Identification of a membrane-bound, glycol-stimulated phospholipase A2 located in the secretory granules of the adrenal medulla

Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca2(+)-stimulated phospholipase A2 (PLA2) activity when assayed at pH 9.0 with phosphatidylcholine containing an [14C]-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA2 activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycol, or poly(ethylene glycol). This glycol-stimulated PLA2 activity codistributed with membrane-bound dopamine beta-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA2 of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different 14C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethanolamine, and distearoylphosphatidylcholine was not hydrolyzed.     

Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

Using [U-14C]phosphatidylinositol as substrate, Ca2+-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca2+ ("chromobindins," Creutz, C. E., Dowling, L. G., Sando, J. J., Villar-Palasi, C., Whipple, J. H., and Zaks, W. J. (1983) J. Biol. Chem. 258, 14664-14674). The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca2+ was complex; no activity was present in the absence of Ca2+, 25% activation occurred at 1 microM Ca2+, and full activation at 5 mM Ca2+. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca2+ but was released at 40 microM Ca2+, suggesting that intrinsic enzyme activity may be regulated by [Ca2+] at 1 microM, but additional activation at higher concentrations of Ca2+ is seen in vitro as a result of Ca2+-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.     

Characterization and partial purification of soluble, lysosomal phospholipase(s) A2 from adrenal medulla

Soluble, cation-dependent, lysosomal phospholipase A2 in bovine adrenal medulla has been biochemically characterized and partially purified, and its unique pH-dependent modulation by cations has been investigated. Chromatographically distinct activities with somewhat broad pI ranges centered at 7.8, 8.1, and 8.4 have been purified 83-, 1900- and 4400-fold, respectively, from the soluble fraction of tissue homogenates. With a specific activity of 4.2 mumol phospholipid hydrolyzed per mg protein per min, the fraction of pI 8.4 is the most highly purified lysosomal phospholipase A2 reported to date; yet silver staining of isoelectric focusing gels indicates that all three species are still only minor components of the protein mixtures with which they co-purify. Lysosomal phospholipase(s) A2 has an apparent molecular weight of 30,600, as determined by gel permeation chromatography; and is probably an oligomannose-containing glycoprotein as indicated by binding to concanavalin A-Sepharose and elution by methyl alpha-D-mannopyranoside. Cation concentrations modulate hydrolysis of biomembranous phospholipid, but not neat liposomal phospholipids, in a complex manner over a broad pH range (pH 4.0-8.0). Triton X-100 stabilizes the enzyme(s) but is inhibitory when present during assay; consequently, detergent-phospholipid mixed micelles are poor substrates. Thus, experimental results are dramatically dependent on the physicochemical nature of the substrate. The role of this phospholipase(s) A2 in the membrane fusion and lysis events of catecholamine secretion, as well as its regulation by cellular proteins, can now be investigated utilizing this partially purified enzyme(s).     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

It was the purpose of this study to define the chromogranin A-processing proteinases present in highly purified preparations of bovine chromaffin granules. The most active enzyme had a pH optimum of 5.0 and was inhibited by pepstatin. It could be identified immunologically as a cathepsin D-like enzyme and subcellular fractionation established its lysosomal origin. After removal of this enzyme the remaining activity at pH 5.0 was mainly due to a cathepsin B-like proteinase. The presence of this enzyme could also be attributed to lysosomal contamination. In the presence of calcium, a further proteolytic activity became apparent at pH 5.0. This enzyme which was inhibited by rho-chloromercuriphenylsulfonic acid was localized in chromaffin granules. A trypsin-like peptidase, most active at pH 8.2, was enriched in a membrane wash of chromaffin granules. Subcellular fractionation indicated that this enzyme is preferentially bound to the membranes of very dense particles probably representing a subpopulation of chromaffin granules. This study establishes that the most active chromogranin A-degrading proteinases present in highly purified chromaffin granules are attributable to lysosomal contamination. Two enzymes with low activity (a Ca2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.     

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.     

Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary.

Thiol and aspartyl proteolytic activities in isolated secretory vesicles of neural (NL) and intermediate (IL) lobes of bovine pituitary were characterized with heterologous enkephalin and tachykinin precursor substrates, 35S-(Met)-preproenkephalin and 35S-(Met)-beta-preprotachykinin. IL and NL secretory vesicles contained thiol-dependent proteolytic activity that cleaved the enkephalin precursor with a pH optimum of 4.5; this activity resembled a novel "prohormone thiol protease' previously purified and characterized from adrenal medulla chromaffin granules. IL and NL vesicles also demonstrated aspartyl proteolytic activity with acidic pH optimum, as shown by pepstatin A inhibition of tachykinin and enkephalin precursor cleaving activity. This activity may be related to a previously characterized chromaffin granule aspartyl protease (CGAP) related to cathepsin D (2), as indicated by the presence of immunoreactive CGAP in NL secretory vesicles by anti-CGAP immunoblots. These results show that pituitary secretory vesicles, like chromaffin granules, may contain similar thiol-dependent and aspartyl proteolytic activities.     

Inhibition of liver lysosomal acid phospholipase A1 by blood serum proteins

Pathophysiological conditions may lead to a release of lysosomal acid phospholipase A1 like that of other lysosomal enzymes into the blood stream. As shown here, various serum protein fractions, obtained by dye-ligand affinity chromatography, inhibit phosphoglyceride hydrolysis by lysosomal acid phospholipase A1 in vitro. Their inhibitory potencies vary considerably, and the degree of inhibition depends on the substrate concentration. A delayed phospholipid flotation rate in sucrose gradients in the presence of one of the more potent inhibitory serum proteins, serum albumin, suggests that the inhibition is due to inhibitor-substrate interactions. Although lysosomal phospholipase A1 activity at blood pH is extremely low, serum proteins may contribute to protect biomembranes which are exposed to the vascular lumen against uncontrolled destruction by this enzyme.     

Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization

We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location.     

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.     

The presence of phospholipase A2 in bovine adrenal medulla: arguments for more than one type of phospholipase A2.

Since phospholipase A2 (PLA2) is expected to play a role in the mechanism of exocytosis, the presence and subcellular localization of PLA2 in bovine adrenal medulla have been studied. The results of this study reveal that, although a large part of the PLA2 activity in chromaffin cells is due to a lysosomal PLA2, a cytoplasmic PLA2 is also present. This finding is supported by experiments in which the influence of pH, CaCl2 and NaCl on cytoplasmic PLA2 as well as the binding capacity to concanavalin A are investigated. According to these results the properties of a cytoplasmic PLA2 are clearly different from those reported by other authors for the lysosomal PLA2. For this reason, in chromaffin cells a PLA2 could be present which remains in the cytosol when the cell is in rest. Future experiments will have to prove whether this PLA2 becomes associated with the plasma membrane upon stimulation of the cell, thus mediating exocytosis.     

Phospholipid metabolism by phagocytic cells. Phospholipases A2 associated with rabbit polymorphonuclear leukocyte granules

Polymorphonuclear leukocytes obtained from sterile peritoneal exudates in rabbits contain two phospholipid-splitting activities (phosphatidylacylhydrolases EC 3.1.1.4), one most active at pH 5.5 and the other between pH 7.2 and 9.0. Hydrolysis of phospholipid was demonstrated using Escherichia coli labeled during growth with [1-(14)C]oleate and then autoclaved to inactivate E. coli phospholipases and to increase the accessibility of the microbial phospholipid substrates. The acid and alkaline phospholipase activities are both membrane bound, calcium dependent, and heat stable, and they appear to be specific for the 2-acyl position of phospholipids. Evidence was also obtained suggesting that the E. coli envelope phospholipids with oleate in position 2 are more readily degraded than those with palmitate. The two activities are associated with azurophilic as well as specific granules (obtained by zonal centrifugation) and with phagosomes (isolated after ingestion of paraffin particles by the granulocytes). Phospholipase A activities at pH 5.5 and pH 7.5 degrade the two major phospholipids of E. coli, phosphatidylethanolamine and phosphatidylglycerol, to the same extent, but the phospholipase activity at acid pH does not hydrolyze micellar dispersions of phosphatidylethanolamine. By contrast, phospholipase A(2) activity at pH 7.5 degrades both types of phosphatidylethanolamine substrates. Heparin and chondroitin sulfate inhibit phospholipase activity at pH 5.5 but have little effect on activity at pH 7.5. All detergents tested inhibited phospholipase activity, and both activities are inhibited by reaction products, free fatty acid and lysophosphatidylethanolamine. This product inhibition is only partially prevented by addition of albumin. Supernatant fractions of granulocyte homogenates contain a heat-labile inhibitor of granule phospholipase activity at pH 7.5. Boiling the fraction not only removes the inhibition but actually results in stimulation of hydrolysis at pH 7.5 as well as pH 5.5. These granule-associated phospholipase A activities of polymorphonuclear leukocytes differ in several of their properties from granule or lysosomal phospholipases of other phagocytic cells.     

Characterization of Proenkephalin-Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [35S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.     

Kex2-like proteolytic activity in adrenal medullary chromaffin granules.

This study demonstrates the presence of boc-Gln-Arg-Arg-MCA cleaving activity in bovine chromaffin granule membranes that resembles yeast Kex2 proteolytic activity. The chromaffin granule boc-Gln-Arg-Arg-MCA cleaving activity, like Kex2 proteolytic activity, shows calcium dependence, optimum activity at pH 7.5-8.2, inhibition by serine protease inhibitors, and preference for cleavage at the COOH-terminal side of Arg-Arg and Lys-Arg, over Lys-Lys, paired basic residues. Potent inhibition by the active-site directed inhibitor [D-Tyr]-Glu-Phe-Lys-Arg-CK (20 microM) provided further evidence for dibasic residue cleavage site specificity. These results are the first report of endogenous mammalian Kex2-like proteolytic activity that may be related to PC1/PC3 and PC2 enzymes, the newly discovered mammalian homologues of Kex2 protease. It will be important to determine the role of this Kex2-like proteolytic activity in processing the precursors of adrenal medullary neuropeptides.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

The role of negatively charged lipids in lysosomal phospholipase A2 function

Lysosomal phospholipase A2 (LPLA2) is characterized by increased activity toward zwitterionic phospholipid liposomes containing negatively charged lipids under acidic conditions. The effect of anionic lipids on LPLA2 activity was investigated. Mouse LPLA2 activity was assayed as C2-ceramide transacylation. Sulfatide incorporated into liposomes enhanced LPLA2 activity under acidic conditions and was weakened by NaCl or increased pH. Amiodarone, a cationic amphiphilic drug, reduced LPLA2 activity. LPLA2 exhibited esterase activity when p-nitro-phenylbutyrate (pNPB) was used as a substrate. Unlike the phospholipase A2 activity, the esterase activity was detected over wide pH range and not inhibited by NaCl or amiodarone. Presteady-state kinetics using pNPB were consistent with the formation of an acyl-enzyme intermediate. C2-ceramide was an acceptor for the acyl group of the acyl-enzyme but was not available as the acyl group acceptor when dispersed in liposomes containing amiodarone. Cosedimentation of LPLA2 with liposomes was enhanced in the presence of sulfatide and was reduced by raising NaCl, amiodarone, or pH in the reaction mixture. LPLA2 adsorption to negatively charged lipid membrane surfaces through an electrostatic attraction, therefore, enhances LPLA2 enzyme activity toward insoluble substrates. Thus, anionic lipids present within lipid membranes enhance the rate of phospholipid hydrolysis by LPLA2 at lipid-water interfaces.—Abe, A., and J. A. Shayman. The role of negatively charged lipids in lysosomal phospholipase A2 function.     

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.     

Phospholipid-deacylating enzymes of rat small intestinal mucosa.

1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.     

The selective release of phospholipase A2 by resident mouse peritoneal macrophages.

Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.     

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.