A.J. Aarsman

Studies on lysophospholipases: I. Purification and some properties of a lysophospholipase from beef pancreas

Abstract 1. 1. Lysophospholipase was purified from homogenates of fresh beef pancreas by acid treatment, (NH 4 ) 2 SO 4 precipitation in the presence of n -butanol and ion-exchange chromatography using SE-Sephadex C-50 and DEAE-Sephadex A-50. 2. 2. The molecular weight of the nearly homogeneous enzyme was estimated to be 65000 from sodium dodecyl sulfate disc electrophoresis and Sephadex G-200 filtration. 3. 3. The products of the hydrolysis of 1-acyl-glycerylphosphorylcholine containing long chain acyl groups were shown to be free fatty acid and glycerylphosphorylcholine, whereas long chain phosphatidylcholine was not attacked at all. 4. 4. The enzyme did not require bivalent metal ions and was unaffected by SH-group reagents. Diisopropylfluorophosphate in I mM concentration completely abolished enzymatic 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.

Isolation and properties of a phospholipase A1 activity from beef pancreas

Abstract 1. 1. The phospholipase A1 activity of beef pancreas was purified till near homogeneity using 1[9,10-3H2]-palmitoyl-2[1-14C]-linoleoylphosphatidylethanolamine as substrate. 2. 2. The phospholipase A1 activity was found to be different from pancreatic lipase. 3. 3. Evidence is provided indicating that phospholipase A1 activity in beef pancreas is caused by an esterolytic enzyme that also exhibits lysophospholipase activity. 4. 4. Phospholipase A1 activity is greatly stimulated by sodium deoxycholate, whereas lysophospholipase activity is inhibited by this detergent. 5. 5. Under certain conditions this enzyme exhibits phospholipase B activity i.e. catalyses the complete deacylation of phosphatidylcholine. 6. 6. Some similarities between this enzyme and other phospholipases A1 described in the literature are discussed.

A review on methods of phospholipase a determination

A short review is presented on methods to determine phospholipase A activities. On purpose, only those methods are covered which can, at least potentially, be used for assaying the low activities generally associated with intracellular phospholipases A. A selection was made of procedures which apply non-radioactive or radioactive substrates or chemically modified substrate analogs which can be used for spectrophotometric assays.

On the specificity of rat-liver lysophospholipase

Abstract 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 enzymes 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.

Studies on lysophospholipases: VII. Synthesis of acylthioester analogs of lysolecithin and their use in a continuous spectrophotometric assay for lysophospholipases, a method with potential applicability to other lipolytic enzymes

Abstract The synthesis of acylthioester analogs of lysolecithins, i.e., 2-hexadecanoylthio-1-ethyl-phosphorylcholine and 3-hexadecanoylthio-1-propyl-phosphorylcholine is described. Both compounds were found to be hydrolysed by a homogeneous lysophospholipase from beef liver, a spectrophotometric assay for the activity of which was developed by continuous measurement of the released thiol groups in the presence of dithionitrobenzoic acid. Phospholipase A 2 from pig pancreas effected hydrolysis of the acylthioester bond in 2-hexadecanoylthio-1-ethyl-phosphorylcholine, the enzymatic action of which could also be monitored spectrophotometrically. Lipase from pig pancreas was found to hydrolyse acylthioester bonds in 2-hexadecanoylthio-1-ethanol. The tributyryl ester of 3-mercapto-1,2-propanediol was synthesized and used to compare the release of total acid and thiol groups during hydrolysis with lipase. A ratio of about 2:1 was found for these releases. These findings clearly indicate the potential applicability of acylthioester analogs of substrates for phospholipases, lysophospholipases, and lipases in continuous spectrophotometric assays for lipolytic enzymes.

Synthetic peptide from lipocortin I has no phospholipase A2 inhibitory activity.

Two anti‐inflammatory peptides corresponding to a high amino acid similarity region between lipocortins were synthesized and tested on their ability to inhibit porcine pancreatic phospholipase A2. Kinetic assays using monomeric and aggregated phospholipids did not reveal any phospholipase A2 inhibitory activity. The peptides did not inhibit phospholipase A2 activity on monolayers of negatively charged substrate and did not prevent phospholipase A2 action on mixed micelles of 1‐stearoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine and sodiumdeoxycholate. Ultraviolet difference spectroscopy did not show binding of the peptides to phospholipase A2. Therefore we conclude that these anti‐inflammatory peptides do not inhibit pancreatic phospholipase A2 in vitro, in contrast to the results recently published [(1988) Nature 335,726–730].

Calcium-independent phospholipase A2 in rat tissue cytosols.

Cytosols (105,000 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-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 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 A2 isolated from rat liver mitochondria.

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.

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells.

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).