We show for the first time that secreted low-molecular weight phospholipase A2 (EC 3.1.1.4) catalyzes the deacylation of monoacylphosphoglycerides directly from the sn-1 position, although at a very low rate: purified phospholipase A2 enzymes from bee venom, crotalus atrox venom, and porcine pancreas hydrolyze the sn-1 ester bond in 1-palmitoyl-2-O-methyl-sn-glycero-3-phosphorylcholine. Hydrolytic rates with the corresponding isomer, 1-O-methyl-2-palmitoyl-sn-glycero-3-phosphorylcholine, are about 3-4 orders of magnitude higher. The similarities in Ca2+ requirement and inactivation profiles suggest that deacylation, albeit with different rates, from both sn-1 and sn-2 positions is catalyzed by the same catalytic site of phospholipase A2. Furthermore, evidence is provided that phospholipase A2-catalyzed 1-acyl lysophospholipid deacylation is mediated by sn-1-directed action, but above pH 7 acyl migration with subsequent enzyme-catalyzed hydrolytic cleavage from the sn-2 position contributes to the overall deacylation of monoacylphosphoglycerides, acyl migration becoming eventually the rate-limiting factor.

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.

A simple radiometric procedure is presented for the separation and determination of amino alcohol-labelled zwitterionic phospholipids (phosphatidylcholine and phosphatidylethanolamine) and their hydrolytic metabolites. The protocol allows the determination of all potential amino alcohol-containing metabolites, in a range of at least 0.2–500 nmol, in the absence or presence of non-ionic (Triton X-100) and ionic (sodium deoxycholate, hexadecyltrimethylammonium bromide) detergents. The discontinuous assay is based on the determination of both water-soluble and lipid-soluble metabolites, i.e., on lipid extraction and thin-layer and ion-exchange chromatography. In addition, simplified and less time-consuming modifications of the procedure have been developed for specifically monitoring phospholipase C and D activities in the course of enzyme purification. The validity of the methods is documented by employing various model phospholipases.

A member of the annexin family (the heterotetrameric annexin II2p11(2) complex purified from porcine intestinal epithelium) was tested for its ability to affect different calcium-dependent intrinsic lipolytic activities of rat liver hepatic lipase (HL). Whereas annexin II in the presence of calcium failed to interfere with HL triacyl glycerol lipase (EC 3.1.1.3) activity, it inhibited HL phospholipase A1 (EC 3.1.1.32) and lysophospholipase (EC 3.1.1.5) activities. Inhibition could be overcome by increasing the substrate concentration. Under phospholipase A1 assay conditions, annexin II did not bind to the purified HL enzyme. These results therefore suggest that only inhibitor/substrate interactions lead to inhibition of HL phospholipase A1 and lysophospholipase activities, an obviously general mechanism of phospholipase inhibition by annexins. Possible implications of HL inhibition in vivo by annexins are discussed.

The effects of the antiinflammatory drug benzydamine (Tantum) on phospholipase activities were determined in vitro, employing various enzyme preparations (rat liver plasma membranes, endoplasmic reticulum, lysosomes; human seminal plasma) and stereospecifically radiolabeled phosphatidylethanolamines as substrates. Fatty acid release from the sn-2 position was inhibited at drug concentrations above 10(-5) mol/l. Concerning the mode of inhibition, a mixed type was found for the soluble phospholipase A2. Impaired fatty acid release from the sn-2 position might contribute to the mechanism of antiinflammatory action of benzydamine by rendering less free precursor acid available for the synthesis of eicosanoids. Fatty acid release from the sn-1 position was inhibited at benzydamine concentrations from 10(-6)-10(-2) mol/l only in lysosomes, whereas in plasma membranes and endoplasmic reticulum it was stimulated, maximally (at 10(-3) mol/l) about 25% and 50%, respectively.

Activities of membrane-associated phospholipases A1 and A2, and membrane-associated as well as soluble lysophospholipases were measured in different subcellular fractions of rat liver, using suspensions of stereospecifically labelled radioactive phospholipids as substrates. Plasma membranes and endoplasmic reticulum were shown to contain phospholipase A1 and lysophospholipase activities, both of which could be stimulated by Ca2+, mitochondria Ca2+-dependent phospholipase A2 and cytosol Ca2+-independent lysophospholipase activities. Each of these lipolytic enzymes could be inhibited by antimalarial drugs (chloroquine, mepacrine, primaquine) at concentrations above 1 · 10−4 M. Inhibition of the alkaline cytosolic lysophospholipase by these drugs was noncompetitive with respect to the substrate, and the inhibitory potency increased, when the pH was raised.

Phospholipase A2 activity of polymorphonuclear leukocytes (PMNLs) towards radioactively labelled phosphatidylethanolamine is inhibited by FOY (ethyl 4-(6-guanidinohexanoyloxy)-benzoate methanesulfonate) in concentrations above 10−4M. The inhibition is noncompetitive with respect to the substrate employed and depends on the pH. Impairment of .PMNL phospholipase A2 activity could play a role in the anti-inflammatory effect of FOY: the release of fatty acids from mebrane phospholipids is reduced and consequently also the biosynthesis of bioactive oxygenated arachidonate metabolites, among them inflammatory mediators.

Rat-liver lysosomal phospholipase A1 activity is unspecifically inhibited in a pH dependent and competitive manner by the cationic amphiphilic protease inhibitors FOY (ethyl 4 — (6-guanidinohexanoyloxy) benzoate methanesulfonate) and FOY 305 (N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate methanesulfonate) in concentrations above 10−4M. This inhibition may contribute to anti-inflammatory effects of these drugs in that they reduce the release by lysosomal enzymes of fatty acids from membrane phospholipids and consequently decrease the biosynthesis of bioactive oxygenated arachidonate metabolites, among them inflammatory mediators.

The effects of three cationic amphiphilic antimalarial drugs (chloroquine, mepacrine and primaquine) on the intralysosomal catabolism of phosphatidylethanolamine and several of its metabolites were studied with rat-liver lysosomes which had been isolated from animals previously treated with Triton WR-1339. The activities of each of the various enzymes involved in the main pathways of intralysosomal phosphatidylethanolamine degradation (Kunze, H., Hesse, B. and Bohn, E. (1982) Biochim. Biophys. Acta 711, 10-18) exhibited almost identical inhibitory sensitivities towards mepacrine and primaquine. In contrast, chloroquine inhibited the activities of the various enzymes to different extents, lysophospholipid acylhydrolase (EC 3.1.1.5) being the most sensitive enzyme, followed by phospholipase A1 (EC 3.1.1.32) and monoacylglycerol lipase, and eventually lysophospholipid monoacylglycerol hydrolase as the least sensitive enzyme. The relative inhibitory potencies towards phospholipase A1 activity of chloroquine were increased with increasing pH, and the mode of inhibition was competitive. In contrast, the inhibitory potencies towards monoacylglycerol lipase activity of chloroquine increased only up to pH 5 but decreased above this value, and the mode of inhibition was noncompetitive.

Lysosomal catabolism of radioactively labelled phosphatidylethanolamine, phosphatidylcholine and several potential metabolites of these diacylphospholipids was studied using rat-liver lysosomes which had been isolated from Triton WR-1339-treated animals. Hydrolysis of these lipids seems to be restricted to the soluble lysosomal compartment. The initial intralysosomal degradation is predominantly catalysed by phospholipase A1 (EC 3.1.1.32) followed by lysophospholipase (EC 3.1.1.5). The end products of this pathway are free fatty acids and glycerophosphorylethanolamine or glycerophosphorylcholine. These phosphodiesters are not hydrolysed further in lysosomes, as has been shown previously (Fowler, S. and De Duve, C. (1969) J. Biol. Chem. 144, 471-481). The intermediary lysophospholipids, however, are also hydrolysed by an alternative pathway, i.e. by a lysophospholipase which catalyses the hydrolysis of the glycerophosphate ester bond, followed by a monoacylglycerol lipase and a phosphomonoesterase (EC 3.1.3.2), respectively. Besides these two catabolic routes of intralysosomal hydrolysis of phosphatidylethanolamine and phosphatidylcholine, additional pathways are possible, which seem, however, to be of minor importance, at least in the substrate concentration ranges employed in these studies. These additional reactions include attack by a phospholipase A2 (EC 3.1.1.4) and--as discovered recently (Matsuzawa, Y. and Hostetler, K.Y. (1980) J. Biol. Chem. 255, 646-652)--by a phospholipase C (EC 3.1.4.3). Cations such as Mg2+, Ca2+, K+ and Na+ inhibit preferentially deacylation reactions.

Lipolytic activity toward phospholipids in human seminal plasma had been ascribed to phospholipase A2 (17). The enzyme is soluble, heat stable at pH 5, requires Ca2+ for optimal activity, inhibited by ionic and nonionic detergents, and catalyzes the hydrolysis of the fatty acids in the 2 position of various sonicated phospholipids. In 12 healthy fertile and 20 subfertile individuals, the total phospholipase A2 activity toward radioactively labeled phosphatidylethanolamine has been compared with the total amounts of prostaglandins E and F, which have been determined by specific radioimmunoassay. There is a statistically significant correlation (p is less than 0.01) between total phospholipase A2 activity and prostaglandin E (and F) contents in the seminal plasma. It is concluded that phospholipase A2 is secreted along with prostaglandins into seminal plasma. This seminal phospholipase A2 possibly reflects the initial step of substrate release for prostaglandin biosynthesis in the human male reproductive system.

Prostaglandins E1 (PGE1) and E2 (PGE2) have been coupled with the amine group of phosphatidylethanolamine (PE) by means of dicyclohexylcarbodiimide. These complexes basically mimic the relaxant and contractile effects of the corresponding free prostaglandins (PGs) on various smooth muscle preparations, but exhibit a delayed onset of action and a lower affinity for the PG receptors. The complexes are comparable with the free, parent PGs, in their intrinsic activities. The same holds true for the effects on blood pressure and on the motility of the uterus in situ. The PGE2-PE complex is hydrolysed to release obviously free PGE2 by cell-free homogenates prepared from various tissues, but not by blood plasma. The PGE2-PE complex is immunologically indistinguishable from the free PGE2.

Journal Article Effects of psychotropic drugs on prostaglandin biosynthesis in vitro Get access H Kunze, H Kunze Department of Biochemical Pharmacology, Max-Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 34 Göttingen, GFR Search for other works by this author on: Oxford Academic Google Scholar E Bohn, E Bohn Department of Biochemical Pharmacology, Max-Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 34 Göttingen, GFR Search for other works by this author on: Oxford Academic Google Scholar G Bahrke G Bahrke Department of Biochemical Pharmacology, Max-Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 34 Göttingen, GFR Search for other works by this author on: Oxford Academic Google Scholar Journal of Pharmacy and Pharmacology, Volume 27, Issue 11, November 1975, Pages 880–881, https://doi.org/10.1111/j.2042-7158.1975.tb10239.x Published: 12 April 2011 Article history Received: 04 March 1975 Published: 12 April 2011

1.1. The spontaneous formation of prostaglandins in homogenates of bovine seminal vesicles was stimulated by Ca2+ and inhibited by EDTA. The effects of various local anaesthetics were studied over a concentration range of 0.01–20 mM. The local anaesthetics used inhibited prostaglandin formation, except cocaine, lidocaine and mepivacaine. The order of potency was benzocaine > chlorpromazine > dibucaine > tetracaine > procaine > butacaine.2.2. Phospholipase A2 activity in homogenates of bovine seminal vesicles, estimated with sonicated radioactive phosphatidylethanolamine as substrate, was stimulated by Ca2+ and inhibited by EDTA. At a concentration of 1 mM benzocaine, lidocaine and mepivacaine had no significant effect. The other local anaesthetics inhibited in the order chlorpromazine > dibucaine > tetracaine > butacaine > cocaine > procaine.3.3. In acetone-pentane powder preparations of sheep seminal vesicles the prostaglandin synthetase activity was studied by adding arachidonic acid as substrate. Ca2+ inhibited and EDTA stimulated the prostaglandin synthetase activity. At 1 mM final concentrations only chlorpromazine > benzocaine > dibucaine > butacaine inhibited the conversion to prostaglandin, whereas procaine, tetracaine, cocaine, lidocaine and mepivacaine had no effect or even stimulated the conversion.4.4. Thus chlorpromazine, dibucaine and butacaine inhibit prostaglandin biosynthesis in a non-specific manner, whereas benzocaine acts only on the prostaglandin synthetase. Tetracaine and possibly procaine specifically inhibit prostaglandin biosynthesis by interference with phospholipase A2 activity.5.5. The present work does not imply that local anaesthesia is mediated by inhibition of prostaglandin biosynthesis. This inhibition may be a side effect of local anaesthetics.

The Mg2+-dependent, Na+-K+-activated ATPase of ox brain was inhibited by the direct lytic factor of cobra venom at concentrations of 10−7 g/ml or higher. Only weak inhibition was seen in ghosts of human red cells. Haemolysis of guinea-pig red cells by phospholipase A was not enhanced when the erythrocyte ATPase had been blocked by ouabain. It is concluded that direct lytic factor-induced haemolysis is not dependent on an ATPase inhibiting effect.

Different methods are compared for extraction of prostaglandins as free acids from AgNO3-impregnated silica gel layers for biological assay. A simple and quick method is described which gives 80–100% recovery of prostaglandins E1, E2, F1α and F2α.