Phospholipase activities of the P388D1 macrophage-like cell line

The murine macrophage (M phi) cell line, P388D1, was employed as a source of M phi phospholipases in order to characterize the enzymatic properties and subcellular localization of these enzymes because of their importance for prostaglandin biosynthesis. Phospholipase activity was assessed with dipalmitoylphosphatidylcholine (DPPC) as substrate. Phospholipases were characterized with respect to divalent cation dependence, pH optima, and localization in subcellular compartments using linear sucrose gradients. By these criteria a number of different phospholipases were identified. Most importantly, a single Ca2+-dependent activity with a pH optimum of 8.8 was identified in membrane-rich fractions (plasma membrane, mitochondria, and endoplasmic reticulum) and could be clearly separated from the remaining activities, which are Ca2+ independent and exhibit pH optima of 7.5, 5.1, and 4.2. The phospholipases with acidic pH optima may be associated with subcellular components containing lysosomal enzymes and both phospholipase A1 and phospholipase A2 activities are observed. In contrast, the phospholipase activity with a pH optimum of 7.5 sediments with the cytosolic proteins and is inhibited by 5 mM Ca2+. No significant phospholipase C activity was detected in assays performed with or without added Ca2+ at pH's 4.2, 5.1, 7.5, or 8.8 using DPPC as substrate. However, the P388D1 cells do contain a lysophospholipase that is at least 20 times more active than the phospholipase A activities identified. Its presence must be taken into account in evaluating the positional specificities and properties of the macrophage phospholipases.     

Solubilization and partial characterization of phospholipase A from rat heart sarcoplasmic reticulum

Phospholipase A has been solubilized from the sarcoplasmic reticulum of rat heart by treatment with Tris buffer, potassium chloride, taurodeoxycholate or octyl glucoside. On HPLC gel permeation, two phospholipases were identified at the void volume of a TSK 3000 column and at an apparent molecular mass of 60 kDa. The two activity peaks exhibited a predominance of phospholipase A1 activity (83-91%) and a lesser phospholipase C activity (4-9%) using sonicated 1-palmitoyl-2[1-14C]oleoylphosphatidylcholine liposomes as substrate. The voiding phospholipase A peak, which represented the bulk of the recovered activity, exhibited a requirement for calcium ions in the 0.3-3 microM range. The heat stability and response to mercuric ions was studied and some similarities were noted between the solubilized sarcoplasmic reticulum phospholipases A and the cytosolic phospholipases A of rat heart. It is speculated that the cytosolic phospholipase A which we reported earlier may represent in part phospholipase A released from sarcoplasmic reticulum during isolation of the subcellular membrane fractions.     

Phospholipases A1 and A2 in bovine thyroid.

In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.     

Subcellular distribution and kinetic parameters of HS mouse liver aldehyde dehydrogenase

1. Aldehyde dehydrogenase subcellular distribution studies were performed in a heterogeneous stock (HS) of male and female mice (Mus musculus) with propionaldehyde (5 mM and 50 microM) and formaldehyde (1 mM) and NAD+ or NADP+. 2. The relative percents of distribution were: cytosolic 55-68%, mitochondrial 12-20%, microsomal 9-18% and lysosomal 3-15% for both propionaldehyde concentrations and NAD+. 3. Kinetic experiments using propionaldehyde and acetaldehyde with NAD+ revealed two separate enzymes, Enzyme I (low Km) and Enzyme II (high Km) in the cytosolic and mitochondrial fractions. 4. The kinetic data also indicated a spectrum of cytosolic low Km values that exhibited a bimodal distribution with one congruent to 40 microM and one congruent to 5 microM. 5. It was concluded that there was no significant difference in aldehyde-metabolizing capability between male and female HS mice, compared on a per gram of liver basis. The cytosolic low Km enzyme plays a major role in aldehyde oxidation at moderate to low aldehyde concentrations.     

Effects of antimalarial drugs on phospholipase A and lysophospholipase activities in plasma membrane, mitochondrial, microsomal and cytosolic subcellular fractions of rat liver

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 x 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.     

Fractionation, biochemical characterization and lysosomal phospholipases of human liver

Human liver was homogenised and fractionated by differential centrifugation, and the subcellular fractions were characterised biochemically. Absolute values and distribution patterns of protein and marker enzyme activities obtained from human liver have also been compared with those from rat liver. In addition, acid phospholipase activities have been studied in human liver. On the basis of product formation from stereo-specifically radiolabeled phosphatidylethanolamine substrates, lysosomal phospholipases A1 and A2 with optimal activities at pH 4.7 have been identified in human liver. Acid phospholipase C and lysophospholipase activities, however, were not found in human liver. Cationic amphiphilic drugs inhibited the activities of the acid phospholipases A in human and rat liver lysosomes to about the same extent.     

Selective release of phospholipase A2 and lysophosphatidylserine-specific lysophospholipase from rat platelets

Rat platelets released phospholipase A2 and lysophospholipase upon activation with thrombin or ADP. The release of phospholipases was energy-dependent and was not in parallel with that of a known lysosomal marker enzyme, N-acetyl-beta-D-glucosaminidase. The phospholipases are derived from other granules (dense granules or alpha-granules) rather than lysosomal granules of the cells. All of the activities of both phospholipases in the cell free fraction obtained from the activated platelet reaction mixture was recovered in the supernatant after centrifugation at 105,000 X g. The degree of hydrolysis of phospholipids by the phospholipase A2 followed the order: phosphatidylethanolamine (PE) greater than phosphatidylserine (PS) greater than phosphatidylcholine (PC). Phospholipase A2 shows a broad pH optimum (greater than pH 7.0) and absolutely requires Ca2+. Lysophospholipase was specific to lysophosphatidylserine (lysoPS), and neither lysophosphatidylethanolamine (lysoPE) nor lysophosphatidylcholine (lysoPC) was hydrolyzed appreciably. Both 1-acyl- and 2-acyl-lysophosphatidylserine were equally hydrolyzed. Lysophospholipase activity shows similar pH optimum to phospholipase A2. The lysophospholipase activity was lost easily at 60 degrees C. The activity was reduced by the presence of EDTA, though low but distinct activity was observed even in the presence of EDTA. Addition of Ca2+ to the mixtures restores the full activity.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

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.     

Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.     

Lysosomal phospholipase A activities of rat ovarian tissue.

1.1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rats ovaries. Acid phospholipase A activities were characterized with [U-14C]diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[9,10-3H]- or [1-14C]oleoyl-sn-glycero-3-phosphocholine as substrates. Acid phospholipase A1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. Phospholipase A1 and A2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca2+ and slightly activated by EDTA. A Ca2+- stimulated phospholipase A2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinizatin, suggesting a hormonal influence on lysosomal enzyme activities.     

Regulation of lysophosphatidylcholine-metabolizing enzymes in isolated myocardial cells from rat heart

Enzymatic pathways involved in the metabolism of lysophosphatidylcholine were investigated in rat heart myocardial cells. Acyl CoA-dependent acyltransferase activity was localized in microsomes, and was much greater than lysophospholipase activity in either cytosolic or microsomal fractions. The cytosolic lysophospholipase was more sensitive to inhibition by palmitylcarnitine in comparison to free fatty acids. In contrast, free fatty acids (oleate and palmitate) produced a greater inhibition of the microsomal acyltransferase and lysophospholipase than did palmitylcarnitine. A reduction in the assay pH to 6.5 resulted in an increase in microsomal acyltransferase and cytosolic lysophospholipase activities, but brought about a marked reduction in the microsomal lysophospholipase activity. At pH 6.5, the percentage inhibition of the microsomal acyltransferase by palmitylcarnitine was reduced, whereas the inhibition by palmitic acid was enhanced. The inhibition of the microsomal lysophospholipase by both palmitylcarnitine and palmitic acid was reduced at pH 6.5. With respect to myocardial ischemia, the inhibition of microsomal acyltransferase by free fatty acids and the reduction in microsomal lysophospholipase activity due to acidosis may contribute to the elevation of cellular lysophosphoglycerides which are arrhythmogenic.     

The catabolism of plasmenylcholine in the guinea pig heart.

The hydrolysis of the alkenyl bonds of plasmenylcholine and plasmenylethanolamine by plasmalogenase, followed by hydrolysis of the resultant lysophospholipid by lysophospholipase, has been postulated as the major pathway for the catabolism of these plasmalogens. However, the postulation was based solely on the presence of plasmalogenase activity towards plasmenylethanolamine and plasmenylcholine in the brain. In this study we have demonstrated the absence of plasmalogenase activity for plasmenylcholine in the guinea pig heart under a wide range of experimental conditions. Plasmenylcholine was hydrolysed by phospolipase A2 activities in cardiac microsomal, mitochondrial and cytosolic fractions. Phospholipase A2 activities in these fractions had an alkaline pH optimum and were enhanced by Ca2+. The enzymes also displayed high specificity for plasmenylcholine with linoleoyl or oleoyl at the C-2 position. Lysoplasmalogenase activity for lysoplasmenycholine was also detected and characterized in the microsomal and mitochondrial fractions. Since the cardiac plasmalogenase is only active towards plasmenylethanolamine but not plasmenylcholine, the catabolism of these two plasmalogens must be different from each other. We postulate that the major pathway for the catabolism of plasmenycholine involves the hydrolysis of the C-2 fatty acid by phospholipase A2, and hydrolysis of the vinyl ether group of the resultant lysoplasmenylcholine by lysoplasmalogenase.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Sialidase in the guinea pig pulmonary parenchyma. Increased activity in the cytosolic and microsomal subcellular fractions after stimulation with Bacillus Calmette Guérin

The sialidase activity was assayed in the guinea pig pulmonary parenchyma after removal of bronchoalveolar cells by washing. After differential centrifugation of the crude tissue homogenate, sialidase activities were measured in the subcellular fractions using the fluorogenic substrate 2-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminate. Sialidase activities were found in the lysosomal-enriched (17,000 x g pellet), in the microsomal (105,000 x g pellet) and in the cytosolic (105,000 x g supernatant) fractions. Microsomal and lysosomal forms of sialidase had an optimum activity at pH 3.6-3.8, whereas the optimum for the cytosolic form was pH 4.6. The activity of all three forms was inhibited by Cu2+, whereas 1 mM Zn2+ and 0.5 mM Ca2+ activated the lysosomal and the cytosolic forms, respectively. In the crude homogenate taken from lungs of Bacillus Calmette Guérin-(BCG-) stimulated guinea pigs, the sialidase activity was increased by 43% (p = 0.025) 3 weeks after the end of the treatment. The cytosolic (+246%) and microsomal (+51%) sialidase activities were significantly increased, whereas the lysosomal sialidase activity was not changed significantly by BCG stimulation.     

Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin

Phospholipase A2 (EC 3.1.1.4) activity appeared to be unevenly distributed among the subcellular fractions of rabbit lung homogenates. The mitochondrial/lysosomal fraction, which possessed the highest specific activity, was the second most abundant source of enzyme, following the 1000 x g pellet. Crude microsomes, which were the poorest source of enzyme, had a specific activity intermediate between that of crude mitochondria and of cytosol. Despite these observations, in view of the putative role of microsomal phospholipase A2 in remodelling phosphatidylcholines for pulmonary surfactant biosynthesis, the purification of phospholipase A2 from microsomal membranes was investigated. The activity was solubilized from rabbit lung microsomes with 1 M KCl and resolved into two distinct peaks by ion-exchange chromatography. The larger peak (95% of the recovered activity) was subjected to a combination of hydroxyapatite and gel-filtration chromatography, resulting in a purification factor in excess of 70,000 relative to the microsomal membranes. There was no indication for the removal of endogenous inhibitor(s) during the purification. Application of the same purification protocol to a 1 M KCl extract of lung mitochondria resulted in phospholipase A2 profiles in each of the four columns employed that had exactly the same elution characteristics as those generated by the microsomal extracts. The purified enzyme is specific for the sn-2 ester bond of phosphatidylcholine, requires Ca2+ for activity and has an alkaline pH optimum. It is heat-labile and susceptible to treatment by p-bromophenacyl bromide and by 2-mercaptoethanol but remains unaffected by NaF, diisopropylfluorophosphate and thiol reagents.     

Hydrolysis of phospholipids by a lysosomal enzyme

The phospholipid-hydrolyzing activity of rat liver lysosomes has been studied. These lysosomes contain a phospholipase that cleaves both fatty acid ester linkages of lecithin and of phosphatidyl ethanolamine and releases free fatty acids from both positional isomers of lysolecithin. The enzyme does not require calcium for maximum activity, and is inhibited by diethyl ether and sodium deoxycholate. Mercuric ions and cetyltrimethyl ammonium bromide also inhibit the hydrolysis. Compared with lipase activity, this enzyme is relatively stable to heat. The specific activity of the hydrolysis of lecithin by the lysosomal enzyme is considerably higher than those reported for mitochondrial and microsomal phospholipases. The enzyme resembles other hydrolases of the lysosome in that it has an acid pH optimum (pH 4.5). This enzymic activity is present in both the lysosomal soluble enzyme fraction and in the lysosomal membrane fraction. The enzyme may participate in the intracellular digestion of mitochondria that is carried out by the intact lysosome in vivo. Localized inflammation and changes in vascular permeability following tissue damage could be catalyzed by this phospholipase.     

Acid phospholipase A activities in rat hepatocytes

Cultured rat hepatocytes exhibit acid phospholipase A activity. On the basis of product formation from stereospecifically radiolabeled phosphatidylethanolamine substrates, phospholipases A1 and A2 have been identified with optimal activities at pH 4.5. According to subcellular fractionation studies, the acid phospholipases in hepatocytes appear to be located in the lysosomal compartment. Application of specific inhibitors of the biosynthesis, glycosylation, and translocation of lysosomal enzymes in hepatocyte cultures suggests a half-life of approx. 1 day for the acid lysosomal phospholipase A1. About the same value for the half-life was obtained for the lysosomal marker enzymes, acid phosphatase and beta-N-acetyl-D-hexosaminidase.     

Modulation of epididymal delta 4-steroid 5 alpha-reductase activity in vitro by the phospholipid environment

Epididymal 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal membranes, and using two approaches, we investigated the relationship between 5 alpha-reductase activity and the membrane environment. In the first, nuclear and microsomal membrane fractions were treated with phospholipases to modify specifically the structure of the phospholipid component of the membranes, and the effects of these treatments on the kinetic parameters of 5 alpha-reductase were examined. The second approach was to observe the effects of phospholipids of known structure on solubilized 5 alpha-reductase activity. Treatment of the membrane fractions with phospholipase C increased the Km(app) of both the nuclear and microsomal 5 alpha-reductases for testosterone. Phospholipase A2 treatment also increased the Km(app) of the microsomal enzyme, but in contrast, the Km(app) of the nuclear 5 alpha-reductase for testosterone was unaffected. This demonstrated a fundamental difference in the role of the membrane environment in the expression of 5 alpha-reductase activity in these subcellular compartments. The ability of phospholipids to enhance the activity of solubilized 5 alpha-reductase was highly specific and structure related. Only phosphatidylcholines containing either unsaturated acyl chains or saturated acyl chains of 12 carbon atoms were found to activate 5 alpha-reductase. The most potent activator was dilauroyl phosphatidylcholine, which reduced the Km(app) values of both nuclear and microsomal 5 alpha-reductases for testosterone, without affecting the concentration of active 5 alpha-reductase (Vmax(app) ). This is the first time that an activator of 5 alpha-reductase has been found. These findings suggest that epididymal 5 alpha-reductase activity may be regulated by changes in the phospholipid environment.     

Phospholipid metabolism in the rat renal inner medulla

In view of the importance of phospholipids as a source of precursor fatty acids for the high prostaglandin synthesis in the renal inner medulla, we studied pathways of phospholipid esterification and degradation in the rat inner medulla. De novo acylation of [14C]arachidonate occurred predominantly in position 2 of phosphatidylcholine in the microsomal fraction. This newly esterified [14C]arachidonate was accessible to deacylation by a microsomal phospholipase A2 (EC 3.1.1.4) with alkaline optimum which was Ca2+-dependent and resistant to 0.1% deoxycholate. No phospholipase A1 (EC 3.1.1.32) activity against endogenous labeled phosphatidylcholine could be demonstrated in the microsomal fraction. When exogenous phosphatidylcholine labeled at position 2 was deacylated by renomedullary homogenates, labeled free fatty acid but no labeled lysophosphatidylcholine was recovered in the reaction products. This could be attributed to further degradation of generated lysophosphatidylcholine by a cytosolic lysophospholipase (EC 3.1.1.5). Sodium deoxycholate at a concentration of 0.1% or higher inhibited the lysophospholipase and allowed the demonstration of both A2 and A1 alkaline phospholipase activities in the homogenate. The major in vitro pathway of lysophosphatidylcholine disposition is further degradation by a cytosolic lysophospholipase, while reutilization for phosphatidylcholine synthesis through the action of a predominantly microsomal acyltransferase appears to be a minor pathway. In the presence of several acyl-CoAs, reutilization of lysophosphatidylcholine is significantly increased by an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) but there is no preferential transfer of arachidonyl-CoA compared to other acyl-CoAs.