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Journal of Dairy Science Jun 2021Dairy fat intake has been considered as a risk factor for cardiovascular disease. Rodent models show that trans fatty acids in industrial hydrogenated oil and ruminant...
Trans triacylglycerols from dairy products and industrial hydrogenated oil exhibit different effects on the function of human umbilical vein endothelial cells via modulating phospholipase A2/arachidonic acid metabolism pathways.
Dairy fat intake has been considered as a risk factor for cardiovascular disease. Rodent models show that trans fatty acids in industrial hydrogenated oil and ruminant milk have different effects on cardiovascular diseases. One of the main reasons is that the distributions of trans fatty acids in triacylglycerols from dairy products and from industrial hydrogenated oil are different, which affects lipid absorption and metabolism. This study investigated the effects of 1,3-olein-2-elaidin (OEO, representing industrial hydrogenated oil triacylglycerols) and 1-vaccenic-2,3-olein (OOV, representing ruminant triacylglycerols in dairy products) on the function of human umbilical vein endothelial cells (HUVEC), including cell viability, lactate dehydrogenase (LDH) exudation rate, and nitric oxide secretory and nitric oxide synthase relative activity. We found that the detrimental effect of OEO on HUVEC was significantly greater than that of OOV. The results also showed that the absorption rate of OEO in HUVEC (78.25%) was significantly greater than that of OOV (63.32%). Mechanistically, based on phospholipidomics analysis, we found that calcium-independent phospholipase A2 (iPLA2) played a key role with regard to the OOV-mediated arachidonic acid (ARA)/COX-2/PG pathway, whereas secretory phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) are responsible for the OEO-mediated ARA/COX-2/PG pathway. Moreover, OEO had a greater effect on the protein expression of COX-2 and PG secretion than OOV. In addition, iPLA2, sPLA2, and cPLA2 could mediate the ARA/CYP4A11 pathway in OOV-treated HUVEC, but only iPLA2 could mediate this pathway in HUVEC treated with OEO. We also found that sPLA2 could mediate the ARA/5-LOX pathway in HUVEC treated with OOV, but none of these 3 forms of PLA2 could mediate this pathway in HUVEC treated with OEO. On the other hand, after OOV treatment, trans-11 C18:1 was converted to beneficial forms of fatty acids in HUVEC, including conjugated linoleic acid (CLA) and trans-9 C16:1. In conclusion, we elucidated the potential mechanisms that might account for the diverse effects of triacylglycerols from industrial hydrogenated oil and ruminant milk on the function of HUVEC.
Topics: Animals; Arachidonic Acids; Human Umbilical Vein Endothelial Cells; Phospholipases A2; Phospholipases A2, Cytosolic; Triglycerides
PubMed: 33773784
DOI: 10.3168/jds.2020-19715 -
The Journal of Biological Chemistry Feb 1978
Topics: Colloids; Elapid Venoms; Micelles; Phospholipases; Substrate Specificity
PubMed: 624729
DOI: No ID Found -
Autophagy Sep 2021All membrane-bound organelles are degraded during the terminal differentiation of lens fiber cells. How these organelles are degraded has been a long-standing question...
All membrane-bound organelles are degraded during the terminal differentiation of lens fiber cells. How these organelles are degraded has been a long-standing question in biology. We recently revealed that PLAAT (phospholipase A and acyltransferase)-family phospholipases degrade organelles in the lens independently of macroautophagy. Here, we discuss the mechanism and physiological relevance of this new mode of intracellular degradation.
Topics: Autophagy; Cytosol; Lens, Crystalline; Organelles; Phospholipases
PubMed: 34233574
DOI: 10.1080/15548627.2021.1950372 -
Journal of Bacteriology Dec 1988From a genomic library of Serratia liquefaciens, a cloned DNA fragment comprising a two-gene operon was isolated and expressed in Escherichia coli. One of the gene...
From a genomic library of Serratia liquefaciens, a cloned DNA fragment comprising a two-gene operon was isolated and expressed in Escherichia coli. One of the gene products was identified as a phospholipase A1, and the enzyme was found to be excreted to the outer environment from S. liquefaciens as well as from E. coli. Both genes were sequenced, and the relationship between open reading frames in the DNA sequence and in vitro-expressed polypeptides was established. The length of the phospholipase polypeptide was found to be 319 amino acids. In the amino-terminal end of the coding sequence was a stretch of about 20 hydrophobic amino acids, but, in contrast to consensus signal peptides, no basic residues were present. The length of the second polypeptide was 227 amino acids. It was found that expression of the phospholipase gene in both E. coli and S. liquefaciens was growth phase regulated (late expression).
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Escherichia coli; Genes; Genes, Bacterial; Molecular Sequence Data; Operon; Phospholipases; Phospholipases A; Phospholipases A1; Plasmids; Serratia
PubMed: 3056919
DOI: 10.1128/jb.170.12.5855-5862.1988 -
The Journal of Biological Chemistry Jun 1985Two novel phospholipase activities have been identified in the cytosolic fraction of canine myocardium. Neutral active phospholipase C activity was partially purified by...
Two novel phospholipase activities have been identified in the cytosolic fraction of canine myocardium. Neutral active phospholipase C activity was partially purified by anion exchange, hydroxylapatite, chromatofocusing, and gel filtration chromatographies. The partially purified enzyme had similar maximum velocities (237 versus 241 nmol/mg X h) and apparent Michaelis constants (20 versus 14 microM) utilizing either plasmenylcholine or phosphatidylcholine as substrate. Myocardial phospholipase C had a pH optimum between 7 and 8, required divalent cations for maximal activity, and did not hydrolyze phosphatidylinositol or sphingomyelin. Myocardial cytosol contained a potent inhibitor of phospholipase C which masked enzymic activity until it was removed during the purification procedure. A plasmalogen selective phospholipase A2 activity was also identified in the cytosolic fraction of canine myocardium. The protein catalyzing this activity was partially purified by DEAE-Sephacel-hydroxylapatite tandem chromatography and exhibited a maximum velocity of 5 nmol/mg X h for plasmenylcholine but only 1 nmol/mg X h for phosphatidylcholine, had a pH optimum between 6 and 7 for both substrates, and did not require calcium ion for activity. These results constitute the first demonstration of a neutral active phospholipase C specific for choline and ethanolamine glycerophospholipids and a plasmalogen selective phospholipase A2 in mammalian tissue.
Topics: Animals; Cell Fractionation; Cytosol; Dogs; Indicators and Reagents; Isoenzymes; Kinetics; Lysophospholipids; Microsomes; Myocardium; Phosphatidylcholines; Phospholipases; Phospholipases A; Phospholipases A2; Plasmalogens; Substrate Specificity; Type C Phospholipases
PubMed: 3997869
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2021Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated...
Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.
Topics: Enzyme Assays; Fluorescein-5-isothiocyanate; Hydrolysis; Isoenzymes; Lipid Metabolism; Lipids; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C gamma; Protein Binding; Type C Phospholipases
PubMed: 33481244
DOI: 10.1007/978-1-0716-1142-5_17 -
European Journal of Biochemistry Dec 19891. Five increasingly anionic variants (Pa1-Pa5) of Ca2+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila...
Purification and characterization of five variants of phospholipase A2 and complete primary structure of the main phospholipase A2 variant in Heloderma suspectum (Gila monster) venom.
1. Five increasingly anionic variants (Pa1-Pa5) of Ca2+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila monster). The purification procedure was based on semi-preparative reverse-phase HPLC followed by anion-exchange HPLC and analytical reverse-phase HPLC. 2. Their Mr were 17,000-18,000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 greater than Pa5 greater than Pa4 greater than Pa1 greater than Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase A2 type and were lost upon p-bromophenacyl bromide treatment. 3. All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa2 greater than Pa1 approximately equal to Pa4 greater than Pa3 approximately equal to Pa5. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4. The five variants, Pa1-Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Pa2 and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N-terminal amino acid sequence (31 residues) of Pa1-Pa5 was exactly the same. 5. The full sequence of the major variant, Pa5, showed that this 142-amino-acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class II secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa5 displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half-cystine residues only, an incomplete N-terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C-terminal extension never seen in other phospholipases A2, with the only exception being bee venom.
Topics: Amino Acid Sequence; Amylases; Animals; Antibody Formation; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Lizards; Phosphatidylcholines; Phospholipases; Phospholipases A; Phospholipases A2; Protein Conformation; Rabbits; Substrate Specificity; Venoms
PubMed: 2480893
DOI: 10.1111/j.1432-1033.1989.tb15172.x -
The Journal of Biological Chemistry Jun 1990Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable...
Purification and characterization of canine myocardial cytosolic phospholipase A2. A calcium-independent phospholipase with absolute f1-2 regiospecificity for diradyl glycerophospholipids.
Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable phospholipase A2 activity in myocardial homogenates (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). We now report the 154,000-fold purification of this phospholipase A2 to homogeneity through utilization of sequential anion exchange, chromatofocusing, affinity, Mono Q, and hydroxylapatite chromatographies. The purified enzyme had a molecular mass of 40 kDa, possessed a specific activity of 227 mumol/mg min, had a pH optimum of 6.4, and catalyzed the regiospecific cleavage of the sn-2 fatty acid from diradyl glycerophospholipids. The purified polypeptide was remarkable for its ability to selectively hydrolyze plasmenylcholine in homogeneous vesicles (subclass rank order: plasmenylcholine greater than alkyl-ether choline glycerophospholipid greater than phosphatidylcholine) as well as in mixed bilayers comprised of equimolar plasmenylcholine/phosphatidylcholine. Purified myocardial phospholipase A2 also possessed selectivity for hydrolysis of phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic or palmitic acid. Taken together, these results constitute the first purification of a calcium-independent phospholipase with absolute regiospecificity for cleavage of the sn-2 acyl linkage in diradyl glycerophospholipids and demonstrate that myocardial phospholipase A2 has kinetic characteristics which are anticipated to result in the selective hydrolysis of sarcolemmal phospholipids during myocardial ischemia.
Topics: Animals; Calcium; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cytosol; Dogs; Kinetics; Myocardium; Phospholipases; Phospholipases A; Phospholipases A2; Phospholipids; Substrate Specificity
PubMed: 2355013
DOI: No ID Found -
The Journal of Antibiotics Jul 2000A series of novel inhibitors of lipoprotein associated phospholipase A2 were isolated from the culture broths of Pseudomonas fluorescens strain DSM11579. The inhibitors...
SB-253514 and analogues: novel inhibitors of lipoprotein associated phospholipase A2 produced by Pseudomonas fluorescens DSM 11579. II. Physico-chemical properties and structure elucidation.
A series of novel inhibitors of lipoprotein associated phospholipase A2 were isolated from the culture broths of Pseudomonas fluorescens strain DSM11579. The inhibitors fall into two structurally isomeric classes each of which comprise compounds incorporating glycosylated hydrocarbon chains. The structure elucidation for the major member of each structural class is reported. The crystal structure of a non-glycosylated analogue of the 5,5-series, produced through biotransformation, is also reported.
Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Bridged Bicyclo Compounds, Heterocyclic; Crystallography, X-Ray; Enzyme Inhibitors; Magnetic Resonance Spectroscopy; Molecular Structure; Phospholipases A; Phospholipases A2; Pseudomonas fluorescens; Pyrans
PubMed: 10994808
DOI: No ID Found -
Journal of the American Chemical Society Mar 2010Phospholipases are a large and diverse set of enzymes that metabolize the phospholipid components of cell membranes and function in key lipid-signaling pathways. The...
Phospholipases are a large and diverse set of enzymes that metabolize the phospholipid components of cell membranes and function in key lipid-signaling pathways. The molecular characterization of novel phospholipases would benefit from chemical probes that selectively target these enzymes on the basis of their distinct substrate specificities and catalytic properties. Here we present the synthesis and characterization of a set of activity-based protein profiling (ABPP) probes that contain key recognition and reactivity elements for targeting phospholipases of the serine hydrolase superfamily. We show that these probes accurately report on the sn-1 and sn-2 substrate specificities of phospholipases in cell and tissue proteomes, including the sn-1-selective phospholipase DDHD1 and a calcium-dependent transacylase activity implicated in endocannabinoid biosynthesis. We anticipate that these phospholipase-directed ABPP probes will facilitate the discovery of new lipid-metabolizing enzymes and provide valuable insights into their substrate preferences.
Topics: Alkynes; Animals; Mice; Organophosphonates; Phospholipases; Proteome; Rats; Substrate Specificity
PubMed: 20178358
DOI: 10.1021/ja1000505