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The Journal of Biological Chemistry Jun 1989Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified... (Comparative Study)
Comparative Study
Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.
Topics: Amino Acid Sequence; Animals; Chromatography, Affinity; Chromatography, Gel; Liver; Microsomes, Liver; Molecular Sequence Data; Organ Specificity; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Species Specificity; Subcellular Fractions; Swine
PubMed: 2722857
DOI: No ID Found -
Journal of Biochemistry Sep 1989During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption...
During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, beta,gamma-methylene adenosine 5'-triphosphate, prevented both the release of Ca2+ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca2+ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A2 suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca2+, which activate phospholipase A2, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H+ of the inner membrane. The results also indicate another event; activation of phospholipase A2 by release Ca2+ which results in H+ leakiness of the inner membrane.
Topics: Animals; Calcium; Enzyme Activation; Hypoxia; Intracellular Membranes; Male; Mitochondria, Liver; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains
PubMed: 2606905
DOI: 10.1093/oxfordjournals.jbchem.a122887 -
The Plant Journal : For Cell and... Apr 2021Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids....
Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids. Arabidopsis NPC2 and NPC6 are essential in gametogenesis, but their underlying role in the lipid remodeling remains elusive. Here, we show that these NPCs are required for triacylglycerol (TAG) production in pollen tube growth. NPC2 and NPC6 are highly expressed in developing pollen tubes and are localized at the endoplasmic reticulum. Mutants of NPC2 and NPC6 showed reduced rate of pollen germination, length of pollen tube and amount of lipid droplets (LDs). Overexpression of NPC2 or NPC6 induced LD accumulation, which suggests that these NPCs are involved in LD production. Furthermore, mutants defective in the biosynthesis of TAG, a major component of LDs, showed defective pollen tube growth. These results suggest that NPC2 and NPC6 are essential in gametogenesis for a role in hydrolyzing phospholipids and producing TAG required for pollen tube growth. Thus, lipid remodeling from phospholipids to TAG during pollen tube growth represents an emerging role for the NPC family in plant developmental control.
Topics: Arabidopsis; Arabidopsis Proteins; Flowers; Phospholipases; Pollen Tube; Triglycerides; Type C Phospholipases
PubMed: 33506578
DOI: 10.1111/tpj.15172 -
Journal of Medicinal Chemistry Jan 2021-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of -acylethanolamines (NAEs), a family of...
-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of -acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported -(cyclopropylmethyl)-6-(()-3-hydroxypyrrolidin-1-yl)-2-(()-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (, ) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [Mock , 2020, 16, 667-675]. Here, we describe the structure-activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of . A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an -methylphenethylamine group by replacement with an ()-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an ()-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording as a nanomolar potent inhibitor with drug-like properties. is a suitable pharmacological tool compound to investigate NAPE-PLD function and .
Topics: Amides; Carboxylic Acids; Enzyme Inhibitors; Phosphatidylethanolamines; Phospholipases; Pyrimidines; Structure-Activity Relationship
PubMed: 33382264
DOI: 10.1021/acs.jmedchem.0c01441 -
FEBS Letters Jun 1989Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C...
Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.
Topics: Amnion; Antiviral Agents; Cells, Cultured; Enzyme Activation; Fatty Acids; Humans; Interferon Type I; Neomycin; Phospholipases; Phospholipases A; Phospholipases A2; Phospholipids; Protein Kinase C; Quinacrine; Type C Phospholipases; Vesicular stomatitis Indiana virus; Virus Replication
PubMed: 2544450
DOI: 10.1016/0014-5793(89)80635-0 -
FEBS Letters Jul 2004In eukaryotes some surface proteins are attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. A GPI-specific phospholipase D (GPI-PLD) activity...
In eukaryotes some surface proteins are attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. A GPI-specific phospholipase D (GPI-PLD) activity has been characterized and implicated in the regulation of anchoring, thereby influencing the dispersal of anchored proteins or their maintenance on the cell surface, and possibly in cell signalling. Despite its biological and medical importance, little is known of the structure of GPI-PLD. Here, a distant relationship between the catalytic domains of GPI-PLD and some bacterial phospholipases C is demonstrated. A model of the GPI-PLD catalytic site sheds light on catalysis and highlights possibilities for design of improved and more specific GPI-PLD inhibitors. The databases contain hitherto unnoticed close homologues of GPI-PLD from yeast and Dictyostelium discoideum.
Topics: Amino Acid Sequence; Animals; Bacteria; Biological Evolution; Conserved Sequence; Dictyostelium; Molecular Sequence Data; Phosphatidylcholines; Phospholipase D; Protein Structure, Secondary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Type C Phospholipases
PubMed: 15225639
DOI: 10.1016/j.febslet.2004.05.071 -
Journal of Biochemistry Jul 1985Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed...
Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.
Topics: Animals; Cattle; Cell Membrane; Hot Temperature; Hydrogen-Ion Concentration; Lysosomes; Macromolecular Substances; Microsomes; Mitochondria; Phospholipases; Phospholipases A; Phospholipases A1; Phospholipases A2; Protein Binding; Sodium Chloride; Substrate Specificity; Trypsin; Type C Phospholipases
PubMed: 4044547
DOI: 10.1093/oxfordjournals.jbchem.a135254 -
The Journal of Biological Chemistry Jun 1999We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta, that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding... (Comparative Study)
Comparative Study
We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta, that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding domain as well as in the catalytic domain. Transient expression of cPLA2-beta cDNA in COS cells results in an increase in calcium-dependent phospholipase A1 (PLA1) and PLA2 activities compared with vector-transfected cells. cPLA2-beta is markedly less selective for cleavage at sn-2 as compared with cPLA2-alpha and cPLA2-gamma. Northern analysis reveals a cPLA2-beta transcript of 8 kilobase pairs that is expressed in all the human tissues examined. With the identification of cPLA2-beta, the newly defined cPLA2 family now comprises three members that may have dramatically different mechanisms for regulation of expression and enzymatic activation.
Topics: Amino Acid Sequence; Calcium; Cloning, Molecular; Cytosol; DNA, Complementary; Gene Library; Group IV Phospholipases A2; Humans; Molecular Sequence Data; Phospholipases A; Phospholipases A1; Phospholipases A2; Recombinant Proteins; Sequence Homology, Amino Acid; Tissue Distribution; U937 Cells
PubMed: 10358058
DOI: 10.1074/jbc.274.24.17063 -
Rat pancreatic phospholipase A2: purification, characterization, and N-terminal amino acid sequence.Journal of Biochemistry Sep 1984Phospholipase A2 was purified from rat pancreas by heat treatment of the homogenate and the sequential use of DEAE-Sepharose chromatography, CM-Sepharose chromatography,... (Comparative Study)
Comparative Study
Phospholipase A2 was purified from rat pancreas by heat treatment of the homogenate and the sequential use of DEAE-Sepharose chromatography, CM-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (HPLC). Prophospholipase A2 was not separated from the phospholipase A2 by CM-Sepharose chromatography under the conditions used, but it was well resolved by the reverse-phase HPLC. The enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and on analytical HPLC, and its molecular weight was estimated to be 14,000. The enzyme specifically hydrolyzed an acylester bond at the sn-2-position of the phospholipid examined. The purified enzyme has a pH optimum in the range of pH 9.5 to 10.5 and requires the presence of Ca2+ (3 mM) and sodium deoxycholate (0.1%) for optimum activity. The amino acid sequence of the first 32 residues in the N-terminal region of the enzyme was determined. The sequence revealed a marked homology with those of pancreatic phospholipases A2 of man, pig, ox, and horse, and porcine intestinal phospholipase A2 reported previously.
Topics: Amino Acid Sequence; Amino Acids; Animals; Cattle; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Horses; Humans; Intestines; Pancreas; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Species Specificity; Substrate Specificity; Swine
PubMed: 6501264
DOI: 10.1093/oxfordjournals.jbchem.a134896 -
Microbiology and Immunology 1984The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro.... (Comparative Study)
Comparative Study
The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A1 was identified by the formation of 32P- and 14C-labeled lysoderivatives from 32P-phosphatidylcholine, 32P-phosphatidylethanolamine, or 1-acyl-2-[1-14C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A1 activity was independent of lipase in the microorganism since 14C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using 32P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A1 was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-14C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A1 and/or A2?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.
Topics: Hot Temperature; Hydrogen-Ion Concentration; Leptospira; Lysophospholipase; Phosphatidylethanolamines; Phospholipases; Phospholipases A; Phospholipases A1
PubMed: 6493072
DOI: 10.1111/j.1348-0421.1984.tb00730.x