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.     

Mechanism of cytosol phospholipase C and sphingomyelinase-induced lysosome destabilization

Lysosomal disintegration may cause apoptosis, necrosis and some diseases. However, mechanisms for these events are still unclear. In this study, we measured lysosomal beta-hexosaminidase free activity, membrane potential and intralysosomal pH. The results revealed that the cytosolic extracts of rat hepatocytes could increase the lysosomal permeability to both potassium ions and protons, and osmotically destabilize lysosomes via K(+)/H(+) exchange. The effects of cytosol on lysosomes could be completely abolished by D609, which inhibited both phospholipase C and sphingomyelinase, and partly prevented by sphingomyelinase inhibitor Ara-AMP, but not by the inhibitors of PLA(2). Moreover, purified phospholipase C could destabilize the lysosomes while phospholipase A(2) and phospholipase D did not produce such effects. The cytosolic phospholipases hydrolyzed lysosomal membrane phospholipids by 50%, which could be prevented by D609. Disintegration of the cytosol-treated lysosomes biphasically depended on the cytosolic [Ca(2+)]. The cytosol did not disintegrate lysosomes below 100 nM or above 10 muM cytosolic [Ca(2+)], but markedly destabilized lysosomes at about 340 nM [Ca(2+)]. The results suggest that cytosolic phospholipase C and sphingomyelinase may be responsible for the alterations in lysosomal stability by increasing the ion permeability.     

Role of phospholipids in changes in lysosomal membrane stability in conditions of chronic alcoholic intoxication

Pronounced destabilization of liver lysosomal membranes has been revealed in rats in conditions of 30-day-long alcohol intoxication. Noticeable fractional changes in phospholipid composition of lysosomal membranes have been found. Significant increase in lysophosphatidylethanolamine and lysophosphatidylcholine levels have been observed. Type A2 phospholipase activity was found in lysosomal fractions, with the enzyme activity Ca2+-dependent, optimal at pH 8 and increasing many-fold following alcohol intoxication. The changes in lysosomal membrane phospholipids appear to be related to phospholipase A2 activation.     

Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A2 had an optimum pH at 4.5, and enzyme activation was observed in Ca++-free medium; but the maximum activity was found at 0.5 mM Ca++ concentration. The Km value for PC of acidic PLase A2 was 4.2 microM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A2 in light of the uncompetitive manner of Ca++ action. Furthermore, the release of [3H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca++ at pH 4.5. These data suggest that the acid PLase A2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A2 properties are opposite to those found for lysosomal PLase A2.     

Interrelationship between growth factor-induced activation of the Na+/H(+)-antiporter and mobilization of intracellular Ca2+ in NIH3T3-fibroblasts

Addition of serum growth factors or bombesin to quiescent NIH3T3-fibroblasts leads to a simultaneous mobilization of intracellular Ca2+ and an increase in cytosolic pH which is inhibitable by dimethylamiloride. The mobilization of intracellular Ca2+ is a pH-dependent process with an optimum at pH 7.1. In quiescent cells with a pHi greater than or equal to 6.8, inhibition of the Na+/H(+)-antiporter by dimethylamiloride or reduction of extracellular Na+ attenuates the growth factor induced Ca2(+)-response. It is concluded that the growth factor induced activation of the Na+/H(+)-antiporter facilitates the mobilization of Ca2+ by shifting the internal pH towards the optimum for the Ca2(+)-release.     

Properties of canine myocardial phosphatidylethanolamine N-acyltransferase

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.     

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.     

Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.     

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 the mitochondrial Na+/Ca2+ antiport by matrix pH

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.     

Differentiation of phospholipases A in mitochondria and lysosomes of rat liver

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).     

Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes.

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.     

Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle

We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.     

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.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Biomembrane-modulated, lysosomal phospholipase A2 contamination of chromaffin granule ghosts

It was reported that subcellular fractionation of bovine adrenal medulla results in the separation of distinct, non-calcium-dependent phospholipases A2--one associated with chromaffin granule ghosts, another with lysosomes. The basis of this distinction is pH optimum: in routine assays utilizing neat liposomal substrates, the chromaffin granule ghost-associated enzyme is alkaline-active whereas the lysosomal enzyme is acid-active (Husebye, E.S. and Flatmark, T. (1987) Biochim. Biophys. Acta 920, 120-130). We now report that biomembranes after liposomal substrates and/or lysosomal phospholipase A2 such that the enzyme now hydrolyzes them (at low cation concentration) with an alkaline pH optimum. In a lysosomal membrane fraction, phospholipase A2 activity at pH 7.5 relative to activity at pH 5.0 increases as increasing amounts of lysosomal membranes are assayed. The pH optimum of chromaffin granule ghost-associated phospholipase A2 toward liposomal substrates is likewise biomembrane-dependent and, when assayed carefully, is indistinguishable on the basis of optimal pH from the lysosomal enzyme. Although chromaffin granule ghost-associated phospholipase A2 is most likely a lysosomal contaminant, its broad, biomembrane-modulated pH range may still allow it to participate in catecholamine secretion. More importantly, however, sensitivity of adrenal medullary lysosomal phospholipase A2 to biomembranes broadens its potential physiologic pH range and may also play a role in the regulation of this potentially deleterious activity.     

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.     

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.     

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane Ca2(+)-ATPase

The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.     

Isolation and characterization of a phospholipase A2 from an inflammatory exudate.

Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed.