Does de novo synthesis of lysophosphatidylcholine occur in rat lung microsomes?

Incubation of rat lung microsomes with CDP[Me-14C]choline resulted in formation of radioactive lysophosphatidylcholine and phosphatidylcholine. Evidence is provided which suggests that lysophosphatidylcholine formation cannot be ascribed completely to phospholipase A degradation of phosphatidylcholine. Lysophosphatidylcholine production can be stimulated by addition of monoacylglycerol or diacylglycerol. It is suggested that diacylglycerol is partly hydrolyzed to monoacylglycerol and subsequently converted to lysophosphatidylcholine. A direct transfer of phosphocholine from CDPcholine to monoacylglycerol is demonstrated by equimolar incorporation of 1(3)-[9,10-3H2]palmitoylglycerol and phospho[Me-14C]choline into lysophosphatidylcholine

A comparison of acyl-oxyester and acyl-thioester substrates for some lipolytic enzymes

1. 1. A comparison of 2-hexadecanoylthio-ethane-1-phosphocholine and 3-hexadecanoylthio-propane-1-phosphocholine and their oxyester counterparts as substrates for some lipolytic enzymes was made. 2. 2. The critical micelle concentration and the transition temperature of the synthetic substrates were compared with the values for 1-hexadecanoyl-sn-glycero-3-phosphocholine. 3. 3. All above-mentioned compounds were deacylated by lysophospholipases. Phospholipase A2 hydrolyzed only the acyl- sulfur- and oxygenester bond in 2-hexadecanoyl-ethane-1-phosphocholine. 4. 4. Kinetic parameters, Km and V, for hydrolysis of these substrates were determined. Km values for thioester substrates were 5–10-fold lower than for the corresponding oxyesters. Maximal hydrolysis rates were 2–5 times higher for the thioesters. 5. 5. Hydrolysis of thioesters by phospholipase A2, lipase and lysophospho-lipase was shown to proceed by an S-acyl cleavage mechanism. 6. 6. Beef liver lysophospholipase II was rapidly and stoichiometrically inactivated by diisopropylfluorophosphate and bis(p-nitrophenyl) phosphate. Inactivation by the latter inhibitor showed burst-like kinetics. 7. 7. Attempts to show burst-kinetics during the pre-steady state hydrolysis of 2-hexadecanoylthio-ethane-1-phosphocholine by lysophospholipase II were negative. These results are interpreted to indicate that a step prior to deacylation of the enzyme is rate-determining

Calcium-independent phospholipase A2 in rat tissue cytosols

Cytosols (105000 X g supernatant) from seven rat tissues were assayed for Ca²⁺-independent phospholipase A₂ activity with either 1-acyl-2-[1-¹⁴C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[l-¹⁴C]linoleoyl-snglycero- 3-phosphoethanohunine or 1-0-hexadecyl-2-[9,10-³H₂]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from l0-120 pmol /min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca²⁺-independent phospholipase A₂ originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca²⁺-dependent phospholipase A₂ activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca²⁺ -dependent phospholipase A₂ activity, however, came from lung cells. Neither this enzyme nor the Ca²⁺-independent phospholipase A₂ from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca²⁺-dependent phospholipase A ₂ isolated from rat liver mitochondria

On the specificity of rat-liver lysophospholipase

1. 1. A study on the specificity of rat-liver lysophospholipase activity (EC 3.1.1.5) revealed that both 1-acyl-sn-glycero-3-phosphorylcholine and 2-acyl-sn-glycero-3-phosphorylcholine are deacylated. From both positional isomers the unsaturated analogs appeared to be degraded at higher rates. 2. 2. Circumstantial evidence is presented indicating that 2-acyl-sn-glycero-3-phosphorylcholine can be attacked directly by this lysophospholipase activity without a prior migration of the fatty acyl constituent. 3. 3. Compounds lacking the free hydroxyl group present in lysophosphatidyl-cholines, e.g. acyl-ethylene glycolphosphorylcholine and 1-acyl-propane diol-3-phosphorylcholine, also fall in the enzyme's range of specificity. 4. 4. Mono-acyl derivatives of sn-glycero-1-phosphorylcholine, sn-glycero-2-phosphorylcholine, as well as sn-glycero-3-phosphorylcholine, were found to be degraded. 5. 5. Inhibition of lysophospholipase activity by various agents exhibited the same effect on the deacylation of both 1-acyl- and 2-acyl-sn-glycero-3-phosphoryl-choline. 6. 6. The degradation of mono-acyl-phosphatidylcholine appeared to be strongly inhibited in the presence of phosphatidylcholine

Some aspects of rat platelet and serum phospholipase A2 activities

Rat platelet lysate contained appreciable phospholipase A2 activity. In agreement with literature data this enzymatic activity eluted in the void volume of a Sephadex G-100 column. When the void volume peak was chromatographed over a Matrex gel blue A column, part of the phospholipase A2 activity ran through, whereas the remainder was bound to the gel. The latter activity could be eluted with buffers containing a high salt concentration. In contrast, phospholipase A2 activity solubilized from rat platelet lysates by treatment with high salt eluted from Sephadex G-100 columns with an apparent molecular weight of 10–15 kDa. This solubilized enzyme completely bound to Matrex gel blue A and, in the presence of Ca2+ also to an alkylphosphocholine-AH Sepharose affinity column. No indications were obtained for the presence of inactive phospholipase A2 and activator proteins in rat platelet lysates as described by Etienne, J., Grüber, A. and Polonovski, J. ((1980) Biochim. Biophys. Acta 619, 693–698; (1982) Biochemie 64, 377–380). Phospholipase A2 activity, both the associated form in platelet lysate and the monomeric form as eluted from Sephadex G-100 was slightly inhibited by trifluoperazine but calmodulin exerted no stimulation. Likewise, phospholipase A2 activity from rat serum eluted in the void volume of a Sephadex G-100 column. Rather than indicating the presence of high molecular weight forms of the enzyme, this is apparently caused by association with lipids or other proteins, in that chromatography in the presence of high salt revealed a molecular weight similar to that found for solubilized platelet phospholipase A2 activity

Lipocortin inhibition of extracellular and intracellular phospholipases A2 is substrate concentration dependent

Hydrolysis of Escherichia coli membrane phospholipids by pancreatic phospholipase A2 was inhibited by lipocortin from human monocytes in a substrate dependent manner. Inhibition was completely overcome at substrate concentrations above 250 μM. Lipocortin also inhibited partially purified preparations of two intracellular phospholipases A2, isolated from rat liver mitochondria and rat platelets when these enzymes were assayed at low micromolar concentrations of phosphatidylethanolamine. Inhibition gradually decreased with increasing substrate concentrations both for pancreatic and platelet phospholipase A2 and became completely abolished above 15 and 50 μM phosphatidylethanolamine, respectively