; Long, Jonathan Z.; Nomura, Daniel K.; Pan, Bin; Kinsey, Steven G.; Nguyen, Peter T.; Ramesh, Divya; Booker, Lamont; Burston, James J.; Thomas, Elizabeth A.; Selley, Dana E.; Sim-Selley, Laura J.; Liu, Qingsong; Lichtman, Aron H.; Cravatt, Benjamin F.

    2010-01-01

    Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. Here we show that a similar form of functional antagonism is produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of MAGL. Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity, and desensitization of brain CB1 receptors. These data contrasted with blockade of fatty acid amide hydrolase (FAAH), an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively. PMID:20729846

  12. Protein Kinase Cα (PKCα) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation.

    PubMed

    Lum, Michelle A; Barger, Carter J; Hsu, Alice H; Leontieva, Olga V; Black, Adrian R; Black, Jennifer D

    2016-03-18

    Sustained activation of PKCα is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKCα desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC8) resulted in sustained plasma membrane association of PKCα in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCα, prolonged activation by DiC8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC8-activated PKCα did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCα desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCδ and PKCϵ, differences in Ca(2+) sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCα. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCα to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC8 treatment failed to desensitize PKCα in several cell types and did not affect PKCβI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCα is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCα deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCα from

  13. Protein Kinase Cα (PKCα) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation*

    PubMed Central

    Lum, Michelle A.; Barger, Carter J.; Hsu, Alice H.; Leontieva, Olga V.; Black, Adrian R.; Black, Jennifer D.

    2016-01-01

    Sustained activation of PKCα is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKCα desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC8) resulted in sustained plasma membrane association of PKCα in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCα, prolonged activation by DiC8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC8-activated PKCα did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCα desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCδ and PKCϵ, differences in Ca2+ sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCα. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCα to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC8 treatment failed to desensitize PKCα in several cell types and did not affect PKCβI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCα is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCα deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCα from

  14. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    PubMed Central

    Bansal, Sunil; Durrett, Timothy P.

    2016-01-01

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

  15. Biochemical characterization of the surface-associated lipase of Staphylococcus saprophyticus.

    PubMed

    Sakinç, Türkân; Kleine, Britta; Gatermann, Sören G

    2007-09-01

    Staphylococcus saprophyticus, an important cause of urinary tract infections, produces a surface-associated lipase, Ssp. In contrast to other lipases, Ssp is a protein that is present in high amounts on the surface of the bacteria and it was shown that it is a true lipase. Characterization of S. saprophyticus lipase (Ssp) showed that it is more similar to Staphylococcus aureus lipase and Staphylococcus epidermidis lipase than to Staphylococcus hyicus lipase and Staphylococcus simulans lipase. Ssp showed an optimum of lipolytic activity at pH 6 and lost its activity at pH>8 or pH<5. The present results show that Ssp activity is dependent on Ca(2+). Consequently, activity increased c. 10-fold in the presence of 2 mM Ca(2+). Optimal activity was reached at 30 degrees C. It was also observed that the enzymatic activity of Ssp depends strongly on the acyl chain length of the substrate molecule.

  16. Immobilizing Yarrowia lipolytica Lipase Lip2 via Improvement of Microspheres by Gelatin Modification.

    PubMed

    Xie, Rong; Cui, Caixia; Chen, Biqiang; Tan, Tianwei

    2015-10-01

    The purpose of this study was to investigate the feasibility of immobilizing Yarrowia lipolytica lipase lip2 on epoxy microspheres with or without gelatin modifications. The activity of lipase immobilized on gelatin-modified supports was twofold higher than those immobilized on native supports. There was no significant difference in the Michaelis-Menten constant (K M ) between the two immobilized lipases. However, lipase immobilized on gelatin modified supports showed an approximately fourfold higher V max than lipase immobilized on native supports. Lipase immobilization on the gelatin-modified support exhibited a significantly improved operational stability in an esterification system. After it was reused for a total of 35 batches, the ester conversion of lipase immobilized on gelatin-modified and native microspheres was 83 and 60 %, respectively. Furthermore, the immobilized lipase could be stored at 4 °C for 12 months without any loss of activity.

  17. No-carrier-added carbon-11-labeled sn-1,2- and sn-1,3-diacylglycerols by (11C)propyl ketene method

    SciTech Connect

    Imahori, Y.; Fujii, R.; Ueda, S.; Ido, T.; Nishino, H.; Moriyama, Y.; Yamamoto, Y.L.; Nakahashi, H. )

    1991-08-01

    This article describes the preparation of sn-1,2-(11C)diacylglycerols and sn-1,3-(11C)diacylglycerols by a no-carrier-added reaction based on a labeling method using (1-11C)propyl ketene, which is one of the most potent acylating agents. (1-11C)Propyl ketene was produced by pyrolytic decomposition of (1-11C)butyric acid and was trapped in pyridine containing L-alpha-palmitoyl-lysophosphatidylcholine, producing L-alpha-palmitoyl-2-(1-11C)butyryl-sn-glycero-3-phosphorylcholine. The authors adopted an enzymatic reaction to remove the phosphorylcholine, in which L-alpha-palmitoyl-2-(1-11C)butyryl-sn-glycero-3-phosphorylcholine was incubated with phospholipase C, hydrolyzing to produce 1-palmitoyl-sn-2-(1-11C)butyrylglycerol. Total synthesis time was about 50 minutes and the specific activity was estimated at 93 GBq/mumol (2.5 Ci/mumol) at end of synthesis. Radiochemical yield was 3.8% based on the trapped 11CO2. sn-1,3-(11C)Diacylglycerol was also synthesized by (1-11C)propyl ketene reaction with 1-palmitoyl-sn-glycerol in a single procedure. The regional brain tissue radioactivities obtained in sn-1,2-(11C)diacylglycerol were higher than those of sn-1,3-(11C)diacylglycerol, and the regional values varied widely. In autoradiography of brain slices from conscious rats, sn-1,2-(11C)diacylglycerol incorporation sites were discretely localized, especially in the amygdala, cerebral cortex, and hippocampus, suggesting that intensive neuronal processing occurred in these areas on the basis of phosphatidylinositol turnover.

  18. Immobilization of active lipase B from Candida antarctica on the surface of polyhydroxyalkanoate inclusions.

    PubMed

    Jahns, Anika C; Rehm, Bernd H A

    2015-04-01

    Polyhydroxyalkanoate (PHA) beads, recombinantly produced in Escherichia coli, were functionalized to display lipase B from Candida antarctica as translational protein fusion. The respective beads were characterized in respect to protein content, functionality, long term storage capacity and re-usability. The direct fusion of the PHA synthase, PhaC, to lipase B yielded active PHA lipase beads capable of hydrolyzing glycerol tributyrate. Lipase B beads showed stable activity over several weeks and re-usability without loss of function.

  19. Endothelial dysfunction in adipose triglyceride lipase deficiency

    PubMed Central

    Schrammel, Astrid; Mussbacher, Marion; Wölkart, Gerald; Stessel, Heike; Pail, Karoline; Winkler, Sarah; Schweiger, Martina; Haemmerle, Guenter; Al Zoughbi, Wael; Höfler, Gerald; Lametschwandtner, Alois; Zechner, Rudolf; Mayer, Bernd

    2014-01-01

    Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~ 50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease. PMID:24657704

  20. Estolides synthesis catalyzed by immobilized lipases.

    PubMed

    Aguieiras, Erika C G; Veloso, Cláudia O; Bevilaqua, Juliana V; Rosas, Danielle O; da Silva, Mônica A P; Langone, Marta A P

    2011-01-01

    Estolides are vegetable-oil-based lubricants obtained from oleic acid or any source of hydroxy fatty acids. In this work, the estolides synthesis from oleic acid and methyl ricinoleate (biodiesel from castor oil), using immobilized commercial lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM) in a solvent-free medium was investigated. Acid value was used to monitor the reaction progress by determining the consumption of acid present in the medium. Novozym 435 showed the best performance. Water removal improved the conversion. Novozym 435 was more active at atmospheric pressure. Novozym 435 was reused four times with conversion reaching 15% after the fourth reaction at 80°C. Estolides produced under the reaction conditions used in this work presented good properties, such as, low temperature properties as pour point (-24°C), viscosity (23.9 cSt at 40°C and 5.2 cSt at 100°C), and viscosity index (153).

  1. Estolides Synthesis Catalyzed by Immobilized Lipases

    PubMed Central

    Aguieiras, Erika C. G.; Veloso, Cláudia O.; Bevilaqua, Juliana V.; Rosas, Danielle O.; da Silva, Mônica A. P.; Langone, Marta A. P.

    2011-01-01

    Estolides are vegetable-oil-based lubricants obtained from oleic acid or any source of hydroxy fatty acids. In this work, the estolides synthesis from oleic acid and methyl ricinoleate (biodiesel from castor oil), using immobilized commercial lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM) in a solvent-free medium was investigated. Acid value was used to monitor the reaction progress by determining the consumption of acid present in the medium. Novozym 435 showed the best performance. Water removal improved the conversion. Novozym 435 was more active at atmospheric pressure. Novozym 435 was reused four times with conversion reaching 15% after the fourth reaction at 80°C. Estolides produced under the reaction conditions used in this work presented good properties, such as, low temperature properties as pour point (−24°C), viscosity (23.9 cSt at 40°C and 5.2 cSt at 100°C), and viscosity index (153). PMID:21755040

  2. Crowding enhances lipase turnover rate on surface-immobilized substrates.

    PubMed

    Balevicius, Zigmas; Ignatjeva, Dalia; Niaura, Gediminas; Ignatjev, Ilja; Vaicikauskas, Viktoras; Babonas, Gintautas Jurgis; Valincius, Gintaras

    2015-07-01

    Utilizing surface-immobilized synthetic lipid substrates containing the redox-active ferrocene groups, the enzymatic activity of lipase from Thermomyces lanuginosus was measured by the cyclic voltammetry method. The activity was correlated with the surface density of the protein by the ATR-IR spectroscopy and the total internal reflection ellipsometry. It was found that the lipase turnover rate significantly increases with its surface density. Despite expected hindrance effects due to the crowding of the enzyme molecules in the near surface-saturation range of concentrations, the turnover rate was consistently higher compared with the values measured at low concentrations. The effect was explained by the change in the surface arrangement of the enzyme. In the low concentration range, lipase adsorbs onto a surface adopting a predominantly horizontal position. At high concentrations, as the surface density approaches saturation, the enzyme molecules due to crowding are forced into the predominantly vertical position, which is more favorable for the activation of the lipase through the interaction between the "hydrophobic lid" of the lipase and the hydrophobic adsorbate surface.

  3. Lipase-catalyzed polyester synthesis – A green polymer chemistry

    PubMed Central

    Kobayashi, Shiro

    2010-01-01

    This article is a short comprehensive review describing in vitro polyester synthesis catalyzed by a hydrolysis enzyme of lipase, most of which has been developed for these two decades. Polyesters are prepared by repeated ester bond-formation reactions; they include two major modes, ring-opening polymerization (ROP) of cyclic monomers such as cyclic esters (lactones) and condensation polymerization via the reaction between a carboxylic acid or its ester group and an alcohol group. Polyester synthesis is, therefore, a reaction in reverse way of in vivo lipase catalysis of ester bond-cleavage with hydrolysis. The lipase-catalyzed polymerizations show very high chemo-, regio-, and enantio-selectivities and involve various advantageous characteristics. Lipase is robust and compatible with other chemical catalysts, which allows novel chemo-enzymatic processes. New syntheses of a variety of functional polyesters and a plausible reaction mechanism of lipase catalysis are mentioned. The polymerization characteristics are of green nature currently demanded for sustainable society, and hence, desirable for conducting ‘green polymer chemistry’. PMID:20431260

  4. Exploring the Conformational States and Rearrangements of Yarrowia lipolytica Lipase

    PubMed Central

    Bordes, Florence; Barbe, Sophie; Escalier, Pierre; Mourey, Lionel; André, Isabelle; Marty, Alain; Tranier, Samuel

    2010-01-01

    We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding. PMID:20923657

  5. High milk lipase activity associated with breast milk jaundice.

    PubMed

    Poland, R L; Schultz, G E; Garg, G

    1980-12-01

    Human milk samples that inhibit bilirubin-UDP-glucuronyl transferase (UDPGT) activity in vitro have been associated with prolonged unconjugated hyperbilirubinemia in newborn infants. We measured the concentration of nonesterified fatty acids (total and individual fatty acids), total fat and protein, and lipase activities (with and without bile salt stimulation) in milk samples from two groups of women. Women whose infants had prolonged unconjugated hyperbilirubinemia and whose milk inhibited the activity of UDPGT were in the first group (N = 9). Volunteers with healthy infants acted as controls. Inhibitory milk contained significantly more nonesterified fatty acids (total, palmitic, and oleic) than did controls. Fat and protein concentrations and bile salt-stimulated lipase activities were similar in the two groups. Unstimulated lipase activity was higher in the inhibitory milks (11.9 +/- 0.8 mM x min-1 x ml-1) than in the controls (6.0 +/- 0.1 mM x min-1 x ml-1) (P less than 0.01). The specific activity (mM x min-1 x mg protein) of unstimulated lipase was also significantly higher in the inhibitory milks (P less than 0.0001). The high nonesterified fatty acid levels in inhibitory milks is accounted for by the elevated unstimulated lipase activities. How these circumstances lead to jaundice in the infants remains to be shown.

  6. Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., ...

  7. A Cutinase from Trichoderma reesei with a lid-covered active site and kinetic properties of true lipases.

    PubMed

    Roussel, Alain; Amara, Sawsan; Nyyssölä, Antti; Mateos-Diaz, Eduardo; Blangy, Stéphanie; Kontkanen, Hanna; Westerholm-Parvinen, Ann; Carrière, Frédéric; Cambillau, Christian

    2014-11-11

    Cutinases belong to the α/β-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.

  8. Castor Phospholipid:Diacylglycerol Acyltransferase Facilitates Efficient Metabolism of Hydroxy Fatty Acids in Transgenic Arabidopsis1[W][OA

    PubMed Central

    van Erp, Harrie; Bates, Philip D.; Burgal, Julie; Shockey, Jay; Browse, John

    2011-01-01

    Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expression of castor (Ricinus communis) fatty acid hydroxylase (RcFAH) in Arabidopsis (Arabidopsis thaliana) resulted in only 17% hydroxy fatty acids (HFAs) in the seed oil. In order to increase HFA levels, we investigated castor phospholipid:diacylglycerol acyltransferase (PDAT). We cloned cDNAs encoding three putative PDAT enzymes from a castor seed cDNA library and coexpressed them with RcFAH12. One isoform, RcPDAT1A, increased HFA levels to 27%. Analysis of HFA-triacylglycerol molecular species and regiochemistry, along with analysis of the HFA content of phosphatidylcholine, indicates that RcPDAT1A functions as a PDAT in vivo. Expression of RcFAH12 alone leads to a significant decrease in FA content of seeds. Coexpression of RcPDAT1A and RcDGAT2 (for diacylglycerol acyltransferase 2) with RcFAH12 restored FA levels to nearly wild-type levels, and this was accompanied by a major increase in the mass of HFAs accumulating in the seeds. We show the usefulness of RcPDAT1A for engineering plants with high levels of HFAs and alleviating bottlenecks due to the production of unusual FAs in transgenic oilseeds. PMID:21173026

  9. Nanosized self-emulsifying lipid vesicles of diacylglycerol-PEG lipid conjugates: Biophysical characterization and inclusion of lipophilic dietary supplements

    SciTech Connect

    Koynova, Rumiana; Tihova, Mariana

    2010-04-12

    Hydrated diacylglycerol-PEG lipid conjugates, glyceryl dioleate-PEG12 (GDO-PEG12) and glyceryl dipalmitate-PEG23 (GDP-PEG23), spontaneously form uni- or oligolamellar liposomes in their liquid crystalline phase, in distinct difference from the PEGylated phospholipids which form micelles. GDP-PEG23 exhibits peculiar hysteretic phase behavior and can arrange into a long-living hexagonal phase at ambient and physiological temperatures. Liposomes of GDO-PEG12 and its mixture with soy lecithin exchange lipids with the membranes much more actively than common lecithin liposomes; such an active lipid exchange might facilitate the discharging of the liposome cargo upon uptake and internalization, and can thus be important in drug delivery applications. Diacylglycerol-PEG lipid liposome formulations can encapsulate up to 20-30 wt.% lipophilic dietary supplements such as fish oil, coenzyme Q10, and vitamins D and E. The encapsulation is feasible by way of dry mixing, avoiding the use of organic solvent.

  10. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed Central

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-01-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo. PMID:9001227

  11. A role for ceramide, but not diacylglycerol, in the antagonism of insulin signal transduction by saturated fatty acids.

    PubMed

    Chavez, Jose Antonio; Knotts, Trina A; Wang, Li-Ping; Li, Guibin; Dobrowsky, Rick T; Florant, Gregory L; Summers, Scott A

    2003-03-21

    Multiple studies suggest that lipid oversupply to skeletal muscle contributes to the development of insulin resistance, perhaps by promoting the accumulation of lipid metabolites capable of inhibiting signal transduction. Herein we demonstrate that exposing muscle cells to particular saturated free fatty acids (FFAs), but not mono-unsaturated FFAs, inhibits insulin stimulation of Akt/protein kinase B, a serine/threonine kinase that is a central mediator of insulin-stimulated anabolic metabolism. These saturated FFAs concomitantly induced the accumulation of ceramide and diacylglycerol, two products of fatty acyl-CoA that have been shown to accumulate in insulin-resistant tissues and to inhibit early steps in insulin signaling. Preventing de novo ceramide synthesis negated the antagonistic effect of saturated FFAs toward Akt/protein kinase B. Moreover, inducing ceramide buildup recapitulated and augmented the inhibitory effect of saturated FFAs. By contrast, diacylglycerol proved dispensable for these FFA effects. Collectively these results identify ceramide as a necessary and sufficient intermediate linking saturated fats to the inhibition of insulin signaling.

  12. Molecular Basis for Failure of “Atypical” C1 Domain of Vav1 to Bind Diacylglycerol/Phorbol Ester*

    PubMed Central

    Geczy, Tamas; Peach, Megan L.; El Kazzouli, Saïd; Sigano, Dina M.; Kang, Ji-Hye; Valle, Christopher J.; Selezneva, Julia; Woo, Wonhee; Kedei, Noemi; Lewin, Nancy E.; Garfield, Susan H.; Lim, Langston; Mannan, Poonam; Marquez, Victor E.; Blumberg, Peter M.

    2012-01-01

    C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu9, Glu10, Thr11, Thr24, and Tyr26) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1. PMID:22351766

  13. Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.).

    PubMed

    Sun, Li; Ouyang, Chao; Kou, Shanglong; Wang, Shenghua; Yao, Yunyi; Peng, Tong; Xu, Ying; Tang, Lin; Chen, Fang

    2011-01-01

    A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).

  14. Regulation of Cellular Diacylglycerol through Lipid Phosphate Phosphatases Is Required for Pathogenesis of the Rice Blast Fungus, Magnaporthe oryzae

    PubMed Central

    Mir, Albely Afifa; Choi, Jaeyoung; Choi, Jaehyuk; Lee, Yong-Hwan

    2014-01-01

    Considering implication of diacylglycerol in both metabolism and signaling pathways, maintaining proper levels of diacylglycerol (DAG) is critical to cellular homeostasis and development. Except the PIP2-PLC mediated pathway, metabolic pathways leading to generation of DAG converge on dephosphorylation of phosphatidic acid catalyzed by lipid phosphate phosphatases. Here we report the role of such enzymes in a model plant pathogenic fungus, Magnaporthe oryzae. We identified five genes encoding putative lipid phosphate phosphatases (MoLPP1 to MoLPP5). Targeted disruption of four genes (except MoLPP4) showed that MoLPP3 and MoLPP5 are required for normal progression of infection-specific development and proliferation within host plants, whereas MoLPP1 and MoLPP2 are indispensable for fungal pathogenicity. Reintroduction of MoLPP3 and MoLPP5 into individual deletion mutants restored all the defects. Furthermore, exogenous addition of saturated DAG not only restored defect in appressorium formation but also complemented reduced virulence in both mutants. Taken together, our data indicate differential roles of lipid phosphate phosphatase genes and requirement of proper regulation of cellular DAGs for fungal development and pathogenesis. PMID:24959955

  15. Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress

    PubMed Central

    Liu, Xiao-Yu; Ouyang, Long-Ling; Zhou, Zhi-Gang

    2016-01-01

    In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress. PMID:27216435

  16. Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

    PubMed

    Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Yamaoka, Masakazu

    2013-08-01

    Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is

  17. Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism.

    PubMed Central

    Gordon, S. E.; Downing-Park, J.; Tam, B.; Zimmerman, A. L.

    1995-01-01

    The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated second messenger, cellular levels of other substances, including Ca2+ and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting that DAG did not act by competitive inhibition of cGMP binding. Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8--1,3-dioctanoyl-sn-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective. Our results suggest that DAG allosterically interferes with channel opening, and raise the question of whether DAG is involved in visual transduction. PMID:8527654

  18. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

    PubMed

    Wang, Haikuan; Wang, Xiaojie; Li, Xiaolu; Zhang, Yehong; Dai, Yujie; Guo, Changlu; Zheng, Heng

    2012-09-28

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters.

  19. Purification and partial characterization of nonspecific lipase from rat pancreas.

    PubMed

    Albron, P W; Corbett, B J; Latimer, A D

    1976-03-26

    Nonspecific lipase (also referred to as micelle lipase and secondary ester hydrolase) has been purified to electrophoretic homogeneity starting from acetone powder of rat pancreas. The purified enzyme is found to have a molecular weight (gel filtration) of 64 000 +/- 2000, and an equivalent weight (titration with E-600) of 65 000. Nonspecific lipase is seen to be very sensitive to inhibition by organophosphates but resistant to quinine. Evidence for the presence of sulfhydryl and imidazole groups essential for activity is presented, and some observations on substrate specificity are made. The purified enzyme appears to lack phosphate groups and lipids, and is unstable under conditions of low ionic strength and/or exposure to 2-mercaptoethanol.

  20. Lipoprotein metabolism and lipoprotein lipase in severe cystic acne.

    PubMed

    Pigatto, P; Altomare, G F; Negri, M; Finzi, A F; Vigotti, G; Vergani, C

    1985-01-01

    In severe cystic acne we found low levels of high density lipoprotein-cholesterol (HDL-C) and apolipoprotein A (Apo-A) in the presence of normal total lipids. In a larger number of patients, we always observed significantly lower levels of HDL-C and Apo-A than in either age-matched controls or subjects with acne vulgaris. Since lipoprotein lipase is one major determinant of HDL concentration, we assayed the lipase activity in liver and extra-hepatic tissues by the method of Krauss et al. There was highly significant less total and hepatic lipase activity than in age-matched controls. HDL distribution was examined by zonal ultracentrifugation and a decrease in the HDL2 subclass was discovered. Since HDL are inversely correlated to atherosclerosis, cystic acne is one risk factor for atherosclerosis. The linkage between low HDL levels and severe cystic acne should be further investigated.

  1. Biodiesel production from microalgae oil catalyzed by a recombinant lipase.

    PubMed

    Huang, Jinjin; Xia, Ji; Jiang, Wei; Li, Ying; Li, Jilun

    2015-03-01

    A recombinant Rhizomucor miehei lipase was constructed and expressed in Pichia pastoris. The target enzyme was termed Lipase GH2 and it can be used as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system. Conversion rates of two major types of biodiesel, fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE), reached maximal values (>90%) after 24h. The process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE, and this system provides efficiency and reduced costs in biodiesel production.

  2. Lactobacillus sps. lipase mediated poly (ε-caprolactone) degradation.

    PubMed

    Khan, Imran; Ray Dutta, Jayati; Ganesan, Ramakrishnan

    2017-02-01

    Polymer degradation through lipase appears to be an enthralling alternative to bulk chemical routes. Poly (ε-caprolactone) (PCL) is an artificial polyester that can be degraded by microbes and enzymes like lipases and esterases. The environmental degradation of PCL is dependent on the activity of bacteria that characterization techniques such as thermogravimetric analysis, differential thermal are widely present in the ecosystem. In this study, three different lipases derived from Lactobacillus brevis, Lactobacillus plantarum and their co-culture have been utilized to explore their efficiency towards PCL enzymatic degradation. The effect of parameters such as enzyme loading and degradation time has been explored to understand the efficiency of the enzymes used in this study. Various analysis, scanning electron microscopy and Fourier transform infrared spectroscopy have been employed to study the enzymatic degradation and its possible mechanistic insight.

  3. [Structure and Activity of Fungal Lipases in Bile Salt Solutions].

    PubMed

    Bogdanova, L R; Bakirova, D R; Valiullina, Yu A; Idiyatullin, B Z; Faizullin, D A; Zueva, O S; Zuev, Yu F

    2016-01-01

    The changes in structure and catalytic properties of fungal lipases (Candida rugosa, Rhizomucor miehei, Mucor javanicus) were investigated in micellar solutions of bile salts that differ in hydrophilic-lypophilic balance and reaction medium properties. The methods of circular dichroism and tryptophan fluorescence were applied to estimate the changes in peptide structure within complexes with bile salt micelles. Bile salts do not exert a significant influence on the structure of the enzymes under study: in Rh. miehei and M. javanicus lipases the alpha helix content slightly decreased, the influence of bile salts on the C. rugosa structure was not revealed. Despite negligible structural modifications in the enzymes, in bile salt solutions a considerable change in their catalytic properties was observed: an abrupt decrease in catalytic effectiveness. Substrate-bile salts micelles complex formation was demonstrated by the NMR self-diffusion method. The model of a regulation of fungal lipase activity was proposed.

  4. The immobilization of lipase on PVDF-co-HFP membrane

    NASA Astrophysics Data System (ADS)

    Kayhan, Naciye; Eyüpoǧlu, Volkan; Adem, Şevki

    2016-04-01

    Lipase is an enzyme having a lot of different industrial applications such as biodiesel production, biopolymer synthesis, enantiopure pharmaceutical productions, agrochemicals, etc. Its immobilized form on different substances is more conventional and useful than its free form. Supporting material was prepared using PVDF-co-HFP in laboratory conditions and attached 1,4-diaminobutane (DA) and epichlorohydrin (EPI) ligands to the membrane to immobilize lipase enzyme. The immobilization conditions such as enzyme amount, pH, the concentration of salt, thermal stability and activity were stabilized for our experimental setup. Then, biochemical characterizations were performed on immobilized lipase PVDF-co-HFP regarding optimal pH activity, temperature and thermal stability. Also, the desorption ratios of immobilized enzyme in two different pathway were investigated to confirm immobilization stability for 24 hours.

  5. Studies of the in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities of selected Thai medicinal plants

    PubMed Central

    2012-01-01

    , a known lipase inhibitor. The highest antimicrobial activity was observed in the extracts from S. alba and S. caseolaris against Pseudomonas aeruginosa and Candida albicans, respectively. Conclusion The Thai medicinal plant B. strychnifolia is first reported to exert strong in vitro cytotoxic activities against human cancer cell lines and warrants further enrichment and characterization. The broad spectrum of the biological activities from the studied plant extracts can be applied as the guideline for the selection of Thai medicinal plant species for further pharmacological and phytochemical investigations. PMID:23145786

  6. Interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates: kinetics, mechanism, and analogy to the acylenzyme mechanism.

    PubMed

    Shin, H C; Quinn, D M

    1992-01-28

    The interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates [PNPOC(=O)-NHCnH2n+1; n = 4, 8, and 12] proceeds by the three-stage mechanism shown below. After reversible [formula: see text] formation of the enzyme-carbamate complex (EC), rapid carbamylation (kc) precedes slow decarbamylation. Therefore, in short-term assays (less than or equal to 30 min) of lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl butyrate, activity is rapidly lost. The inhibition by p-nitrophenyl N-butylcarbamate follows saturation kinetics, which allows determination of Kc = 5.4 +/- 0.9 microM and kc = (4.9 +/- 0.7) x 10(-2)s-1. Saturation kinetics are not observed for the longer inhibitors p-nitrophenyl N-octylcarbamate and p-nitrophenyl N-dodecylcarbamate. Rather, plots of the pseudo-first-order rate constant for activity loss versus inhibitor concentration are concave upward, consistent with inhibitor binding to two sites on the enzyme. The inhibition phase is sufficiently rapid that p-nitrophenyl N-octylcarbamate can be used to titrate enzyme active sites. On the other hand, long-term assays (greater than 5 h) show sequential inhibition and activity return phases, and from the activity return phase kd is calculated. The long-term activity time course is accurately simulated by Runge-Kutta integration of the differential equations for the three-stage mechanism. These approaches have been used to characterize the kinetics of interaction of the enzyme with the carbamate inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. A comparison of currently used serum lipase and amylase procedures in the serial detection of enzyme elevations in acute pancreatitis.

    PubMed

    Hathaway, J A; Kitt, D; Wingate, B

    1983-10-14

    Twenty-eight patients having acute pancreatitis were followed during convalescence with serum amylase and lipase determinations. Starch and p-nitrophenyl-oligosaccharide substrates were used for amylase. Dimercaptotributyrate and triolein were employed for lipase. The extreme sensitivity of the lipase procedure using the tributyrate detected a persistent elevation of lipase when other parameters of measurement had returned to normal.

  8. Enzymatic Synthesis of Structured Lipids using a Novel Cold-Active Lipase from Pichia lynferdii NRRL Y-7723

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were s...

  9. Comparison of immunoreactive serum trypsinogen and lipase in Cystic Fibrosis

    SciTech Connect

    Lloyd-Still, J.D.; Weiss, S.; Wessel, H.; Fong, L.; Conway, J.J.

    1984-01-01

    The incidence of Cystic Fibrosis (CF) is 1 in 2,000. Early detection and treatment of CF may necessitate newborn screening with a reliable and cost-effective test. Serum immunoreactive trypsinogen (IRT) an enzyme produced by the pancreas, is detectable by radioimmunoassay (RIA) techniques. Recently, it has been shown that IRT is elevated in CF infants for the first few months of life and levels become subnormal as pancreatic insufficiency progresses. Other enzymes produced by the pancreas, such as lipase, are also elevated during this time. The author's earlier work confirmed previous reports of elevated IRT levels in CF infants. The development of a new RIA for lipase (nuclipase) has enabled comparison of these 2 pancreatic enzymes in C.F. Serum IRT and lipase determinations were performed on 2 groups of CF patients; infants under 1 year of age, and children between 1 and 18 years of age. Control populations of the same age groups were included. The results showed that both trypsin (161 +- 92 ng/ml, range 20 to 400) and lipase (167 +- 151 ng/ml, range 29 to 500) are elevated in CF in the majority of infants. Control infants had values of IRT ranging from 20 to 29.5 ng/ml and lipase values ranging from 23 to 34 ng/ml. IRT becomes subnormal in most CF patients by 8 years of age as pancreatic function insufficiency increases. Lipase levels and IRT levels correlate well in infancy, but IRT is a more sensitive indicator of pancreatic insufficiency in older patients with CF.

  10. Lipase inactivation in wheat germ by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Jha, Pankaj Kumar; Kudachikar, V. B.; Kumar, Sourav

    2013-05-01

    An attempt was made to improve the shelf life of wheat germ by optimizing processing conditions involving γ-irradiation. Studies were carried out to investigate the effect of γ-irradiation (0-30 kGy doses) on the chemical composition of wheat germ with respect to variation in moisture, total ash, crude fat, free fatty acid, protein and lipase activity. The results demonstrate that shelf stability of wheat germ was achieved by inactivation of lipase at doses of γ-irradiation greater than 12 kGy.

  11. JCL Roundtable: Hypertriglyceridemia due to defects in lipoprotein lipase function.

    PubMed

    Brown, W Virgil; Goldberg, Ira J; Young, Stephen G

    2015-01-01

    In this Roundtable, our intent is to discuss those rare genetic disorders that impair the function of lipoprotein lipase. These cause severe hypertriglyceridemia that appears in early childhood with Mendelian inheritance and usually with full penetrance in a recessive pattern. Dr Ira Goldberg from New York University School of Medicine and Dr Stephen Young from the University of California, Los Angeles have agreed to answer my questions about this topic. Both have done fundamental work in recent years that has markedly altered our views on lipoprotein lipase function. I am going to start by asking them to give us a brief history of this enzyme system as a clinical entity.

  12. Genomic organization of the human lysosomal acid lipase gene (LIPA)

    SciTech Connect

    Aslandis, C.; Klima, H.; Lackner, K.J.; Schmitz, G. )

    1994-03-15

    Defects in the human lysosomal acid lipase gene are responsible for cholesteryl ester storage disease (CESD) and Wolman disease. Exon skipping as the cause for CESD has been demonstrated. The authors present here a summary of the exon structure of the entire human lysosomal acid lipase gene consisting of 10 exons, together with the sizes of genomic EcoRI and SacI fragments hybridizing to each exon. In addition, the DNA sequence of the putative promoter region is presented. The EMBL accession numbers for adjacent intron sequences are given. 7 refs., 2 figs., 1 tab.

  13. Clinical Features of Lysosomal Acid Lipase Deficiency

    PubMed Central

    Burton, Barbara K.; Deegan, Patrick B.; Enns, Gregory M.; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K.; Lobritto, Steve J.; Malinova, Vera; McLin, Valerie A.; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B.; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G.

    2015-01-01

    Abstract Objective: The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Methods: Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. Results: A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0–42); mean age at diagnosis was 15.2 years (range 1–46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n = 31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ∼1.15 multiples of normal (MN) and median spleen volume was ∼2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9–43.5 years). Conclusion: This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation. PMID:26252914

  14. Phenolic antioxidants in some Vigna species of legumes and their distinct inhibitory effects on α-glucosidase and pancreatic lipase activities.

    PubMed

    Sreerama, Yadahally N; Takahashi, Yoko; Yamaki, Kohji

    2012-09-01

    Phenolic extracts of 4 Vigna species of legumes (mung bean, moth bean, and black and red varieties of adzuki beans) were evaluated for phenolic contents, antioxidant activities, and inhibitory properties against α-glucosidase and pancreatic lipase. Results showed that adzuki bean varieties contain higher phenolic indexes than mung bean and moth beans. Adzuki bean (black) variety was found to be the most active 2,2'-diphenyl-1-picrylhydrazyl and superoxide anion scavenger. However, the hydrogen peroxide scavenging and metal chelating abilities were significantly higher in adzuki bean (red) variety. Mung bean exhibited least antioxidant activities in all the methods tested. Phenolic extracts from these legumes also showed distinct variations in the inhibition of enzymes associated with hyperglycemia and hyperlipidemia. Inhibitory activities of all the extracts against lipase were found to be more potent than α-glucosidase. Although, α-glucosidase inhibitory activity was superior in the black variety of adzuki bean (IC(50,) 26.28 mg/mL), both adzuki bean varieties (black and red) along with moth bean showed strong inhibitory activities on lipase with no significant difference in their IC(50) values (7.32 to 9.85 mg/mL). These results suggest that Vigna species of legumes are potential source of antioxidant phenolics and also great sources of strong natural inhibitors for α-glucosidase and lipase activities. This information may help for effective utilization of these legumes as functional food ingredients for promoting health. Practical Application:  Vigna species of legumes are good sources of phenolic antioxidants and strong natural inhibitors of enzymes associated with diabetes and obesity. Therefore, utilization of these legumes in the development of functional foods with increased therapeutic value would be a significant step toward health promotion and wellness.

  15. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

    PubMed Central

    Dutta, Mouparna; Ghosh, Anindya S.; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J.; Dandekar, Abhaya M.; Goñi, Félix M.

    2015-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  16. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

    PubMed

    Chakraborty, Sandeep; Rendón-Ramírez, Adela; Ásgeirsson, Bjarni; Dutta, Mouparna; Ghosh, Anindya S; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J; Dandekar, Abhaya M; Goñi, Félix M

    2013-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  17. Optimum conditions for the production of soy polyol oils and diacylglycerol from soybean oil by Acinetobacter haemolyticus A01-35 NRRL B-59985

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Triacylglycerols (TAG) containing hydroxy fatty acids have many industrial uses such as the manufacture of aviation lubricant, plastic, paint, nylons, and cosmetics, because of the hydroxyl groups on the fatty acid (FA) constituents. Diacylglycerols (DAG) containing hydroxy FA can also be used in th...

  18. Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid.

    PubMed

    Takuwa, Y; Takuwa, N; Rasmussen, H

    1986-11-05

    The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.

  19. Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

    SciTech Connect

    Takuwa, Y.; Takuwa, N.; Rasmussen, H.

    1986-11-05

    The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of (/sup 3/H)inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in (/sup 3/H)inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of (/sup 3/H)inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca/sup 2 +/ and does not appear to be secondary to an increase in intracellular Ca/sup 2 +/. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.

  20. SECRETION OF LIPASES IN THE DIGESTIVE TRACT OF THE CRICKET Gryllus bimaculatus.

    PubMed

    Weidlich, Sandy; Hoffmann, Klaus H; Woodring, Joseph

    2015-12-01

    Little is known concerning the sites and the ratios of the lipase secretions in insects, therefore we undertook an examination of the lipase secretion of fed and unfed adult female Gryllus bimaculatus. The ratio of triacylglyceride lipase, diacylglyceride lipase, and phosphatidylcholine lipase secreted by fed females in the caecum and ventriculus is 1:1.4:0.4. These activities decrease in the caecum by 30-40% in unfed females. The total lipase activity (TLA) in the caecum is about 10 times that in the ventriculus. Minimal lipase secretion occurs before and during the final moult, and remains at this level in unfed crickets, indicating a basal secretion rate. In 2-day-old fed females, about 10% of the TLA in the entire gut is found in the crop, about 70% in the caecum, 20% in the ventriculus, and 3% in the ileum. Lipases in the ventriculus are recycled back to the caecum and little is lost in the feces. Oleic acid stimulated in vitro lipase secretion, but lipids did not. Feeding stimulated lipase secretion, starvation reduced lipase secretion, but this does not prove a direct prandal regulation of secretion, because feeding also induced a size and volume increase of the caecum.

  1. Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F2 alpha in response to oxytocin: role of phospholipase C and diacylglycerol.

    PubMed

    Silvia, W J; Lee, J S; Trammell, D S; Hayes, S H; Lowberger, L L; Brockman, J A

    1994-06-01

    The first objective was to describe and evaluate the relationship between the ability of oxytocin to stimulate the activity of phospholipase (PL) C and its ability to stimulate the release of prostaglandin (PG) F2 alpha in ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes after completion of an 11-day steroid replacement protocol. In experiment 1, explants were incubated either in the presence (10(-6) M) or absence of oxytocin for 0, 1, 3, 10, 30 or 100 min to examine the time-course for activation of PLC and release of PGF2 alpha in response to oxytocin. An increase in the activity of PLC was detected at 3 min while an increase in the release of PGF2 alpha was not detected until 10 min (P < 0.05). In experiment 2, explants were incubated in the presence of various oxytocin analogues (10(-6) M) to compare their abilities to activate PLC and release PGF2 alpha. Oxytocin and three receptor agonists stimulated the activity of PLC and the release of PGF2 alpha (P < 0.05) while two oxytocin receptor antagonists had no effect on either response. In experiment 3, explants were incubated in the presence of oxytocin or arginine vasopressin at 10(-9) to 10(-6) M to establish dose-response curves for the activation of PLC and release of PGF2 alpha. For both hormones, significant increases (P < 0.05) in the release of PGF2 alpha were observed at 10(-8) M while increases in PLC activity were not detected until 10(-7) M was used. In experiment 4, explants were pretreated with either U-73122 (an inhibitor of PLC activity) or U-73343 (an inactive analogue of U-73122). Explants were then treated with control medium, oxytocin or AlF4-. Both oxytocin and AlF4-stimulated the activity of PLC and the release of PGF2 alpha (P < 0.05). U-73122 blocked the ability of oxytocin to stimulate the release of PGF2 alpha (P < 0.05) but had no effect on its ability to stimulate the activity of PLC (P > 0.1). Based on the results from these experiments

  2. Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta.

    PubMed Central

    van Dijk, M; Muriana, F J; van Der Hoeven, P C; de Widt, J; Schaap, D; Moolenaar, W H; van Blitterswijk, W J

    1997-01-01

    The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta. PMID:9169602

  3. Slowing down fat digestion and absorption by an oxadiazolone inhibitor targeting selectively gastric lipolysis.

    PubMed

    Point, Vanessa; Bénarouche, Anais; Zarrillo, Julie; Guy, Alexandre; Magnez, Romain; Fonseca, Laurence; Raux, Brigitt; Leclaire, Julien; Buono, Gérard; Fotiadu, Frédéric; Durand, Thierry; Carrière, Frédéric; Vaysse, Carole; Couëdelo, Leslie; Cavalier, Jean-François

    2016-11-10

    Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity".

  4. Competition of Thermomyces lanuginosus lipase with its hydrolysis products at the oil-water interface.

    PubMed

    Muth, Marco; Rothkötter, Stefanie; Paprosch, Steven; Schmid, Reiner P; Schnitzlein, Klaus

    2017-01-01

    Lipase-catalyzed hydrolysis of triglycerides yields glycerol and free fatty-acids, provided that the enzyme is non-regioselective. For an Sn-1,3 regioselective enzyme, such as lipase from Thermomyces lanuginosus, the final product is no longer glycerol but Sn-2 monoglyceride instead. However, surface active molecules generated by lipolysis may have a detrimental effect on the interfacial biocatalysis since it is known that low molecular weight surfactants can displace proteins from interfaces. By using drop profile analysis tensiometry, we evaluated the interfacial properties of the lipase-generated molecules and their competitive effect on the adsorption behavior of the lipase and on the proceeding lipolysis. Our results show that even at concentration ratios of 8.64×10(-4)M (Sn-2 monoglyceride) to 2.5×10(-7)M (lipase), the final interfacial pressure values are very similar as for the system containing the lipase alone (i.e. ∼26 mN/m). This is a strong indication that monoglycerides, as the most interfacially active products generated during regioselective lipolysis, are expelled from the oil-water interface by the lipase. We attribute this effect to intermolecular lipase-lipase interactions, resulting in a low desorption probability of the lipase. For low oleic acid concentrations, the interfacial tension is solely determined by the lipase, while for higher concentrations, lipase and oleic acid both contribute to the tension values. We propose a hypothesis based on the preferential interaction of oleic acid molecules with hydrophobic sites on the lipase. The pH dependence of the adsorption rate and the interfacial activity of the lipase were also investigated.

  5. Microplate Bioassay for Determining Substrate Selectivity of "Candida rugosa" Lipase

    ERIC Educational Resources Information Center

    Wang, Shi-zhen; Fang, Bai-shan

    2012-01-01

    Substrate selectivity of "Candida rugosa" lipase was tested using "p"-nitrophenyl esters of increasing chain length (C[subscript 1], C[subscript 7], C[subscript 15]) using the high-throughput screening method. A fast and easy 96-well microplate bioassay was developed to help students learn and practice biotechnological specificity screen. The…

  6. Tuning Lipase Reaction for Production of Fatty Acids from Oil.

    PubMed

    Odaneth, Annamma A; Vadgama, Rajeshkumar N; Bhat, Anuradha D; Lali, Arvind M

    2016-10-01

    Fats or oils are split partially or completely to obtain fatty acids that find wide applications in oleo-chemical industries. Lipase-mediated complete splitting (hydrolysis) of oils is a green process having great potential to replace the traditional methods of oil splitting. However, cost of lipases, mechanistic kinetic equilibrium and associated operational limitations prove to be deterrents for scale up of the enzymatic oil splitting process. In the present study, we demonstrate the use of immobilised 1,3-regioselective lipase (HyLIP) for complete hydrolysis of oil in monophasic reaction medium. Incorporation of a polar organic solvent (tert-butanol, 1:5, v/v) homogenises the oil-water mixture and contributes positively towards complete hydrolysis. The monophasic oil hydrolysis reaction with optimised water concentration (0.05 %, v/v) gave Free Fatty Acid (FFA) yield of 88 % (HyLIP and Novozym-435) and 66 % (TLIM and RMIM). Smart reaction engineering and modification of the reaction intermediates to favourable substrate lead to ∼99 % degree of hydrolysis of triglycerides with ∼90 % FFA yield using 1,3-regioselective lipase. The present work becomes basic platform for developing technologies for synthesis of fatty acids, monoglycerides, diglycerides and glycerol.

  7. Safety evaluation of a lipase expressed in Aspergillus oryzae.

    PubMed

    Greenough, R J; Perry, C J; Stavnsbjerg, M

    1996-02-01

    A programme of studies was conducted to establish the safety of a lipase artificially expressed in Aspergillus oryzae to be used in the detergent industry and as a processing aid in the baking industry. Laboratory animal studies were used to assess general and inhalation toxicity, skin sensitization, and skin and eye irritation. Its potential to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies. The pathogenicity of A. oryzae, the organism used to produce the lipase, was also assessed in laboratory animals. Basic ecotoxicity in a variety of test species was studied. General and inhalation toxicity was low. There was evidence of mild skin irritation. There was no evidence of eye irritation, skin sensitization, mutagenic potential, chromosomal aberrations, exotoxicity or notable pathogenicity. Comparison of these results with human exposure levels and previously published data indicates that the lipase appears safe for consumers in the given applications, requires no special occupational health precautions in manufacture and is of low environmental impact. Furthermore, the organism used in production of the lipase hs no notable pathogenicity.

  8. Study of microwave effects on the lipase-catalyzed hydrolysis.

    PubMed

    Chen, Chia-Chen; Reddy, P Muralidhar; Devi, C Shobha; Chang, Po-Chi; Ho, Yen-Peng

    2016-01-01

    The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.

  9. Lipase-catalyzed synthesis of partial acylglycerols of acetoacetate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A commercially available immobilized preparation of Rhizomucor miehei lipase (Lipozyme RMIM) has been employed in the synthesis of partial glycerides of acetoacetate. Due to the chemical reactivitity of the acetoacetyl group, these glycerides could have novel uses in e.g. polymer formation. Both 1...

  10. Burkholderia cepacia lipase is a promising biocatalyst for biofuel production.

    PubMed

    Sasso, Francesco; Natalello, Antonino; Castoldi, Simone; Lotti, Marina; Santambrogio, Carlo; Grandori, Rita

    2016-07-01

    Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.

  11. Physico-chemical properties of various palm-based diacylglycerol oils in comparison with their corresponding palm-based oils.

    PubMed

    Saberi, Amir Hossein; Kee, Beh Boon; Oi-Ming, Lai; Miskandar, Mat Sahri

    2011-08-01

    Palm-based diacylglycerol (P-DAG) oils were produced through enzymatic glycerolysis of palm kernel oil (PKO), palm oil (PO), palm olein (POL), palm mid fraction (PMF) and palm stearin (PS). High purity DAG (83-90%, w/w) was obtained and compared to palm-based oils (P-oil) had significantly (P<0.05) different fatty acid composition (FAC), iodine value (IV) and slip melting point (SMP). Solid fat content (SFC) profiles of P-DAG oils as compared to P-oils had less steep curves with lower SFC at low temperature range (5-10°C) and the higher complete melting temperatures. Also, P-DAG oils in contrast with P-oils showed endothermic as well as exothermic peaks with higher transition temperatures and significantly (P<0.05) higher crystallisation onsets, heats of fusion, and heats of crystallisation. Crystal forms for P-DAG oils were mostly in the β form.

  12. Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: Implications for the mechanisms of lipase action

    SciTech Connect

    Deckelbaum, R.J. ); Hamilton, J.A.; Butbul, E.; Gutman, A. ); Moser, A. ); Bengtsson-Olivecrona, G.; Olivecrona, T. ); Carpentier, Y.A. )

    1990-02-06

    To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, the authors compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of ({sup 13}C)carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T{sub 1}, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, they found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. These results suggest that despite lower enzyme affinity for MCT emulsions, shorter chain triacylglycerols are more readily hydrolyzed by lipoprotein and hepatic lipases than long-chain triacylglycerols because of greater MCT solubility and mobility at the emulsion-water interface.

  13. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds

    PubMed Central

    Zhang, Chunyu; Iskandarov, Umidjon; Cahoon, Edgar B.

    2013-01-01

    Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions. PMID:23814277

  14. Preliminary characterization of diacylglycerol generation in human basophils: temporal relationship to histamine release and resolution of degranulation.

    PubMed

    Oriente, A; Hundley, T; Hubbard, W C; MacGlashan, D W

    1997-05-01

    Purified human basophils were examined for changes in diacylglycerol levels to determine whether the transient nature of a N-formyl-methionyl-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protein kinase C (PKC) activity could be explained by the transient production of diacylglycerol (DAG). In preliminary experiments total DAG levels were measured by the DAG kinase assay. Although elevations followed stimulation with 1 microM fMLP (basal levels of 15 pmol/10(6) basophils vs. 45 pmol/10(6) basophils at the 3-min time point), there were no detectable changes in the first 60 s of the reaction. Histamine release is typically complete by 30-45 s. Measurement of inositol trisphosphate indicated a rapid increase by 5 s of 2.5 pmol/10(6) basophils. If DAG were produced at similar levels, the DAG kinase assay would not have detected the elevation. Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA-DAG) and 1-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negative ion chemical ionization mass spectroscopy). A 5-s elevation in these two species averaged 2 pmol/10(6) basophils, consistent with the inositol trisphosphate levels and occurring during the period of histamine release. However, a much more pronounced second phase to the SA-DAG response also occurred, mirroring the total DAG levels. This second phase of the DAG response, either total or SA-DAG, was transient on a time scale temporally coincident with the appearance and resolution of degranulation sacs as measured by fluorescence microscopy. These data suggest that there is selective generation of DAG species in the early reaction and the later appearance of DAG may be related to the formation and resolution of granule structures that follow the secretion of histamine.

  15. Diacylglycerol kinase ζ (DGKζ) is a critical regulator of bone homeostasis via modulation of c-Fos levels in osteoclasts†

    PubMed Central

    Zamani, Ali; Decker, Corinne; Cremasco, Viviana; Hughes, Lindsey; Novack, Deborah V.; Faccio, Roberta

    2015-01-01

    Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ−/− mice are osteoporotic due to a marked increase in OC numbers. In vitro, DGKζ−/− bone marrow macrophages (BMMs) form more numerous, larger and highly resorptive OCs. Surprisingly, while increased DAG levels do not alter RANK/RANKL osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine M-CSF. We find that M-CSF is responsible for increased DGKζ−/− OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ−/− cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2+/− BMMs, which have reduced DAG levels and form fewer OCs due to impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2+/− mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression. PMID:25891971

  16. Desaturation of Fatty Acids Associated with Monogalactosyl Diacylglycerol: The Effects of San 6706 and San 9785 1

    PubMed Central

    Lem, Nora W.; Williams, John P.

    1981-01-01

    The effects of two substituted pyridazinone herbicides, San 6706 and San 9785, on photosynthesis, dark respiration, and fatty acid metabolism were studied in mature leaf tissue of Vicia faba. Both San 6706 and San 9785 inhibited photosynthesis within 2 hours after initial exposure of leaf tissue to the chemicals although San 9785 was more effective in inhibiting photosynthesis than San 6706. Neither San 6706 nor San 9785 had any marked effect on dark respiration. Kinetic studies using 14CO2 indicated that synthesis of 18:3 esterified to monogalactosyl diacylglycerol (MGDG) was not completely inhibited by San 9785 up to 48 hours after feeding. Radioactivity was observed to accumulate in MGDG (digalactosyl diacylglycerol [DGDG] and sulpholipid [SL]) 18:2 at the expense of 18:3 but the specific radioactivity of MGDG 18:3 continued to increase throughout the experiment indicating only partial inhibition of MGDG 18:3 synthesis. No significant differences were observed in the metabolism of other fatty acids. The metabolism of fatty acids from leaf tissue was not affected by treatment with San 6706. The data indicate that there are at least two sites for 18:2 desaturation to form 18:3, one associated with MGDG in the chloroplast, which is inhibited by San 9785, and one or more sites not inhibited by San 9785. The fatty acid specific radioactivity data support the hypothesis that there is vectorial transfer of fatty acids from phosphatidyl choline to MGDG to produce the large quantities of MGDG 18:3 found in higher plant tissue. PMID:16662031

  17. Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts.

    PubMed

    Bagnato, Carolina; Igal, R Ariel

    2003-12-26

    Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30-40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties.

  18. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds.

    PubMed

    Zhang, Chunyu; Iskandarov, Umidjon; Klotz, Elliott T; Stevens, Robyn L; Cahoon, Rebecca E; Nazarenus, Tara J; Pereira, Suzette L; Cahoon, Edgar B

    2013-08-01

    Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45-50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.

  19. Pyrazole phenylcyclohexylcarbamates as inhibitors of human fatty acid amide hydrolases (FAAH).

    PubMed

    Aghazadeh Tabrizi, Mojgan; Baraldi, Pier Giovanni; Ruggiero, Emanuela; Saponaro, Giulia; Baraldi, Stefania; Romagnoli, Romeo; Martinelli, Adriano; Tuccinardi, Tiziano

    2015-06-05

    Fatty acid amide hydrolase (FAAH) inhibitors have gained attention as potential therapeutic targets in the management of neuropathic pain. Here, we report a series of pyrazole phenylcyclohexylcarbamate derivatives standing on the known carbamoyl FAAH inhibitor URB597. Structural modifications led to the recognition of compound 22 that inhibited human recombinant FAAH (hrFAAH) in the low nanomolar range (IC50 = 11 nM). The most active compounds of this series showed significant selectivity toward monoacylglycerol lipase (MAGL) enzyme. In addition, molecular modeling and reversibility behavior of the new class of FAAH inhibitors are presented in this article.

  20. Solvent-induced lid opening in lipases: a molecular dynamics study.

    PubMed

    Rehm, Sascha; Trodler, Peter; Pleiss, Jürgen

    2010-11-01

    In most lipases, a mobile lid covers the substrate binding site. In this closed structure, the lipase is assumed to be inactive. Upon activation of the lipase by contact with a hydrophobic solvent or at a hydrophobic interface, the lid opens. In its open structure, the substrate binding site is accessible and the lipase is active. The molecular mechanism of this interfacial activation was studied for three lipases (from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosa) by multiple molecular dynamics simulations for 25 ns without applying restraints or external forces. As initial structures of the simulations, the closed and open structures of the lipases were used. Both the closed and the open structure were simulated in water and in an organic solvent, toluene. In simulations of the closed lipases in water, no conformational transition was observed. However, in three independent simulations of the closed lipases in toluene the lid gradually opened. Thus, pathways of the conformational transitions were investigated and possible kinetic bottlenecks were suggested. The open structures in toluene were stable, but in water the lid of all three lipases moved towards the closed structure and partially unfolded. Thus, in all three lipases opening and closing was driven by the solvent and independent of a bound substrate molecule.

  1. Pancreatic lipase and pancreatic lipase-related protein 2, but not pancreatic lipase-related protein 1, hydrolyze retinyl palmitate in physiological conditions.

    PubMed

    Reboul, Emmanuelle; Berton, Amélie; Moussa, Myriam; Kreuzer, Corinne; Crenon, Isabelle; Borel, Patrick

    2006-01-01

    The major sources of vitamin A in the human diet are retinyl esters (mainly retinyl palmitate) and provitamin A carotenoids. It has been shown that classical pancreatic lipase (PL) is involved in the luminal hydrolysis of retinyl palmitate (RP), but it is not known whether pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2), two other lipases recovered in the human pancreatic juice, are also involved. The aim of this study was to assess whether RP acts a substrate for these lipase-related proteins. Pure horse PL, horse PLRP2 and dog PLRP1 were incubated with RP solubilized in its physiological vehicles, i.e., triglyceride-rich lipid droplets, mixed micelles and vesicles. High performance liquid chromatography (HPLC) was used to assess RP hydrolysis by the free retinol released in the incubation medium. Incubation of RP-containing emulsions with horse PL and colipase resulted in RP hydrolysis (0.051+/-0.01 micromol/min/mg). This hydrolysis was abolished when colipase was not added to the medium. PLRP2 and PLRP1 were unable to hydrolyze RP solubilized in emulsions, regardless of whether colipase was added to the medium. PL hydrolyzed RP solubilized in mixed micelles as well (0.074+/-0.014 micromol/min/mg). Again, this hydrolysis was abolished in the absence of colipase. PLRP2 hydrolyzed RP solubilized in micelles but less efficiently than PL (0.023+/-0.005 micromol/min/mg). Colipase had no effect on this hydrolysis. PLRP1 was unable to hydrolyze RP solubilized in micelles, regardless of whether colipase was present or absent. Both PL and PLRP2 hydrolyzed RP solubilized in a vesicle rich-solution, and a synergic phenomenon between the two lipases was enlighten. Taken together, these results show that (1) PL hydrolyzes RP whether RP is solubilized in emulsions or in mixed micelles, (2) PLRP2 hydrolyzes RP only when RP is solubilized in mixed micelles, and (3) PLRP1 is unable to hydrolyze RP regardless of whether RP is solubilized in emulsions or in mixed

  2. Lipase applications in oil hydrolysis with a case study on castor oil: a review.

    PubMed

    Goswami, Debajyoti; Basu, Jayanta Kumar; De, Sirshendu

    2013-03-01

    Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.

  3. Cloning and Expression of a Subfamily 1.4 Lipase from Bacillus licheniformis IBRL-CHS2.

    PubMed

    Reddy, Nidyaletchmy Subba; Rahim, Rashidah Abdul; Ibrahim, Darah; Kumar, K Sudesh

    2016-11-01

    We report on the cloning of the lipase gene from Bacillus licheniformis IBRL-CHS2 and the expression of the recombinant lipase. DNA sequencing analysis of the cloned lipase gene showed that it shares 99% identity with the lipase gene from B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then cloned into the pET-15b(+) expression vector and the construct was transformed into E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDS-PAGE where the lipase was found to have a molecular weight of about 23 kDa.

  4. Cloning and Expression of a Subfamily 1.4 Lipase from Bacillus licheniformis IBRL-CHS2

    PubMed Central

    Reddy, Nidyaletchmy Subba; Rahim, Rashidah Abdul; Ibrahim, Darah; Kumar, K. Sudesh

    2016-01-01

    We report on the cloning of the lipase gene from Bacillus licheniformis IBRL-CHS2 and the expression of the recombinant lipase. DNA sequencing analysis of the cloned lipase gene showed that it shares 99% identity with the lipase gene from B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then cloned into the pET-15b(+) expression vector and the construct was transformed into E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDS-PAGE where the lipase was found to have a molecular weight of about 23 kDa. PMID:27965753

  5. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.

  6. Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03.

    PubMed

    Ogino, Hiroyasu; Katou, Yoshikazu; Akagi, Rieko; Mimitsuka, Takashi; Hiroshima, Shinichi; Gemba, Yuichi; Doukyu, Noriyuki; Yasuda, Masahiro; Ishimi, Kosaku; Ishikawa, Haruo

    2007-11-01

    Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.

  7. Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

    PubMed Central

    Salleh, Abu Bakar; Basri, Mahiran

    2016-01-01

    Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents. PMID:26934700

  8. Medicinal Plants Traditionally Used for Treatment of Obesity and Diabetes Mellitus - Screening for Pancreatic Lipase and α-Amylase Inhibition.

    PubMed

    Buchholz, Tina; Melzig, Matthias F

    2016-02-01

    In order to find new pancreatic lipase (PL) and α-amylase inhibitors from natural sources for the treatment of obesity and related diseases as diabetes mellitus II, 23 medicinal plants with weight-reducing, serum glucose-reducing or related potential were investigated. Methanolic and water extracts of the plants were evaluated by using two in vitro test systems. Our findings have shown that the methanolic extract of Hibiscus sabdariffa L. (Malvaceae) showed high inhibitory activities to PL (IC50 : 35.8 ± 0.8 µg/mL) and α-amylase (IC50 : 29.3 ± 0.5 µg/mL). Furthermore, the methanolic extract of Tamarindus indica L. (Leguminosae) showed a high anti-lipase (IC50 : 152.0 ± 7.0 µg/mL) and the aqueous extract a high anti-amylase (IC50 : 139.4 ± 9.0 µg/mL) activity. This work provides a priority list of interesting plants for further study with respect to the treatment of obesity and associated diseases.

  9. Lipase production by yeasts from extra virgin olive oil.

    PubMed

    Ciafardini, G; Zullo, B A; Iride, A

    2006-02-01

    Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water from the fruits. Some of our recent microbiological research has shown that a rich micro-flora is present in the suspended fraction of the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research however has, for the first time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis californica 1639 were able to hydrolyse different specific synthetic substrates represented by p-nitrophenyl stearate, 4-nitrophenyl palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was confined to the whole cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was influenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the optimal ratio was 1% (v/v). Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples. The discovery of lipase-positive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a positive or negative way towards

  10. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  11. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  12. Combined inhibition of monoacylglycerol lipase and cyclooxygenases synergistically reduces neuropathic pain in mice

    PubMed Central

    Crowe, Molly S; Leishman, Emma; Banks, Matthew L; Gujjar, Ramesh; Mahadevan, Anu; Bradshaw, Heather B; Kinsey, Steven G

    2015-01-01

    Background and Purpose Neuropathic pain is commonly treated with GABA analogues, steroids or non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit one or more COX isozymes but chronic COX inhibition paradoxically increases gastrointestinal inflammation and risk of unwanted cardiovascular events. The cannabinoids also have analgesic and anti-inflammatory properties and reduce neuropathic pain in animal models. The present study investigated the analgesic effects of inhibiting both monoacylglycerol lipase (MAGL) and COX enzymes, using low doses of both inhibitors. Experimental Approach Mice subjected to chronic constriction injury (CCI) were tested for mechanical and cold allodynia after administration of the MAGL inhibitor, JZL184, or the non-selective COX inhibitor diclofenac. Then, both drugs were co-administered at fixed dose proportions of 1:3, 1:1 and 3:1, based on their ED50 values. PGs, endocannabinoids and related lipids were quantified in lumbar spinal cord. Key Results Combining low doses of JZL184 and diclofenac synergistically attenuated mechanical allodynia and additively reduced cold allodynia. The cannabinoid CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the analgesic effects of the JZL184 and diclofenac combination on mechanical allodynia, implying that CB1 receptors were primarily responsible for the anti-allodynia. Diclofenac alone and with JZL184 significantly reduced PGE2 and PGF2α in lumbar spinal cord tissue, whereas JZL184 alone caused significant increases in the endocannabinoid metabolite, N-arachidonoyl glycine. Conclusions and Implications Combining COX and MAGL inhibition is a promising therapeutic approach for reducing neuropathic pain with minimal side effects. PMID:25393148

  13. Inhibition of monoacylglycerol lipase attenuates nonsteroidal anti-inflammatory drug-induced gastric hemorrhages in mice.

    PubMed

    Kinsey, Steven G; Nomura, Daniel K; O'Neal, Scott T; Long, Jonathan Z; Mahadevan, Anu; Cravatt, Benjamin F; Grider, John R; Lichtman, Aron H

    2011-09-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type 2 (CB(2)), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(2) and D(2). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(2) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.

  14. Effects of methanol on lipases: molecular, kinetic and process issues in the production of biodiesel.

    PubMed

    Lotti, Marina; Pleiss, Jürgen; Valero, Francisco; Ferrer, Pau

    2015-01-01

    The biotechnological production of biodiesel is based on transesterification/esterification reactions between a source of fatty acids and a short-chain alcohol, usually methanol, catalysed by enzymes belonging to the class known as lipases. Several lipases used in industrial processes, although stable in the presence of other organic solvents, are inactivated by methanol at or below the concentration optimal for biodiesel production, making it necessary to use stepwise methanol feeding or pre-treatment of the enzyme. In this review article we focus on what is currently know about methanol inactivation of lipases, a phenomenon which is not common to all lipase enzymes, with the goal of improving the biocatalytic process. We suggest that different mechanisms can lead to inactivation of different lipases, in particular substrate inhibition and protein unfolding. Attempts to improve the performances of methanol sensitive lipases by mutagenesis as well as process engineering approaches are also summarized.

  15. Dry fermented sausages elaborated with lipase from Candida cylindracea. Comparison with traditional formulations.

    PubMed

    Zalacain, I; Zapelena, M J; Astiasarán, I; Bello, J

    1995-01-01

    The addition of microbial lipase to fermented sausages was studied. A sausage with lipase from Candida cylindracea and a control sausage with starter (Lactobacillus plantarum and Staphylococcus carnosus) were produced in a pilot plant. The acidity value and the amounts of the different free fatty acids (FFA) showed a higher intensity of lipolytic activity in sausages with lipase than in sausages with starter. In sausages with lipase, the percentage of saturated FFA was greater and that of polyunsaturated FFA was lower than in sausage with starter. Mono-unsaturated FFA percentage was similar in both sausages. TBA and peroxide values indicated that the increase of FFA produced by lipase action did not increase the rancidity. A slight increase in acetic, propionic and butyric acids was observed in sausage with lipase but this was not sufficient to develop excessive acidity in the product.

  16. Covalent immobilization of lipases on monodisperse magnetic microspheres modified with PAMAM-dendrimer

    NASA Astrophysics Data System (ADS)

    Zhu, Weiwei; Zhang, Yimei; Hou, Chen; Pan, Duo; He, Jianjun; Zhu, Hao

    2016-02-01

    This paper reported an immobilization of Candida rugosa lipase (CRL) onto PAMAM-dendrimer-grafted magnetic nanoparticles synthesized by a modified solvothermal reduction method. The dendritic magnetic nanoparticles were amply characterized by several instrumental measurements, and the CRL was covalently anchored on the three generation supports with glutaraldehyde as coupling reagent. The amount of immobilized enzyme was up to 150 mg/g support and the factors related with the enzyme activity were investigated. The immobilization of lipase improved their performance in wider ranges of pH and temperature. The immobilized lipase exhibited excellent thermal stability and reusability in comparison with free enzyme and can be reused 10 cycles with the enzymatic activity remained above 90 %. The properties of lipase improved obviously after being immobilized on the dendritic supports. The inactive immobilized lipase could be regenerated with glutaraldehyde and Cu2+, respectively. This synthetic strategy was facile and eco-friendly for applications in lipase immobilization.

  17. Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

    PubMed

    Liu, Ying; Guo, Chen; Liu, Chun-Zhao

    2015-03-01

    Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates.

  18. Biodiesel production from Jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite.

    PubMed

    You, Qinghong; Yin, Xiulian; Zhao, Yuping; Zhang, Yan

    2013-11-01

    Lipase from Burkholderia cepacia was immobilized on modified attapulgite by cross-linking reaction for biodiesel production with jatropha oil as feedstock. Effects of various factors on biodiesel production were studied by single-factor experiment. Results indicated that the best conditions for biodiesel preparation were: 10 g jatropha oil, 2.4 g methanol (molar ratio of oil to methanol is 1:6.6) being added at 3h intervals, 7 wt% water, 10 wt% immobilized lipase, temperature 35°C, and time 24h. Under these conditions, the maximum biodiesel yield reached 94%. The immobilized lipase retained 95% of its relative activity during the ten repeated batch reactions. The half-life time of the immobilized lipase is 731 h. Kinetics was studied and the Vmax of the immobilized lipases were 6.823 mmol L(-1). This immobilized lipase catalyzed process has potential industrial use for biodiesel production to replace chemical-catalyzed method.

  19. Lipase entrapment in PVA/Chitosan biodegradable film for reactor coatings.

    PubMed

    Batista, Karla A; Lopes, Flavio Marques; Yamashita, Fabio; Fernandes, Kátia Flávia

    2013-04-01

    This study reports the development and characterization of novel biodegradable film, based on chitosan and polyvinyl alcohol containing lipase entrapped. The films showed a thickness of 70.4 and 79 μm to PVA/Chitosan and PVA/Chitosan/Lipase, respectively. The entrapment of lipase in PVA/Chitosan film resulted in increasing of 69.4% tensile strength (TS), and 52.4% of elongation. SEM images showed the formation of a continuous film, without pores or cracks. The lipase entrapment efficiency was estimated in 92% and the films were repeatedly used for 25 hydrolytic cycles, maintaining 62% of initial activity. The PVA/Chitosan/Lipase film was used for olive oil hydrolysis of high performance. These results indicate that PVA/Chitosan/Lipase is a promising material for biotechnology applications such as triacylglycerol hydrolysis and biodiesel production.

  20. Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis.

    PubMed

    Dewei, Song; Min, Chen; Haiming, Cheng

    2016-11-01

    Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.

  1. Amylase and Lipase Detection in Hemorrhaged Animals Treated with HBOC-201

    DTIC Science & Technology

    2011-01-01

    4184 online DOl: 10.3109/10731199.2010.516260 inform a healthcare Amylase and Lipase Detection in Hemorrhaged Animals Treated with HBOC-201 Fran...Health Sciences, Bethesda, MD, USA Abstract: HBOC-201 may alter lipase and amylase detection on chemistry analyzers using optical methods and affect...pancreatic function after trauma. Amylase and lipase measurements were correlated against HBOC-201 to evaluate interference on samples spiked with

  2. New member of the hormone-sensitive lipase family from the permafrost microbial community.

    PubMed

    Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Gapizov, Sultan Sh; Spirina, Elena V; Durdenko, Ekaterina V; Rivkina, Elizaveta M

    2016-10-18

    Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family.

  3. Biodiesel production with special emphasis on lipase-catalyzed transesterification.

    PubMed

    Bisen, Prakash S; Sanodiya, Bhagwan S; Thakur, Gulab S; Baghel, Rakesh K; Prasad, G B K S

    2010-08-01

    The production of biodiesel by transesterification employing acid or base catalyst has been industrially accepted for its high conversion and reaction rates. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods. Recently, enzymatic transesterification involving lipases has attracted attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. The use of immobilized lipases and immobilized whole cells may lower the overall cost, while presenting less downstream processing problems, to biodiesel production. The present review gives an overview on biodiesel production technology and analyzes the factors/methods of enzymatic approach reported in the literature and also suggests suitable method on the basis of evidence for industrial production of biodiesel.

  4. Cell-bound lipase and esterase of Brevibacterium linens.

    PubMed

    Sorhaug, T; Ordal, Z J

    1974-03-01

    The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.

  5. Enzymatic modification of cassava starch by bacterial lipase.

    PubMed

    Rajan, Akhila; Abraham, T Emilia

    2006-06-01

    Enzymatic modification of starch using long chain fatty acid makes it thermoplastic suitable for a myriad of industrial applications. An industrial lipase preparation produced by Burkholderia cepacia (lipase PS) was used for modification of cassava starch with two acyl donors, lauric acid and palmitic acid. Reactions performed with palmitic acid by liquid-state and microwave esterification gave a degree of substitution (DS) of 62.08% (DS 1.45) and 42.06% (DS 0.98), respectively. Thermogravimetric analysis showed that onset of decomposition is at a higher temperature (above 600 degrees Celsius) for modified starch than the unmodified starch (280 degrees Celsius). Modified starch showed reduction in alpha-amylase digestibility compared to native starch (76.5-18%). Swelling power lowered for modified starch as esterification renders starch more hydrophobic, making it suitable for biomedical applications as materials for bone fixation and replacements, carriers for controlled release of drugs and bioactive agents. Thus enzymatic esterification is ecofriendly.

  6. Covalent immobilization of Pseudomonas cepacia lipase on semiconducting materials

    NASA Astrophysics Data System (ADS)

    Fernandez, Renny Edwin; Bhattacharya, Enakshi; Chadha, Anju

    2008-05-01

    Lipase from Pseudomonas cepacia was covalently immobilized on crystalline silicon, porous silicon and silicon nitride surfaces. The various stages of immobilization were characterized using FTIR (Fourier transform infrared) spectroscopy. The surface topography of the enzyme immobilized surfaces was investigated using scanning electron microscopy (SEM). The quantity of the immobilized active enzyme was estimated by the para-nitrophenyl palmitate (pNPP) assay. The immobilized lipase was used for triglyceride hydrolysis and the acid produced was detected by a pH sensitive silicon nitride surface as a shift in the C- V (capacitance-voltage) characteristics of an electrolyte-insulator-semiconductor capacitor (EISCAP) thus validating the immobilization method for use as a biosensor.

  7. Lipase cocktail for efficient conversion of oils containing phospholipids to biodiesel.

    PubMed

    Amoah, Jerome; Ho, Shih-Hsin; Hama, Shinji; Yoshida, Ayumi; Nakanishi, Akihito; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

    2016-07-01

    The presence of phospholipid has been a challenge in liquid enzymatic biodiesel production. Among six lipases that were screened, lipase AY had the highest hydrolysis activity and a competitive transesterification activity. However, it yielded only 21.1% FAME from oil containing phospholipids. By replacing portions of these lipases with a more robust bioFAME lipase, CalT, the combination of lipase AY-CalT gave the highest FAME yield with the least amounts of free fatty acids and partial glycerides. A higher methanol addition rate reduced FAME yields for lipase DF-CalT and A10D-CalT combinations while that of lipase AY-CalT combination improved. Optimizing the methanol addition rate for lipase AY-CalT resulted in a FAME yield of 88.1% at 2h and more than 95% at 6h. This effective use of lipases could be applied for the rapid and economic conversion of unrefined oils to biodiesel.

  8. Acid lipase from Candida viswanathii: production, biochemical properties, and potential application.

    PubMed

    de Almeida, Alex Fernando; Tauk-Tornisielo, Sâmia Maria; Carmona, Eleonora Cano

    2013-01-01

    Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (Y L/S = 1.381 g/g), lipase yield (Y L/S = 6.892 U/g), and biomass productivity (P X = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (Y L/S ) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties.

  9. Lipases production by solid-state fermentation: the case of Rhizopus homothallicus in perlite.

    PubMed

    Velasco-Lozano, Susana; Volke-Sepulveda, Tania; Favela-Torres, Ernesto

    2012-01-01

    Lipases are widely used in the industry for different purposes. Although these enzymes are mainly produced by submerged fermentation, lipase production by solid-state fermentation (SSF) has been gaining interest due to the advantages of this type of culture. Major advantages are higher production titers and productivity, less catabolite repression, and use of the dried fermented material as biocatalyst. This chapter describes a traditional methodology to produce fungal (Rhizopus homothallicus) lipases by SSF using perlite as inert support. The use of different devices (glass columns or Erlenmeyer flasks) and type of inoculum (spores or growing mycelium) is considered so that lipase production by SSF could be easily performed in any laboratory.

  10. Acid Lipase from Candida viswanathii: Production, Biochemical Properties, and Potential Application

    PubMed Central

    de Almeida, Alex Fernando; Carmona, Eleonora Cano

    2013-01-01

    Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (YL/S = 1.381 g/g), lipase yield (YL/S = 6.892 U/g), and biomass productivity (PX = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (YL/S) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties. PMID:24350270

  11. Cold-adapted organic solvent tolerant alkalophilic family I.3 lipase from an Antarctic Pseudomonas.

    PubMed

    Ganasen, Menega; Yaacob, Norhayati; Rahman, Raja Noor Zaliha Raja Abd; Leow, Adam Thean Chor; Basri, Mahiran; Salleh, Abu Bakar; Ali, Mohd Shukuri Mohamad

    2016-11-01

    Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.

  12. Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology.

    PubMed

    Gupta, Rani; Kumari, Arti; Syal, Poonam; Singh, Yogesh

    2015-01-01

    Lipase catalyzes hydrolysis of fats in lipid water interphase and perform variety of biotransformation reactions under micro aqueous conditions. The major sources include microbial lipases; among these yeast and fungal lipases are of special interest because they can carry out various stereoselective reactions. These lipases are highly diverse and are categorized into three classes on the basis of oxyanion hole: GX, GGGX and Y. The detailed phylogenetic analysis showed that GX family is more diverse than GGGX and Y family. Sequence and structural comparisons revealed that lipases are conserved only in the signature sequence region. Their characteristic structural determinants viz. lid, binding pocket and oxyanion hole are hotspots for mutagenesis. Few examples are cited in this review to highlight the multidisciplinary approaches for designing novel enzyme variants with improved thermo stability and substrate specificity. In addition, we present a brief account on biotechnological applications of lipases. Lipases have also gained attention as virulence factors, therefore, we surveyed the role of lipases in yeast physiology related to colonization, adhesion, biofilm formation and pathogenesis. The new genomic era has opened numerous possibilities to genetically manipulate lipases for food, fuel and pharmaceuticals.

  13. Formation of an ordered phase by ceramides and diacylglycerols in a fluid phosphatidylcholine bilayer--Correlation with structure and hydrogen bonding capacity.

    PubMed

    Ekman, Peik; Maula, Terhi; Yamaguchi, Shou; Yamamoto, Tetsuya; Nyholm, Thomas K M; Katsumura, Shigeo; Slotte, J Peter

    2015-10-01

    Ceramides and diacylglycerols are lipids with a large hydrophobic part (acyl chains and long-chain base) whereas their polar function (hydroxyl group) is small. They need colipids with large head groups to coexist in bilayer membranes. In this study, we have determined how saturated and unsaturated ceramides and acyl-chain matched diacylglycerols form ordered domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers as a function of bilayer concentration. The formation of ordered domains was determined from lifetime analysis of trans-parinaric acid. Ceramides formed ordered domains with equal average tPA lifetime at lower bilayer concentration when compared to acyl-chain matched diacylglycerols. This was true for both saturated (16:0) and mono-unsaturated (18:1) species. This finding suggested that hydrogen bonding among ceramides contributed to their more efficient ordered phase formation, since diacylglycerols do not form similar hydrogen bonding networks. The role of hydrogen bonding in ordered domain formation was further verified by using palmitoyl ceramide analogs with 2N and 3OH methylated long-chain bases. These analogs do not form hydrogen bonds from the 2NH or the 3OH, respectively. While methylation of the 3OH did not affect ordered phase formation compared to native palmitoyl ceramide, 2NH methylation markedly attenuated ceramide ordered phase formation. We conclude that in addition to acyl chain length, saturation, molecular order, and lack of large head group, also hydrogen bonding involving the 2NH is crucial for efficient formation of ceramide-rich domains in fluid phosphatidylcholine bilayers.

  14. Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport.

    PubMed

    Cleland, P J; Appleby, G J; Rattigan, S; Clark, M G

    1989-10-25

    Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.

  15. High-throughput screening method for lipases/esterases.

    PubMed

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

  16. Lipase-catalyzed hydrolysis of TG containing acetylenic FA.

    PubMed

    Jie, Marcel S F Lie Ken; Fua, Xun; Lau, Maureen M L; Chye, M L

    2002-10-01

    Hydrolysis of symmetrical acetylenic TG of type AAA [viz., glycerol tri-(4-decynoate), glycerol tri-(6-octadecynoate), glycerol tri-(9-octadecynoate), glycerol tri-(10-undecynoate), and glycerol tri-(13-docosynoate)] in the presence of eight microbial lipases was studied. Novozyme 435 (Candida antarctica), an efficient enzyme for esterification, showed a significant resistance in the hydrolysis of glycerol tri-(9-octadecynoate) and glycerol tri-(13-docosynoate). Hydrolysis of acetylenic TG with Lipolase 100T (Humicola lanuginosa) was rapidly accomplished. Lipase PS-D (Pseudomonas cepacia) showed a fair resistance toward the hydrolysis of glycerol tri-(6-octadecynoate) only, which reflected its ability to recognize the delta6 positional isomer of 18:1. Lipase CCL (Candida cylindracea, syn. C. rugosa) and AY-30 (C. rugosa) were able to catalyze the release of 10-undecynoic acid and 9-octadecynoic acid from the corresponding TG, but less readily the 13-docosynoic acid in the case of glycerol tri-(13-docosynoate). The two lipases CCL and AY-30 were able to distinguish the small difference in structure of fatty acyl moieties in the TG substrate. To confirm this trend, three regioisomers of mixed acetylenic TG of type ABC (containing one each of delta6, delta9, and delta13 acetylenic FA in various positions) were prepared and hydrolyzed with CCL and AY-40. The results reconfirmed the observation that AY-30 and CCL were able to distinguish the slight differences in the molecular structure (position of the acetylenic bond and chain length) of the acyl groups in the TG during the hydrolysis of such TG substrates.

  17. Iodine-125-labeled lipoprotein lipase as a tool to detect and study spontaneous lipolysis in bovine milk

    SciTech Connect

    Sundheim, G.; Bengtsson-Olivecrona, G.

    1986-07-01

    The distribution of lipoprotein lipase among cream, casein, and milk serum can be evaluated by addition of a trace amount of /sup 125/I-labeled lipoprotein lipase to milk. Radioactive lipase was distributed in parallel to endogenous lipase under several conditions. In some milk samples, binding of lipase to cream increased when the milk was cooled. Correlation was good between bound labeled lipase and degree of cold-induced lipolysis in corresponding milk samples. Binding of lipase to cream or to casein was not saturable by addition of two-to threefold more lipase than is normally present in milk. In milk with a relatively high fraction of lipase bound to cream, a correspondingly lower fraction was associated with casein, whereas the fraction of lipase in milk serum was similar in all milk samples. Cold-induced binding of lipoprotein lipase to cream was not fully reversed when the milk was warmed again. Heparin released lipase from casein and increased the amount of lipase bound to cream after cooling.

  18. Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice

    PubMed Central

    Schweiger, Martina; Romauch, Matthias; Schreiber, Renate; Grabner, Gernot F.; Hütter, Sabrina; Kotzbeck, Petra; Benedikt, Pia; Eichmann, Thomas O.; Yamada, Sohsuke; Knittelfelder, Oskar; Diwoky, Clemens; Doler, Carina; Mayer, Nicole; De Cecco, Werner; Breinbauer, Rolf; Zimmermann, Robert; Zechner, Rudolf

    2017-01-01

    Elevated circulating fatty acids (FAs) contribute to the development of obesity-associated metabolic complications such as insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD). Hence, reducing adipose tissue lipolysis to diminish the mobilization of FAs and lower their respective plasma concentrations represents a potential treatment strategy to counteract obesity-associated disorders. Here we show that specific inhibition of adipose triglyceride lipase (Atgl) with the chemical inhibitor, Atglistatin, effectively reduces adipose tissue lipolysis, weight gain, IR and NAFLD in mice fed a high-fat diet. Importantly, even long-term treatment does not lead to lipid accumulation in ectopic tissues such as the skeletal muscle or heart. Thus, the severe cardiac steatosis and cardiomyopathy that is observed in genetic models of Atgl deficiency does not occur in Atglistatin-treated mice. Our data validate the pharmacological inhibition of Atgl as a potentially powerful therapeutic strategy to treat obesity and associated metabolic disorders. PMID:28327588

  19. Specificity in lipases: A computational study of transesterification of sucrose

    PubMed Central

    Fuentes, Gloria; Ballesteros, Anthonio; Verma, Chandra S.

    2004-01-01

    Computational conformational searches of putative transition states of the reaction of sucrose with vinyl laurate catalyzed by lipases from Candida antarctica B and Thermomyces lanuginosus have been carried out. The dielectric of the media have been varied to understand the role of protein plasticity in modulating the observed regioselective transesterification. The binding pocket of lipase from Candida adapts to the conformational variability of the various substates of the substrates by small, local adjustments within the binding pocket. In contrast, the more constrained pocket of the lipase from Thermomyces adapts by adjusting through concerted global motions between subdomains. This leads to the identification of one large pocket in Candida that accommodates both the sucrose and the lauroyl moieties of the transition state, whereas in Thermomyces the binding pocket is smaller, leading to the localization of the two moieties in two distinct pockets; this partly rationalizes the broader specificity of the former relative to the latter. Mutations have been suggested to exploit the differences towards changing the observed selectivities. PMID:15557256

  20. Preliminary studies on immobilization of lipase using chicken eggshell

    NASA Astrophysics Data System (ADS)

    Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

    2016-06-01

    A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

  1. Isolation and characterization of novel thermophilic lipase-secreting bacteria.

    PubMed

    Rabbani, Mohammed; Bagherinejad, Mohammad Reza; Sadeghi, Hamid MirMohammad; Shariat, Ziaedin Samsam; Etemadifar, Zahra; Moazen, Fatemeh; Rahbari, Manizheh; Mafakher, Ladan; Zaghian, Saeideh

    2013-12-01

    The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

  2. Lipase assay in soils by copper soap colorimetry.

    PubMed

    Saisuburamaniyan, N; Krithika, L; Dileena, K P; Sivasubramanian, S; Puvanakrishnan, R

    2004-07-01

    A simple and sensitive method for the estimation of lipase activity in soils is reported. In this method, 50mg of soil is incubated with emulsified substrate, the fatty acids liberated are treated with cupric acetate-pyridine reagent, and the color developed is measured at 715 nm. Use of olive oil in this protocol leads to an estimation of true lipase activity in soils. The problem of released fatty acids getting adsorbed onto the soil colloids is obviated by the use of isooctane, and separate standards for different soils need not be developed. Among the various surfactants used for emulsification, polyvinyl alcohol is found to be the most effective. Incubation time of 20 min, soil concentration of 50 mg, pH 6.5, and incubation temperature of 37 degrees C were found to be the most suitable conditions for this assay. During the process of enrichment of the soils with oil, interference by the added oil is avoided by the maintenance of a suitable control, wherein 50 mg of soil is added after stopping the reaction. This assay is sensitive and it could be adopted to screen for lipase producers from enriched soils and oil-contaminated soils before resorting to isolation of the microbes by classical screening methods.

  3. Isolation and characterization of novel thermophilic lipase-secreting bacteria

    PubMed Central

    Rabbani, Mohammed; Bagherinejad, Mohammad Reza; Sadeghi, Hamid MirMohammad; Shariat, Ziaedin Samsam; Etemadifar, Zahra; Moazen, Fatemeh; Rahbari, Manizheh; Mafakher, Ladan; Zaghian, Saeideh

    2013-01-01

    The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered. PMID:24688500

  4. Synthesis of naringin 6"-ricinoleate using immobilized lipase

    PubMed Central

    2012-01-01

    Abstract Background Naringin is an important flavanone with several biological activities, including antioxidant action. However, this compound shows low solubility in lipophilic preparations, such as is used in the cosmetic and food industries. One way to solve this problem is to add fatty acids to the flavonoid sugar unit using immobilized lipase. However, there is limited research regarding hydroxylation of unsaturated fatty acids as an answer to the low solubility challenge. In this work, we describe the reaction of naringin with castor oil containing ricinoleic acid, castor oil's major fatty acid component, using immobilized lipase from Candida antarctica. Analysis of the 1H and 13 C NMR (1D and 2D) spectra and literature comparison were used to characterise the obtained acyl derivative. Results After allowing the reaction to continue for 120 hours (in acetone media, 50°C), the major product obtained was naringin 6″-ricinoleate. In this reaction, either castor oil or pure ricinoleic acid was used as the acylating agent, providing a 33% or 24% yield, respectively. The chemical structure of naringin 6″-ricinoleate was determined using NMR analysis, including bidimensional (2D) experiments. Conclusion Using immobilized lipase from C. antarctica, the best conversion reaction was observed using castor oil containing ricinoleic acid as the acylating agent rather than an isolated fatty acid. Graphical abstract PMID:22578215

  5. Accumulation of diacylglycerol in the liver membrane of the Long-Evans Cinnamon (LEC) rat with hepatitis: FT-IR spectroscopic and HPLC detection.

    PubMed

    Yoon, S; Kazusaka, A; Fujita, S

    2000-04-03

    Long-Evans Cinnamon (LEC) rats develop severe hepatitis and subsequent hepatoma with excess accumulation of copper in the liver with increasing age. Lipids extracted from the LEC rat liver membrane were studied using FT-IR spectroscopy and an HPLC technique at the stages of pre-hepatitis and hepatitis, i.e. at 10 and 16 weeks of age, respectively. The 10-week-old rats exhibited an IR spectrum characteristic of a phosphatidylcholine and phosphatidylethanolamine mixture with a ratio of 2:1. The 16-week-old rats developed new absorption bands at 1161 and 1070 cm(-1), which were assigned to the spectra of triglyceride, neutral lipid, and diacylglycerol, an endogenous activator of protein kinase C, respectively. The diacylglycerol was estimated to amount to ca. 10% (w/w) of phospholipid extract by comparing the spectrum with those of model compounds. This was confirmed using an HPLC assay. Previously, we found that a serum response factor is activated by copper in the LEC rat liver, and suggested that it must mediate proto-oncogene c-fos induction. The results obtained here suggest that accumulation of diacylglycerol plays an important role in development of hepatoma in LEC rats by mediating proto-oncogene c-fos induction.

  6. G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

    PubMed

    Violin, Jonathan D; Dewire, Scott M; Barnes, William G; Lefkowitz, Robert J

    2006-11-24

    Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

  7. Diacylglycerol kinase delta and protein kinase C(alpha) modulate epidermal growth factor receptor abundance and degradation through ubiquitin-specific protease 8.

    PubMed

    Cai, Jinjin; Crotty, Tracy M; Reichert, Ethan; Carraway, Kermit L; Stafforini, Diana M; Topham, Matthew K

    2010-03-05

    Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

  8. Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

    PubMed Central

    Bassegoda, Arnau; Pastor, F. I. Javier

    2012-01-01

    Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases. PMID:22226953

  9. Optimization of culture conditions for production of a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipases with abnormal properties such as thermo stability, alkalinity, acidity and cold-activity receive industrial attention because of their usability under restricted reaction conditions. Most microbial cold-active lipases originate from psychrotrophic and psychrophilic microorganisms found in An...

  10. Effect of poly(vinyl acetate-acrylamide) microspheres properties and steric hindrance on the immobilization of Candida rugosa lipase.

    PubMed

    Zhang, Dong-Hao; Yuwen, Li-Xia; Li, Chao; Li, Ya-Qiong

    2012-11-01

    Poly(vinyl acetate-acrylamide) microspheres were synthesized in the absence or presence of isooctane via suspension polymerization and utilized as carriers to immobilize Candida rugosa lipase. When the hydrophobic/hydrophilic surface characteristics of the microspheres were modified by changing the ratio of vinyl acetate (hydrophobic monomer) to acrylamide (hydrophilic monomer) from 50:50 to 86:24, the immobilization ratio changed from 45% to 92% and the activity of the immobilized lipase increased from 202.5 to 598.0 U/g microsphere. Excessive lipase loading caused intermolecular steric hindrance, which resulted in a decline in lipase activity. The maximum specific activity of the immobilized lipase (4.65 U/mg lipase) was higher than that of free lipase (3.00 U/mg lipase), indicating a high activity recovery during immobilization.

  11. New finding and optimal production of a novel extracellular alkaline lipase from Yarrowia lipolytica NRRL Y-2178

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipases are industrially and useful versatile enzymes that catalyze numerous different reactions including hydrolysis of triglycerides, transesterification, and chiral synthesis of esters under natural conditions. Although lipases from various sources have been widely used in industrial application...

  12. Adipose Triglyceride Lipase, Not Hormone-Sensitive Lipase, Is the Primary Lipolytic Enzyme in Fasting Elephant Seals (Mirounga angustirostris).

    PubMed

    Fowler, Melinda A; Costa, Daniel P; Crocker, Daniel E; Shen, Wen-Jun; Kraemer, Fredric B

    2015-01-01

    Little is known about the mechanisms that allow capital breeders to rapidly mobilize large amounts of body reserves. Northern elephant seals (Mirounga angustirostris) utilize fat reserves for maternal metabolism and to create high fat milk for the pup. Hormone-sensitive lipase (HSL) has been hypothesized to be an important lipolytic enzyme in fasting seals, but the activity of HSL and adipose triglyceride lipase (ATGL) has not been quantified in fasting adult seals, nor has their relationship to milk lipid content been assessed. Blubber and milk samples were obtained from 18 early lactation and 19 late lactation females, as well as blubber from five early and five late molting female seals. Blubber lipolytic activity was assessed with radiometric assays. HSL activity was negligible in seal blubber at all fasting stages. Total triglyceride lipase activity was stable among early and late lactation and early molt but increased in late molting seals. Relative abundance of ATGL protein increased across fasting, but neither activity nor relative protein levels were related to circulating nonesterified fatty acids or milk lipid content, suggesting the possibility of other regulatory pathways between lipolytic activity and milk lipid content. These results demonstrate that HSL is not the primary lipolytic enzyme in fasting adult female seals and that ATGL contributes more to lipolysis than HSL.

  13. Isolation and identification of a novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110

    PubMed Central

    Mobarak-Qamsari, E; Kasra-Kermanshahi, R; Moosavi-nejad, Z

    2011-01-01

    Background and Objectives Lipases are particularly important due to the fact that they specifically hydrolyze acyl glycerol, oils and greases, which is of great interest for different industrial applications. Materialst and Methods In this study, several lipase-producing bacteria were isolated from wastewater of an oil processing plant. The strain possessing the highest lipase activity was identified both biochemically and sequencing of 16S rRNA gene. Then we increase lipase activity by improving conditions of production medium. Also, lipase from this strain was preliminarily characterized for use in industrial application. Results The 16S rRNA sequensing revealed it as a new strain of Pseudomonas aeruginosa and the type strain was KM110. An overall 3-fold enhanced lipase production (0.76 U mL−1) was achieved after improving conditions of production medium. The olive oil and peptone was found to be the most suitable substrate for maximum enzyme production. Also the enzyme exhibited maximum lipolytic activity at 45°C where it was also stably maintained. At pH 8.0, the lipase had the highest stability but no activity. It was active over a pH range of 7.0–10.0. The lipase activity was inhibited by Zn2+ & Cu2+ (32 and 27%, respectively) at 1mM. The enzyme lost 29% of its initial activity in 1.0% SDS concentration, whereas, Triton X-100, Tween-80 & DMSO did not significantly inhibit lipase activity. Conclusions Based on the findings of present study, lipase of P. aeruginosa KM110 is a potential alkaline lipase and a candidate for industrial applications such as detergent, leather and fine chemical industries. PMID:22347589

  14. Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.

    SciTech Connect

    Qian, Zhen; Horton, John R.; Cheng, Xiadong; Lutz, Stefan; Emory

    2009-11-02

    Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283{Delta}7) in the apo-form determined at 1.49 {angstrom} resolution and with a bound phosphonate inhibitor at 1.69 {angstrom} resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.

  15. Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

    PubMed Central

    Westers, Helga; Braun, Peter G.; Westers, Lidia; Antelmann, Haike; Hecker, Michael; Jongbloed, Jan D. H.; Yoshikawa, Hirofumi; Tanaka, Teruo; van Dijl, Jan Maarten; Quax, Wim J.

    2005-01-01

    Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor. PMID:15812018

  16. Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma

    PubMed Central

    Zhang, Junyong; Liu, Zuojin; Lian, Zhengrong; Liao, Rui; Chen, Yi; Qin, Yi; Wang, Jinlong; Jiang, Qing; Wang, Xiaobo; Gong, Jianping

    2016-01-01

    Monoacylglycerol lipase (MAGL) is a key enzyme in lipid metabolism that is demonstrated to be involved in tumor progression through both energy supply of fatty acid (FA) oxidation and enhancing cancer cell malignance. The aim of this study was to investigate whether MAGL could be a potential therapeutic target and prognostic indicator for hepatocellular carcinoma (HCC). To evaluate the relationship between MAGL levels and clinical characteristics, a tissue microarray (TMA) of 353 human HCC samples was performed. MAGL levels in HCC samples were closely linked to the degree of malignancy and patient prognosis. RNA interference, specific pharmacological inhibitor JZL-184 and gene knock-in of MAGL were utilized to investigate the effects of MAGL on HCC cell proliferation, apoptosis, and invasion. MAGL played important roles in both proliferation and invasion of HCC cells through mechanisms that involved prostaglandin E2 (PGE2) and lysophosphatidic acid (LPA). JZL-184 administration significantly inhibited tumor growth in mice. Furthermore, we confirmed that promoter methylation of large tumor suppressor kinase 1 (LATS1) resulted in dysfunction of the Hippo signal pathway, which induced overexpression of MAGL in HCC. These results indicate that MAGL could be a potentially novel therapeutic target and prognostic indicator for HCC. PMID:27767105

  17. Self-assembly and lipid interactions of diacylglycerol lactone derivatives studied at the air/water interface.

    PubMed

    Philosof-Mazor, Liron; Volinsky, Roman; Comin, Maria J; Lewin, Nancy E; Kedei, Noemi; Blumberg, Peter M; Marquez, Victor E; Jelinek, Raz

    2008-10-07

    Synthetic diacylglycerol lactones (DAG-lactones) have been shown to be effective modulators of critical cellular signaling pathways. The biological activity of these amphiphilic molecules depends in part upon their lipid interactions within the cellular plasma membrane. This study explores the thermodynamic and structural features of DAG-lactone derivatives and their lipid interactions at the air/water interface. Surface-pressure/area isotherms and Brewster angle microscopy revealed the significance of specific side-groups attached to the terminus of a very rigid 4-(2-phenylethynyl)benzoyl chain of the DAG-lactones, which affected both the self-assembly of the molecules and their interactions with phospholipids. The experimental data highlight the formation of different phases within mixed DAG-lactone/phospholipid monolayers and underscore the relationship between the two components in binary mixtures of different mole ratios. Importantly, the results suggest that DAG-lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases that form, for example, by cholesterol. Moreover, the size and charge of the phospholipid headgroups do not seem to affect DAG-lactone interactions with lipids.

  18. Dissimilar effects of phorbol ester and diacylglycerol derivative on protein kinase activity in the monoblastoid U937 cell.

    PubMed

    Ways, D K; Dodd, R C; Earp, H S

    1987-07-01

    Mechanism, in addition to protein kinase C activation may mediate 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated differentiation of leukemic cells. We compared the effect of pretreating intact monoblastoid U937 cells with TPA or the diacylglycerol derivative, 1-oleoyl-2-acetylglycerol (OAG), by studying the protein kinase C dependent and independent histone phosphotransferase activity, the phosphorylation of endogenous substrates, and the ability to stimulate differentiation. In cellular fractions derived from cells treated with TPA or OAG, cytosolic protein kinase C activity decreased. In the detergent extracted particulate fraction, TPA produced a time and dose dependent decrease in protein kinase C activity. In contrast, OAG increased particulate protein kinase C activity. In addition, the particulate fraction derived from cells treated with TPA exhibited increased phosphatidyl serine and diolein independent histone phosphotransferase activity as well as an increase in the phosphorylation of two endogenous substrates with molecular weights of 120,000 and 80,000. OAG did not mimic these effects. When exposed to 32P-labeled intact cells, OAG and TPA stimulated phosphorylation of three substrates. Thus, the inability of OAG to mimic the effects of TPA was not due to lack of protein kinase C activation. TPA, but not OAG, stimulated differentiation of the U937 cell to a monocyte-like cell. These data demonstrate that TPA and OAG have dissimilar effects on protein kinase activity and differentiation in the U937 monoblastoid cell.

  19. Characterization of a New Trioxilin and a Sulfoquinovosyl Diacylglycerol with Anti-Inflammatory Properties from the Dinoflagellate Oxyrrhis marina

    PubMed Central

    Yoon, Eun Young; Yang, A. Reum; Park, Jaeyeon; Moon, Seung Joo; Jeong, Eun Ju; Rho, Jung-Rae

    2017-01-01

    Two new compounds—a trioxilin and a sulfoquinovosyl diacylglycerol (SQDG)—were isolated from the methanolic extract of the heterotrophic dinoflagellate Oxyrrhis marina cultivated by feeding on dried yeasts. The trioxilin was identified as (4Z,8E,13Z,16Z,19Z) -7(S),10(S),11(S)-trihydroxydocosapentaenoic acid (1), and the SQDG was identified as (2S)-1-O-hexadecanosy-2-O-docosahexaenoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol (2) by a combination of nuclear magnetic resonance (NMR) spectra, mass analyses, and chemical reactions. The two compounds were associated with docosahexaenoic acid, which is a major component of O. marina. The two isolated compounds showed significant nitric oxide inhibitory activity on lipopolysaccharide-induced RAW264.7 cells. Compound 2 showed no cytotoxicity against hepatocarcinoma (HepG2), neuroblastoma (Neuro-2a), and colon cancer (HCT-116) cells, while weak cytotoxicity was observed for compound 1 against Neuro-2a cells. PMID:28264430

  20. Suppressed histamine release from rat peritoneal mast cells by ultraviolet B irradiation: decreased diacylglycerol formation as a possible mechanism

    SciTech Connect

    Danno, K.; Fujii, K.; Tachibana, T.; Toda, K.; Horio, T.

    1988-06-01

    This study was designed to investigate the effect of ultraviolet B (UVB) irradiation on mast cell functions. Purified mast cells obtained from rat peritoneal cavity were irradiated with UVB and subsequently exposed to a degranulator, compound 48/80, or the calcium ionophore A-23187. The amount of histamine released from mast cells measured by the enzyme isotopic assay was significantly decreased by UVB irradiation (100-400 mJ/cm2). Within this dose range, UVB alone was not cytotoxic to the cells because it did not induce histamine release. The suppression was observed when mast cells were subjected to degranulation without intervals after UVB irradiation, and even after 5 h postirradiation. The wavelength of 300 nm from a monochromatic light source showed the maximum effect. When mast cells prelabeled with (/sup 3/H)arachidonate were irradiated and challenged by compound 48/80, label accumulation in diacylglycerol produced by the phosphatidylinositol cycle was considerably decreased by UVB irradiation. From these results, we hypothesize that, within an adequate irradiation dose, UVB irradiation suppresses histamine release from mast cells, probably by causing noncytotoxic damage to the membrane phospholipid metabolism, which is tied to the degranulation mechanisms.

  1. C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia.

    PubMed

    Behler-Janbeck, Friederike; Takano, Tomotsugu; Maus, Regina; Stolper, Jennifer; Jonigk, Danny; Tort Tarrés, Meritxell; Fuehner, Thomas; Prasse, Antje; Welte, Tobias; Timmer, Mattie S M; Stocker, Bridget L; Nakanishi, Yoichi; Miyamoto, Tomofumi; Yamasaki, Sho; Maus, Ulrich A

    2016-12-01

    Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

  2. End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

    PubMed Central

    Ibarguren, Maitane; Bomans, Paul H. H.; Frederik, Peter M.; Stonehouse, Martin; Vasil, Michael L.; Alonso, Alicia; Goñi, Félix M.

    2009-01-01

    A phospholipase C/ sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol, at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230–3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme. PMID:19891956

  3. Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity.

    PubMed Central

    Katavic, V; Reed, D W; Taylor, D C; Giblin, E M; Barton, D L; Zou, J; Mackenzie, S L; Covello, P S; Kunst, L

    1995-01-01

    In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids. PMID:7784510

  4. Anti-inflammatory potential of monogalactosyl diacylglycerols and a monoacylglycerol from the edible brown seaweed Fucus spiralis Linnaeus.

    PubMed

    Lopes, Graciliana; Daletos, Georgios; Proksch, Peter; Andrade, Paula B; Valentão, Patrícia

    2014-03-11

    A monoacylglycerol (1) and a 1:1 mixture of two monogalactosyl diacylglycerols (MGDGs) (2 and 3) were isolated from the brown seaweed Fucus spiralis Linnaeus. The structures were elucidated by spectroscopic means (NMR and MS) and by comparison with the literature. Compound 1 was composed of a glycerol moiety linked to oleic acid (C18:1 Ω9). Compounds 2 and 3 contained a glycerol moiety linked to a galactose unit and eicosapentaenoic acid (C20:5 Ω3) combined with octadecatetraenoic acid (C18:4 Ω3) or linolenic acid (C18:3 Ω3), respectively. The isolated compounds were tested for their cytotoxic and anti-inflammatory activity in RAW 264.7 macrophage cells. All of them inhibited NO production at non-cytotoxic concentrations. The fraction consisting of compounds 2 and 3, in a ratio of 1:1, was slightly more effective than compound 1 (IC₅₀ of 60.06 and 65.70 µg/mL, respectively). To our knowledge, this is the first report of these compounds from F. spiralis and on their anti-inflammatory capacity.

  5. Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments.

    PubMed

    Lanfranconi, Mariana P; Alvarez, Adrián F; Alvarez, Héctor M

    2015-12-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest.

  6. C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia

    PubMed Central

    Behler-Janbeck, Friederike; Maus, Regina; Stolper, Jennifer; Jonigk, Danny; Fuehner, Thomas; Prasse, Antje; Welte, Tobias; Stocker, Bridget L.; Nakanishi, Yoichi; Miyamoto, Tomofumi; Yamasaki, Sho; Maus, Ulrich A.

    2016-01-01

    Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae. PMID:27923071

  7. COORDINATED ACTIVATION OF THE RAC-GAP β2-CHIMAERIN BY AN ATYPICAL PROLINE-RICH DOMAIN AND DIACYLGLYCEROL

    PubMed Central

    Gutierrez-Uzquiza, Alvaro; Colon-Gonzalez, Francheska; Leonard, Thomas A.; Canagarajah, Bertram J.; Wang, HongBin; Mayer, Bruce J.; Hurley, James H.; Kazanietz, Marcelo G.

    2013-01-01

    Chimaerins, a family of GTPase activating proteins (GAPs) for the small G-protein Rac, have been implicated in development, neuritogenesis, and cancer. These Rac-GAPs are regulated by the lipid second messenger diacylglycerol (DAG) generated by tyrosine-kinases such as the epidermal growth factor receptor (EGFR). Here we identify an atypical Pro-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. EGF promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a DAG-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal Pro-rich region. PMID:23673634

  8. Citrus flavonoid, naringenin, increases locomotor activity and reduces diacylglycerol accumulation in skeletal muscle of obese ovariectomized mice

    PubMed Central

    Ke, Jia-Yu; Cole, Rachel M; Hamad, Essam M; Hsiao, Yung-Hsuan; Cotten, Bradley M; Powell, Kimerly A; Belury, Martha A

    2016-01-01

    Scope Estrogen deficiency has been associated with central obesity, muscle loss, and metabolic syndrome in postmenopausal women. This study assessed naringenin accumulation in tissues and investigated the hypothesis that naringenin reverses diet-induced metabolic disturbances in obese ovariectomized mice. Methods and results In study 1, we measured naringenin concentrations in plasma, liver, perigonadal and subcutaneous adipose tissues, and muscle of ovariectomized C57BL/6J female mice after 11 weeks of naringenin supplementation. Naringenin accumulated 5–12 times more in mice fed a 3% naringenin diet than in mice fed a 1% naringenin diet. In study 2, ovariectomized mice were fed a high-fat diet (60 kcal% fat) for 11 weeks and half of the mice were then supplemented with 3% naringenin for another 11 weeks. Dietary naringenin suppressed weight gain, lowered hyperglycemia, and decreased intra-abdominal adiposity evaluated by magnetic resonance imaging. Naringenin-fed mice exhibited elevated locomotor activity monitored by infrared beam breaks, maintained muscle mass, and reduced muscle diacylglycerol content. Real-time PCR analysis in muscle revealed decreased mRNA level for genes involved in de novo lipogenesis, lipolysis, and triglyceride synthesis/storage. Conclusions Long-term 3% naringenin supplementation resulted in significant naringenin accumulation in plasma and tissues, associated with attenuated metabolic dysregulation and muscle loss in obese ovariectomized mice. PMID:26573879

  9. In silico characterization of 1,2-diacylglycerol cholinephosphotransferase and lysophospha-tidylcholine acyltransferase genes in Glycine max L. Merrill.

    PubMed

    Sousa, C S; Barros, B A; Barh, D; Ghosh, P; Azevedo, V; Barros, E G; Moreira, M A

    2016-08-26

    The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.

  10. Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase.

    PubMed

    Rani, Sapa Hima; Krishna, T H Anantha; Saha, Saikat; Negi, Arvind Singh; Rajasekharan, Ram

    2010-12-03

    A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

  11. Effects of antibodies and glycerol as potential inhibitors of ruminal lipase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ruminant-derived foods contain high proportions of saturated fats, a result of ruminal biohydrogenation, which rapidly saturates and thus limits the availability of free unsaturated fatty acids for assimilation. Strategies to enrich ruminant-derived foods with unsaturated fatty acids are desired as...

  12. Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

    PubMed Central

    2013-01-01

    Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme’s function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors. PMID:23731016

  13. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  14. Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability.

    PubMed

    Chen, Guan-Jie; Kuo, Chia-Hung; Chen, Chih-I; Yu, Chung-Cheng; Shieh, Chwen-Jen; Liu, Yung-Chuan

    2012-02-01

    In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization.

  15. The Effect of Storage at Three Different Temperatures on the Activity of Lipase Solution.

    ERIC Educational Resources Information Center

    Bradley, Karen; Mathewman, David

    1984-01-01

    Presented are procedures used to assay the activity of lipase during storage at three different temperatures. Since lipase solutions can decay even when refrigerated, it is recommended that the enzyme be freshly prepared prior to laboratory sessions in which they are used. (JN)

  16. Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013.

    PubMed

    Brabcová, Jana; Zarevúcka, Marie; Macková, Martina

    2010-12-01

    The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell-bound). Both enzymes were tested for their hydrolytic ability to p-nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium- (C12) and long- (C18) chain fatty acids were used as substrates. Cell-bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p-nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p-nitrophenyl decanoate > p-nitrophenyl caprylate > p-nitrophenyl laurate > p-nitrophenyl palmitate > p-nitrophenyl stearate. The cell-bound lipase indicates preference for p-nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell-bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K(m) ) and maximum reaction rate (V(max) ) for p-nitrophenyl palmitate hydrolysis of cell-bound lipase were significantly higher (K(m) 2.462 mM and V(max) 0.210 U/g/min) than those of extracellular lipase (K(m) 0.406 mM and V(max) 0.006 U/g/min).

  17. [Cloning and expression of organic solvent tolerant lipase gene from Staphylococcus saprophyticus M36].

    PubMed

    Tang, Yanchong; Lu, Yaping; Lü, Fengxia; Bie, Xiaomei; Guo, Yao; Lu, Zhaoxin

    2009-12-01

    Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25 degrees C for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.

  18. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  19. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  20. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme... animals. (c) The enzyme is produced by a process which completely removes the organism Mucor miehei...

  1. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  2. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  3. Coconut oil induced production of a surfactant-compatible lipase from Aspergillus tamarii under submerged fermentation.

    PubMed

    Das, Arijit; Bhattacharya, Sourav; Shivakumar, Srividya; Shakya, Sujina; Sogane, Swathi Shankar

    2017-02-01

    Filamentous fungi are efficient producers of lipases. The present study focuses on identification of a potent lipolytic fungus and enhancement of lipase production through optimization of nutritional and cultural conditions under submerged fermentation. Molecular characterization of the fungus by 18S rDNA sequencing revealed its identity as Aspergillus tamarii with 98% homology. Maximum lipase production was noted in mineral salts medium supplemented with coconut oil (2.5%, v/v). A combination of ammonium chloride (2%, w/v) and tryptone (2%, w/v) facilitated maximum lipase production at pH 5 of the production medium. A carbon: nitrogen ratio of 1:4 led to significant (p < 0.00008) increase in the enzyme production in the presence of surfactant cetyltrimethylammonium bromide (0.5%, w/v). Maximum lipase activity (2,32,500 ± 192 U/ml/min) was recorded after 7 days of incubation at 25 °C on a rotary shaker at 120 rpm. A 9.8-fold increase in lipase activity was recorded after optimization of the process parameters. Addition of crude lipase enhanced the oil stain removal activity of a commercially available detergent by 2.2-fold. The current findings suggest the potentiality of this fungal lipase to be used in detergent formulation.

  4. Kinetics of Detergent-Induced Activation and Inhibition of a Minimal Lipase.

    PubMed

    Kübler, Daniel; Bergmann, Anna; Weger, Lukas; Ingenbosch, Kim N; Hoffmann-Jacobsen, Kerstin

    2017-02-16

    Detergents are commonly applied in lipase assays to solubilize sparingly soluble model substrates. However, detergents affect lipases as well as substrates in multiple ways. The effect of detergents on lipase activity is commonly attributed to conformational changes in the lid region. This study deals with the effect of the nonionic detergent, poly(ethylene glycol) dodecyl ether, on a lipase that does not contain a lid sequence, lipase A from Bacillus subtilis (BSLA). We show that BSLA activity depends strongly on the detergent concentration and the dependency profile changes with pH. The interaction of BSLA with detergent monomers and micelles is studied using fluorescence correlation spectroscopy, time-resolved anisotropy decay, and temperature-induced unfolding. Detergent-dependent hydrolysis kinetics of two different substrates at two pH values are fitted with a microkinetic model. This analysis shows that the mechanism of interfacial lipase catalysis is strongly affected by the detergent. It reveals an activation mechanism by monomeric detergent that does not result from structural changes of the lipase. Instead, we propose that interfacial diffusion of the lipase is enhanced by detergent binding.

  5. Cloning, sequencing and characterization of lipase genes from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by PCR-based genome walking. Sequence analyses showed a putative Lip gene-product (...

  6. Enhancing activity and stability of Burkholderia cepacia lipase by immobilization on surface-functionalized mesoporous silicates.

    PubMed

    Kato, Katsuya; Seelan, Sindhu

    2010-06-01

    Burkholderia cepacia lipase was immobilized on various types of phenyl-functionalized mesoporous silicates (MPS). MPS, anchored with a phenyl group on the silica wall and with three dimensional (3D) mesoporosity, showed highest lipase adsorption capacity and best activities both in aqueous and organic reagents.

  7. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  8. Choline acetate enhanced the catalytic performance of Candida rogusa lipase in AOT reverse micelles.

    PubMed

    Xue, Luyan; Zhao, Yin; Yu, Lijie; Sun, Yanwen; Yan, Keqian; Li, Ying; Huang, Xirong; Qu, Yinbo

    2013-05-01

    Choline acetate is an ionic liquid composed of a kosmotropic anion and a chaotropic cation. According to Hofmeister series, a kosmotropic anion and/or a chaotropic cation could stabilize an enzyme, thereby facilitating the retention of the catalytic activity of the enzyme. In this work, we first report the influence of choline acetate on the activity and stability of lipase in AOT/water/isooctane reverse micelles. The indicator reaction is the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate. The results show that a low level of choline acetate does not affect the microstructure of the AOT reverse micelles, but the ionic liquid can improve the catalytic efficiency of lipase. Fluorescence spectra show that a high level of choline acetate has an impact on the conformation of lipase, so the activation is mainly due to the influence of choline acetate on the nucleophilicity of water. Infrared spectra demonstrate that choline acetate can form stronger hydrogen bonds with water surrounding lipase, and therefore enhance the nucleophilicity of the water, which makes it easier to attack the acyl enzyme intermediate, thereby increasing the activity of the lipase-catalyzed hydrolysis of the ester. A study on the stability of lipase in AOT reverse micelles indicates that the ionic liquid is able to maintain the activity of lipase to a certain extent. The effect of choline acetate is consistent with that predicted based on Hofmeister series.

  9. Evaluation of a new lipase from Staphylococcus sp. for detergent additive capability.

    PubMed

    Chauhan, Mamta; Chauhan, Rajinder Singh; Garlapati, Vijay Kumar

    2013-01-01

    Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase.

  10. Development of a lipase fermentation process that uses a recombinant Pseudomonas alc