Biochimica Elsevier
BBALIP
et Biophysics
139
Acta, 1044 (1990) 139-146
53383
Studies on the acyl-chain selectivity of cellular phospholipases A, C.G. Schalkwijk
‘, F. Miirki ’ and H. Van den Bosch’
’ Centre for Biomembranes and Lipid Enzymology,
Utrecht (The Netherlands) and 2 Ciba-Geigy Limited, Pharmaceuticals Division, Base/ (Switzerland) (Received
Key words:
Phospholipase
A,; Acyl chain selectivity;
7 November
(Human
Research Department,
1989)
polymorphonuclear
leukocyte);
(Human
platelet);
(Rat platelet)
selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A *. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A, preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2[ “C]oleoylphosphatidyIcholine and sn-2-(3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A,. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A,. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A 2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-24inoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A 2 are discussed. The
Introduction A selective release of arachidonic acid, as opposed to monoenoic and dienoic fatty acids, from prelabeled human platelets in response to thrombin stimulation of the cells has been reported [1,2]. In subsequent experiments using cells prelabeled with glycerol, this specificity was not observed and all phosphatidylcholine species were hydrolyzed by endogenous phospholipase A, to similar extents [2,3]. These results suggested that preincubation of cells with either fatty acids or glycerol may label specific phospholipid pools that are not representative for total mass distributions. A recent study on the endogenous sources of arachidonate in the human neutrophil also emphasized the major discrepancies between using label or mass to determine cellular sources of arachidonate for eicosanoid formation [4]. To circumvent the problems introduced by prelabeling cells Purdon et al. [5] determined the release of fatty acids
Abbreviations: GPC, nuclear leukocytes.
glycerophosphocholine;
PMN,
polymorpho-
Correspondence: H. Van den Bosch, Centre for Biomembranes and Lipid Enzymology, Transitorium III, Padulaan 8, 3584 CH Utrecht, The Netherlands.
0005-2760/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
from thrombin-stimulated platelets as mass changes in individual molecular species of phospholipids. These investigators arrived at the conclusion that the deacylation was selective for arachidonate species of phosphatidylcholine and phosphatidylethanolamine and explained this finding in intact cells by assuming that a certain proportion of arachidonate species of phospholipids are compartmentalized in the platelet membranes proximal to the site of action of the phospholipase A,. The alternative explanation of specific arachidonate recognition by the enzyme’s active site was considered less likely in view of the fact that acyl-chain selectivity was not observed with partially purified rabbit platelet phospholipase A, and micellar or aggregated phospholipids [6]. However, initial studies with isolated platelet membranes have also provided indications for the selective release of arachidonate from endogenous phospholipids by phospholipase A, [7,8]. This selectivity was best observed with minimal disruption of membrane structure [8] and was lost in platelet homogenates [7] or sonicates [9]. Although the selective release of arachidonate in intact cells can be explained by assuming that the phospholipase A, involved is present in a domain of arachidonate-enriched phospholipid species two other lines of evidence suggest that arachidonate-selective phospholipases A, might exist. First, experiments with rat polymorphonuclear leukocytes have shown a highly Division)
140 reduced rate of platelet activating factor synthesis in cells depleted of arachidonic acid by dietary means [lo]. The role of l-O-alkyl-2-arachidonoylglycerophosphocholine as a common precursor from which lyso-platelet activating factor and arachidonate can be produced by phospholipase A, action is well documented [ll-131. The reduced platelet activating factor synthesis in arachidonate-depleted cells could thus be explained by assuming that lyso-platelet activating factor cannot be formed in sufficient amounts, since other species of l-0-alkyl-2-acylglycerophosphocholine are much less efficiently hydrolyzed by an arachidonate-selective phospholipase A,. Secondly, a number of recent papers have reported evidence for the occurrence of arachidonate-selective phospholipases A 2 in cell-free systems such as those prepared from bone marrow-derived macrophages [14], human neutrophils [15], human platelets [16] and mouse peritoneal macrophages [17]. With the exception of the latter case the in vitro acyl-chain selectivity of these phospholipases A, was assessed by using separate vesicles of phosphatidylcholine species differing in their fatty acyl constituent at the sn-2-position. With this approach the results may be influenced by the physical-chemical properties and structural arrangements of the substrates. For example, the arachidonoyl species may form a less densely packed bilayer, thus allowing better penetration of the enzyme and giving rise to a higher hydrolysis rate that is governed by the properties of the substrate rather than by a true acyl-chain selectivity of the enzyme. In this paper we have used mixed and differentially labeled phospholipid substrates to investigate whether arachidonate specific phospholipases A, are lost during preparation of enzyme extracts from human PMN. In addition, the acyl-chain selectivity of phospholipases AZ in preparations from human PMN and human and rat platelets was investigated using separate substrates and doubly-labeled substrates, in which the two species were mixed before preparation of the phospholipids vesicles in order to present the substrates to the enzyme in a single bilayer. Materials and Methods Materials
[9,10-3H]Oleic acid, [l-i4C]oleic acid, [l-‘4C]linoleic acid, l-palmitoyl-2-[l-‘4C]oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-[5,6,8,9,11,12,14,15-3H]arachidonoyl-sn-glycero-3-phosphocholine and l-acyl-2-[l-i4C]arachidonoyl-sn-glycero-3-phosphoethanolamine were all obtained from Amersham (Buckinghamshire, U.K.). lStearoyl-2-arachidonoylphosphatidylcholine was bought from Sigma (St. Louis, MO, U.S.A.). 1-Palmitoyl-2oleoylphosphatidic acid, disodium salt, was purchased from Avanti Polar Lipids (Birmingham, AL, U.S.A.).
Methods Preparation
of substrates. I-Acyl-2-[ 3H]oleoyland 1-acyl-2-[‘4C]linoleoylphosphatidylethanolamine were biosynthetically prepared using rat liver microsomes as previously described [ 181. Phospholipase A 2 degradation of the purified compounds indicated that 86% of [3H]oleate and 97% of [‘4C]linoleate was located at the sn-Zposition. Escherichia coli membrane substrate labeled with either [3H]oleate or [‘4C]oleate was prepared as described [19,20]. Attempts to doubly label the membrane phospholipids by growing the cells in the presence of [ 3H]oleate and [i4C]arachidonate were unsuccessful due to low incorporation of arachidonic acid. Satisfactory results were obtained by growing cells in the presence of 10 PCi of [3H]oleate and 2.5 PCi of 1-acyl-2-[1i4C]arachidonoy 1p hosphatidylethanolamine in 50 ml medium. Washing the cells two times with saline containing 0.5% bovine serum albumin and autoclaving them to inactivate endogenous phospholipases yielded a double-labeled E. coli membrane substrate with a 3H/‘4C ratio of 0.86 from which pancreatic phospholipase A, released fatty acids with an isotopic ratio of 2.6. Specific radioactivities of the substrate are indicated in the legends to the tables. For the acyl-chain selectivity studies with phosphatidylcholines, l-palmitoyl-2-[l-‘4C]oleoyl-GPC was used at its commercially available specific radioactivity of 52 mCi/mmol. I-Stearoyl-2-[ 3H]arachidonoyl-GPC (120 Ci/mmol) was diluted with the unlabeled species to give a final specific radioactivity of 52 mCi/mmol. Doubly labeled substrates were prepared by mixing equimolar amounts of the two species in organic solvents. After addition of 5 mol% phosphatidic acid the phospholipids were dried under nitrogen and dispersed in distilled water at a concentration of 19 PM by sonication for three times 20 s in an ice bath under nitrogen. Enzyme preparations. Isolation of human polymorphonuclear leukocytes and preparation of cell homogenates and acid extracts were done as previously described [20], except that the stirring after addition of ice-cold 0.18 M H,S0,/2 M NaCl at pH 1.6 was done for 4 h at 0” C rather than overnight. A 1 M KC1 extract from PMN homogenate was prepared by mixing equal volumes of homogenate and 2 M KCl, stirring at 4” C overnight and centrifugation for 30 min at 100 000
x g. Isolation of rat and human platelets and preparation of a 1 M KC1 extract from rat platelets, containing over 90% of the phospholipase A, activity measurable in platelet lysates, was done as described [21,22]. The preparation of human platelet extract was done as described by Kramer et al. [23]. It has previously been shown [16,20,21,23] that release of sn-Zfatty acids from
141 phospholipids in these enzyme preparations is caused by phospholipase A, activity. Phospholipase A, assays. Enzyme activity was measured at 37 o C in a total volume of 0.5 ml containing 5 mM Ca2+ and 0.1 M Tris-HCl buffer and substrates as specified in the legends of tables and figures. E. coli substrates were assayed at the optimal pH of 7.0, whereas phosphatidylethanolamine and phosphatidylcholine assays were run at the optimal pH of 8.5. Where specific activities are given it was ensured that the reactions were linear with time and amount of enzyme preparation. Typically, an amount of enzyme equivalent to (20-40). lo6 PMN cell equivalents was used and the reaction was linear up to at least 80. lo6 cell equivalents. Reactions were stopped after 1 h by a modified Dole extraction procedure [18] and radioactivities in released fatty acids were determined by liquid scintillation spectrometry according to established procedures, Analytical procedures. Protein was determined by the dye binding assay described by Bradford [24]. E. coli membrane phospholipids were extracted according to Bligh and Dyer [25] and quantitated by lipid phosphorus analysis as described by Rouser et al. [26]. Results Previous results have shown [20] that phospholipase A, activity in homogenates of human polymorphonuclear leukocytes is suppressed by endogenous inhibitors that can be separated from the enzyme by acid extraction. Studies on the acyl-chain selectivity of the phospholipase(s) A, in such an extract should preferably include experiments with the original homogenate to exclude the possibility that a putative enzyme with selectivity for a certain fatty acid, e.g., arachidonate, was lost during the acid extraction procedure. In initial experiments we compared the phospholipase A, activities of homogenates with those in extracts and pellets obtained after a harsh extraction procedure using sulfuric acid at pH 1.6 and a mild extraction procedure using 1 M KC1 at pH 7.4. In all cases, phospholipase A, activity was measured both with l-acyl-2-[‘4C]linoleoylphosphatidylethanolamine at the optimal pH of 8.5 and with [14C]oleate-labeled E. coli membranes at the optimal pH of 7.0. Table I shows that the use of E. cofi membranes represents a much more sensitive assay system than pure phosphatidylethanolamine, especially for fractions other than the acid extracts. The total activity with phosphatidylethanolamine as substrate increased 9-fold in the acid extract when compared to homogenate, presumably due to removal of inhibiting substances. Such inhibitors are apparently not separated from the enzyme upon extraction of the homogenate with 1 M KCl. The endogenous suppressors of phospholipase A, activity in PMN homogenates have been identified as unsaturated fatty acids and derivatives
TABLE
I
Phospholipase A, activities in human PMN fractions Enzyme activities with the indicated substrates were determined as described under Materials and Methods. Mean values of two experiments differing by less than 15% are expressed in pmol/min per IO9 cell equivalents. Data in brackets represent specific activities in pmol/min per mg protein. PE substrate, l-acyl-2-[i4C]linoleoylphosphatidylethanolamine (100 yM, 250000 dpm); E. coli substrate, [“‘Cloleate-labeled E. coli membranes (80 PM, 12000 dpm). Fraction
Phospholipase
Homogenate Acid pellet Acid extract KC1 pellet KC1 extract
A 2 activity
PE substrate
E. coli substrate
36 (2) 20 (2) 315 (57)
1250 (55) 425 (32) 1675 (310) 425 (SO) 755 (58)
IO (2) 2 (0.2)
thereof [20]. Attempts to increase the phospholipase A, activity by extraction of the homogenate with diethyl ether prior to assay with phosphatidylethanolamine or by including bovine serum albumin or octyl glucoside yielded activities ranging from 15 to 40% of the original activity. For unknown reasons the effect of removal of endogenous suppressors during acid extraction was much less using E. coli substrate. The specific phospholipase A, activity of 55 pmol. min-’ 1mg protein-’ with E. coli substrate in homogenates is in good agreement with the value of 31 pmol *rnin-’ . mg protein-’ reported by Victor et al. [27]. The activities measured with E. coli substrate in homogenates, acid extract and KC1 extract allowed a comparison of the acyl-chain selectivity of the enzyme(s) in these fractions using double-labeled E. coli membranes containing [ 3H]oleate and [‘4C]arachidonate. The results (Table II), indicate that the enzyme(s) in both acid extract and KC1 extract release the two fatty acids in a ratio that is actually
TABLE
II
Acyl-chain selectivity of human PMN phospholipase A, Enzyme activities with the indicated double-labeled substrates containing [3H]oleate and [‘4C]arachidonate at the sn-2-position were determined as described under Materials and Methods. 3H/‘4C ratios at the sn-2-position of the substrates were determined after complete hydrolysis by porcine pancreatic phospholipase A, and amounted to 1.3 fO.l for PE substrate and 2.6kO.2 for E. cofi substrate. Mean values+S.D. (n = 3) are given. n.c., not conclusive. PE substrate, I-acyl-2-[ 3H]oleoyl/[14C]arachidonoylphosphatidylethanolamine (100 PM, 325000 dpm 3H and 217500 dpm 14C). E. coli substrate, [3H]oleate/[‘4C]arachidonate-labeled E. coli membranes (80 FM, 13 300 dpm ‘H and 15 300 dpm 14C). PMN fraction
3H/‘4C
PE substrate
E. coli substrate
Homogenate Acid extract KC1 extract
n.c. 1.4*0.1
3.4f0.4 1.1*0.1 1.9+0.4
XC.
in released
fatty acids
142 300
200
100
0
0 Time
(min)
Fig. 1. Acyl-chain selectivity of human PMN phospholipase A,. The reaction mixture with a total volume of 0.5 ml contained 0.1 M Tris-HCl buffer (pH 8.5) 10 mM CaCl,, 1.9 nmol of sonicated substrate including 5 mol% phosphatidic acid and an amount of immunopurified human PMN phospholipase A, equivalent to 42.106 cells. (A), l-Stearoyl-2-[3H]arachidonoyl-GPC (o) or l-palmitoyl-2-[‘4C]oleoyl-GPC (+). (B), Cosonicate of 1.9 nmol each of the substrates used in A. After incubation at 37°C for the indicated time periods the reaction was stopped and radioactivities in the released fatty acids were determined as described under Materials and Methods.
somewhat lower than that in which they are present at the sn-2-position of the phospholipids as determined by hydrolysis of the substrate with pancreatic phospholipase A,. Thus, the extracted enzyme(s), while hydrolyzing less than 5% of labeled substrates, certainly show no loss of preference for arachidonate release. In addition, a comparison with the [3H]oleate/[‘4C]arachidonate ratio released by the homogenate enzyme(s) clearly shows no evidence for a specific loss of an arachidonate releasing enzyme (in which case the ‘H/14C ratio should have increased) upon preparation of the acid extract by a harsh procedure or the KC1 extract by a mild procedure. The enzymatic activities in homogenates and KC1 extract with doubly labeled phosphatidylethanolamine as substrate were too low (compare Table I) to give conclusive results. As can be seen in Table II, the acid extract released [ 3H]oleate and [‘4C]arachidonate in a ratio identical to that present at the sn-2-position of this substrate and thus confirmed the lack of acyl-chain selectivity of the phospholipase A,. Further experiments were done with the acid extract and aimed to study acyl-chain selectivities with phosphatidylcholine substrates. Pure rat liver phosphatidylethanolamine adopts a hexagonal H,, organization at the assay conditions at 37 o C in the presence of remain 10 mM Ca 2+ [28], whereas p hosphatidylcholines organized in extended bilayers [28,29]. Because of this difference in lipid polymorphism we decided to perform acyl-chain selectivity experiments with phosphatidylcholine, a substrate representing the natural bilayer membrane as much as possible with respect to lipid organization. Since the phospholipase A, activity in the acid extract towards phosphatidylcholine was lo-fold lower
than that towards phosphatidylethanolamine, a further purification of the enzyme(s) was attempted. This was achieved by immunoprecipitation of the enzyme. As can be seen in Table III, essentially all of the enzyme was bound to Sepharose particles to which a monoclonal antibody against rat liver mitochondrial phospholipase A, had been coupled [22,30]. The enzyme could be recovered in good yield by washing the precipitate with glycine buffer at pH 2.5. Enzyme thus obtained was used to study acyl-chain selectivities with l-palmitoyl2-[i4C]oleoyl-GPC and l-stearoyl-2-[ 3H]arachidonoylGPC. In Fig. 1A the release of oleate and arachidonate from the corresponding phosphatidylcholine membranes in separate tubes is plotted as function of time. Clearly, there is no preferential release of arachidonate TABLE
III
Binding of human PMN phospholipuse A2 to anii-rat liver phospholipase AZ monoclonal antibodies Acid extracts of human PMN were incubated with Sepharose carrying antibodies (spec. either anti-rat liver phospholipase A, monoclonal McAb) or non-related monoclonal antibodies (aspec. McAb) as previously described [22,30]. Phospholipase A z activities were measured in the supernatant remaining after centrifugation and in the supernatant obtained after desorbing the enzyme from the Sepharose pellet with glycine buffer (pH 2.5) and are expressed in pmol phosphatidylethanolamine hydrolyzed per min. Results of two experiments are given. Enzyme
fraction
Acid extract Supernatant Desorbed Bound (8) Total recovery
(W)
Spec. McAb
Aspec. McAb
112; 101 17; 8 73; 64
112; 101 52; 48 4; 1
81; 80;
89 71
7; 50;
2 49
143 chains. This was confirmed by the experiments depicted in Fig. 1B in which the differentially labeled phosphatidylcholines were mixed in organic solvents prior to vesicle formation. Since the reported [14] selectivity for arachidonate release from single-labeled phosphatidylcholine species by a macrophage phospholipase A, was no longer expressed at substrate concentrations above 10 PM, we investigated the acyl-chain selectivity of the PMN phospholipase A, at lower substrate concentrations. As can be seen in Fig. 2 lowering the concentration of either substrate did not result in an apparent selectivity for arachidonate. A similar lack of acyl-chain selectivity as observed for human PMN phospholipase A, (Fig. 1) was also found in studies with an enzyme preparation from rat platelets (Fig. 3). This is in agreement with the lack of acyl-chain selectivity of this enzyme as recently
6-
1
0
4
3
2 Substrate
(JAM1
Fig. 2. Effect of substrate concentration on acyl-chain selectivity of human PMN phospholipase A,. Conditions and symbols as in the legend of Fig. lA, except that substrate concentrations were varied as indicated.
5
600
600
A
B 1
01
I
I
I
I
I
I
I
I
0
2
4
6
0
0
2
4
6
Protein Fig. 3. Acyl-chain
selectivity of rat platelet phospholipase of rat platelets prepared as described
IO 8
lpg )
A,. Conditions and symbols as in the legend of Fig. l.A, except that a 1 M KC1 extract [22] was used as enzyme source and incubation was for 1 h at 37 o C.
Protein
t pg 1
Fig. 4. Acyl-chain selectivity of human platelet phospholipase A,. Conditions and symbols as in the legend of Fig. lA, except that a human platelet supernatant fraction prepared as described [23] was used as enzyme source and incubation was for 1 h at 37 o C. In panel A and B the substrates were as in Fig. 1A and lB, respectively. In panel C the substrate consisted of vesicles composed of 1.9 nmol each of the labeled substrates used in A and B together with 9.5 nmol I-palmitoyl-2-linoleoyl-GPC and 0.5 nmol phosphatidic acid.
144 deduced from experiments utilizing separate vesicles prepared from either sn-2-arachidonoyl-GPC or sn-2linoleoyl-GPC, respectively [16]. In contrast to the rat platelet enzyme, the phospholipase A, in the cytosolic fraction of human platelets showed a lo-fold selectivity for arachidonate versus linoleate release from these substrates [16]. As can be seen in Fig. 4A a similar selectivity was observed for arachidonate versus oleate release when vesicles composed of single phosphatidylcholine species were compared. However, this apparent selectivity for arachidonoyl chains became much reduced when the substrates were presented in a mixed bilayer with a constant quality of the lipid/water interface (Fig. 4B) and was completely lost when the oleateand arachidonate-containing substrates were embedded at a concentration of 14 mol% each in a matrix otherwise composed of 68 mol% of l-palmitoyl-2-linoleoylGPC and 4 mol% phosphatidic acid (Fig. 4C). Exactly the same results as described in Figs. 1 to 4 were obtained when phosphatidic acid, used to introduce a negative charge in the membranes, was omitted from the vesicles, except that the phospholipase activities were about 20% less (data not shown). Discussion As briefly reviewed in the Introduction, the initial indications for the possible existence of arachidonatespecific phospholipases A, were provided by the selective release of this fatty acid after stimulation of prelabeled intact cells. In these cases, other factors such as specific labeling of certain phospholipid pools and compartmentalization of the activated enzyme can contribute to the observed selectivity [5]. The ultimate proof for the existence of an arachidonate-specific phospholipase A, should eventually come from studies with homogeneous enzymes under well-defined conditions. The purification of such an enzyme requires a cell-free system in which the selectivity for arachidonate is maintained in order to trace the enzyme during purification. Only a few cell-free systems have been reported to possibly contain arachidonate-selective phospholipases A *. Flesch et al. [14] described the acyl-chain specificity and kinetic properties of a phospholipase A, in homogenate and supernatant of bone marrow-derived macrophages. The soluble phospholipase A, preparation showed a marked specificity for arachidonate release when incubated with low concentrations of either l-palmitoyl-2-oleoyl-GPC, 1-palmitoyl-2-linoleoyl-GPC or 1 -palmitoyl-2-arachidonoyl-GPC. Thus, at substrate concentrations of 0.76 PM, values of 9.0, 4.0 and 84 pmol/min per mg were measured for oleate, linoleate and arachidonate release, respectively. However, when each substrate was used at its saturating concentration, these differences had largely disappeared due to the fact that the enzyme showed by far the highest affinity for
arachidonate-containing phosphatidylcholine species. Thus, the apparent specificity for arachidonate appears to be mainly caused by a K, effect, which may be influenced by substrate organization rather than being a true acyl-chain specificity due to specific recognition of the arachidonate structure in the active site of the enzyme involved. An arachidonoylphosphatidylcholine-hydrolyzing phospholipase A, has also been described in the cytosolic fraction of human neutrophils [15]. Competitive experiments with unlabeled substrates suggested that the phospholipase showed specificity for arachidonate compared to oleate and linoleate. In these experiments, preformed vesicles of unlabeled phosphatidylcholine species were added to vesicles composed of l-palmitoyl-2-[‘4C]arachidonoyl-GPC. In principle, the results of such an approach to study acyl-chain specificities might also be influenced by differential binding of the enzyme to the different vesicles. The fact that the addition of an equimolar amount of unlabeled arachidonoylphosphatidylcholine caused only 5% inhibition of [i4C]arachidonate release, rather than the expected 50%, stresses this point. As emphasized previously [31], in vitro studies on the acyl-chain selectivity of phospholipases A, should preferably use the phospholipids under investigation in a mixed membrane, rather than as separate lipid aggregates, to minimize the influence of physical-chemical factors and quality of the interfaces on the outcome of the experiments. When this approach was taken utilizing double labeled substrates in a single lipid aggregate, no indication for the existence of an arachidonate-specific phospholipase A, in homogenates or extracts of human PMN was obtained. It can not be excluded that neutrophils may contain several phospholipases A, and that the enzyme isolated by acid treatment is involved in other processes than arachidonate release, such as degradation of ingested bacterial phospholipids and thus might not be expected to exhibit arachidonate specificity. It should be mentioned, however, that no evidence for the selective loss of such an arachidonate-selective enzyme during extract preparation was found (Table II). The immunopurified enzyme hydrolyzed sn-2-oleoyl phosphatidylcholine equally well as the sn-2-arachidonoyl species, irrespective of whether the substrates were presented as pure species or in mixed bilayers (Fig. 1) and independent of the substrate concentration (Fig. 2). Similar results were obtained (Fig. 3) for a rat platelet phospholipase A, preparation that contains at least 90% of the measurable phospholipase A, activity in rat platelet lysates [22]. This confirms previous results comparing pure phospholipid species [9,16] and extends the lack of arachidonate specificity to the use of substrates in a mixed bilayer. An interesting phenomenon was observed with human platelet cytosol. In full agreement with results recently reported by Kim et al. [16] the data obtained
145 with single phosphatidylcholine species (Fig. 4A) were highly suggestive for the occurrence of an arachidonate-selective phospholipase A, in human platelets. This interpretation was strengthened by the fact that a selective release of arachidonate using the same substrates and conditions was not found with rat platelets and, therefore, seemed to exclude substrate effects. Yet, when we extended the studies with human platelet phospholipase A, to substrates in mixed bilayers (Fig. 4B and C), the apparent selectivity for arachidonate was nearly completely lost. This prompted a possible alternative explanation for the initially observed acyl-chain selectivity. This is schematically indicated in Fig. 5. It is quite reasonable to assume that the lipid packing in vesicles composed of 1-saturated-2-oleoyl-GPC is different from that in vesicles composed of 1-saturated-2arachidonoyl-GPC. Monolayer studies [32] have shown that the area per molecule at a constant surface pressure of 12 dynes/cm amounted to 77, 84 and 88 A2 for phosphatidylcholines having a saturated fatty acid at the l-position and oleate, linoleate or arachidonate, respectively, at the 2-position. It can thus be expected that the molecules in an arachidonoylphosphatidylcholine vesicle are more loosely packed than those in an oleoylphosphatidylcholine vesicle and allow for a better penetration of the phospholipases A,. Large differences in the membrane-penetrating capacity of various phospholipases A, have been reported [33]. Porcine pancreatic phospholipase A, hydrolyzed monolayers of lpalmitoyl-2-oleoyl-GPC only below initial surface pressures of 16.5 dynes/cm, whereas these cut-off points were 23.0, 34.8 and 35.3 dynes/cm for Crotalus adamanteus, Naja naja and bee venom phospholipases A,, respectively. It could thus be envisaged that phospholipase A, from human PMN and rat platelet penetrate as efficiently in 1-saturated-2-oleoyl-GPC vesicles as in 1-saturated-2-arachidonoyl-GPC vesicles
and hence show no apparent acyl-chain selectivity. Human platelet phospholipase A, might penetrate much more efficiently in the more loosely packed l-saturated2-arachidonoyl-GPC vesicles resulting in a lower K, value and an apparent selectivity for arachidonate when vesicles composed of single phosphatidylcholine species are compared. Mixing of the phosphatidylcholine species in vesicles may result in less efficient penetration (compare total hydrolyses rates in Fig. 4A and B) and a loss of most of the acyl-chain selectivity. Although such an interpretation could explain the different acyl-chain selectivities observed with single versus mixed-species substrates, it cannot be validated until more insight is obtained in the penetrating capacity of purified cellular phospholipases A,. In the absence of such data it is still felt that these possibilities should be kept in mind before definitive statements on the existence of arachidonate-specific phospholipases A, can be made. On the other hand the model, although emphasizing some of the pitfalls that can be encountered in acyl-chain selectivity studies, is not meant to deny that arachidonate specific phospholipases A, might exist. For example, a recently described 70 kDa phospholipase A, from mouse peritoneal macrophages [17] exhibited a 3.5-fold preference for arachidonate versus oleate release, even though the substrate consisted of an equimolar mixture of the parent phosphatidylcholines. As so often, however, a definitive answer can probably only be obtained by the complete purification of such a putative enzyme or, at least, its physical separation during chromatographic steps from other phospholipases A,. References 1 Bills, T.K., Smith, J.B. and Silver, M.J. (1977) J. Clin. Invest. 60, 1-6. 2 Mahadevappa, V.G. and Holub, B.J. (1984) J. Biol. Chem. 259, 9369-9373.
1 -sat. - 2 - oleoyl - GPC
1 - sat. - 2 - arachidonoyl - GPC
MIXED SPECIES
Fig. 5. Model to explain
the different
acyl-chain
selectivities
of various
phospholipases
A,. Compare
text for details
146 3 Holub, B.J. (1984) Can. _I. Biochem. Cell Biol. 62, 341-351. 4 Chilton, F.H. and Connel, T.R. (1988) J. Biol. Chem. 263, 52605265. 5 Purdon, A.D., Patelunas, D. and Smith, J.B. (1987) Biochim. Biophys. Acta 920, 205-214. 6 Kannagi, R. and Koizumi, K. (1979) Arch. Biochem. Biophys. 196, 534-542. 7 Derksen, A. and Cohen, P. (1975) J. Biol. Chem. 250, 9342-9347. 8 Kannagi, R., Koizumi, K. and Masuda, T. (1981) J. Biol. Chem. 256, 1177-1184. 9 Colard, O., Breton, M. and Bereziat, G. (1987) Biochim. Biophys. Acta 921, 333-334. 10 Ramesha, C.S. and Pickett, W.C. (1986) J. Biol. Chem. 261, 7592-7595. 11 Chilton, F.H., O’Flaherty, J.T., Ellis, J.M., Swendsen, C.L. and Wykle, R.L. (1983) J. Biol. Chem. 258, 726X-7271. 12 Chilton, F.H., Ellis, J.M., Olson, S.C. and Wykle, R.L. (1984) J. Biol. Chem. 259, 12014-12019. 13 Kramer, R.M., Patton, G.M., Pritzker, C.R. and Deykin, D. (1984) J. Biol. Chem. 259, 13316-13320. 14 Flesch. I., Schmidt, B. and Ferber, E. (1985) Z. Naturforsch. 4OC, 356-363. 15 Alonso, F., Henson, P.M. and Leslie. C.C. (1986) Biochim. Biophys. Acta 878, 273-280. 16 Kim, D.K., Kudo, I. and moue, K. (1988) J. Biochem. 104, 492-494. 17 Wijkander. J. and Sundler, R. (1989) FEBS Lett. 244, 51-56. 1X Van den Bosch, H., Aarsman, A.J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 348, 197-207. 19 Franson, R., Patriarca, P. and Elsbach, P. (1974) J. Lipid Res. 15, 3X0-388.
20 MPrki, F. and Franson, R. (1986) Biochim. Biophys. Acta 897, 149-156. 21 Aarsman, A.J., Roosenboom, C.F.P., Van Geffen, G.E.W. and Van den Bosch, H. (1985) Biochim. Biophys. Acta 837, 28X-295. 22 Aarsman, A.J., Leunissen-Bijvelt, J., Van den Koedijk, C.D.M.A.. Neys, F.W., Verkleij, A.J. and Van den Bosch, H. (19X9) J. Lipid Med. 1, 49-61. 23 Kramer, R.M.. Jakubowski, J.A. and Deykin, D. (1988) Biochim. Biophys. Acta 959, 269-279. 24 Bradford, M.M. (1976) Anal. Biochem. 72, 24X-254. 25 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. 26 Rouser, G., Fleischer, S. and Yamamoto, A. (1970) Lipids 5. 494-496. 27 Victor, M., Weiss, J., Klempner, MS. and Elsbach, P. (1981) FEBS Lett. 136, 29X-300. 2X Lenting, H.B.M., Neys, F.W. and Van den Bosch, H. (1987) Biochim. Biophys. Acta 917, 17X-185. 29 Cullis, P.R. and De Kruijff, B. (1979) Biochim. Biophys. Acta 559. 399-420. 30 Aarsman, A.J., De Jong, J.G.N., Arnoldussen, E., Neys. F.W., Van Wassenaar, P.D. and Van den Bosch, H. (1989) J. Biol. Chem. 264, 10008-10014. 31 Van den Bosch, H. (1989) in UCLA Symposia on Molecular and Cellular Biology New Series (Fox, CF., ed.), Vol. 106, pp. 317-321. Alan R. Liss, New York. 32 Demel, R.A., Geurts van Kessel, W.S.M. and Van Deenen. L.L.M. (1972) Biochim. Biophys. Acta 266, 26-40. 33 Demel, R.A., Geurts van Kessel, W.S.M., Zwaal, R.F.A., Roelofsen, B. and Van Deenen, L.L.M. (1975) Biochim. Biophys. Acta 406, 97-107.
BBALIP
et Biophysics
139
Acta, 1044 (1990) 139-146
53383
Studies on the acyl-chain selectivity of cellular phospholipases A, C.G. Schalkwijk
‘, F. Miirki ’ and H. Van den Bosch’
’ Centre for Biomembranes and Lipid Enzymology,
Utrecht (The Netherlands) and 2 Ciba-Geigy Limited, Pharmaceuticals Division, Base/ (Switzerland) (Received
Key words:
Phospholipase
A,; Acyl chain selectivity;
7 November
(Human
Research Department,
1989)
polymorphonuclear
leukocyte);
(Human
platelet);
(Rat platelet)
selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A *. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A, preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2[ “C]oleoylphosphatidyIcholine and sn-2-(3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A,. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A,. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A 2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-24inoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A 2 are discussed. The
Introduction A selective release of arachidonic acid, as opposed to monoenoic and dienoic fatty acids, from prelabeled human platelets in response to thrombin stimulation of the cells has been reported [1,2]. In subsequent experiments using cells prelabeled with glycerol, this specificity was not observed and all phosphatidylcholine species were hydrolyzed by endogenous phospholipase A, to similar extents [2,3]. These results suggested that preincubation of cells with either fatty acids or glycerol may label specific phospholipid pools that are not representative for total mass distributions. A recent study on the endogenous sources of arachidonate in the human neutrophil also emphasized the major discrepancies between using label or mass to determine cellular sources of arachidonate for eicosanoid formation [4]. To circumvent the problems introduced by prelabeling cells Purdon et al. [5] determined the release of fatty acids
Abbreviations: GPC, nuclear leukocytes.
glycerophosphocholine;
PMN,
polymorpho-
Correspondence: H. Van den Bosch, Centre for Biomembranes and Lipid Enzymology, Transitorium III, Padulaan 8, 3584 CH Utrecht, The Netherlands.
0005-2760/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
from thrombin-stimulated platelets as mass changes in individual molecular species of phospholipids. These investigators arrived at the conclusion that the deacylation was selective for arachidonate species of phosphatidylcholine and phosphatidylethanolamine and explained this finding in intact cells by assuming that a certain proportion of arachidonate species of phospholipids are compartmentalized in the platelet membranes proximal to the site of action of the phospholipase A,. The alternative explanation of specific arachidonate recognition by the enzyme’s active site was considered less likely in view of the fact that acyl-chain selectivity was not observed with partially purified rabbit platelet phospholipase A, and micellar or aggregated phospholipids [6]. However, initial studies with isolated platelet membranes have also provided indications for the selective release of arachidonate from endogenous phospholipids by phospholipase A, [7,8]. This selectivity was best observed with minimal disruption of membrane structure [8] and was lost in platelet homogenates [7] or sonicates [9]. Although the selective release of arachidonate in intact cells can be explained by assuming that the phospholipase A, involved is present in a domain of arachidonate-enriched phospholipid species two other lines of evidence suggest that arachidonate-selective phospholipases A, might exist. First, experiments with rat polymorphonuclear leukocytes have shown a highly Division)
140 reduced rate of platelet activating factor synthesis in cells depleted of arachidonic acid by dietary means [lo]. The role of l-O-alkyl-2-arachidonoylglycerophosphocholine as a common precursor from which lyso-platelet activating factor and arachidonate can be produced by phospholipase A, action is well documented [ll-131. The reduced platelet activating factor synthesis in arachidonate-depleted cells could thus be explained by assuming that lyso-platelet activating factor cannot be formed in sufficient amounts, since other species of l-0-alkyl-2-acylglycerophosphocholine are much less efficiently hydrolyzed by an arachidonate-selective phospholipase A,. Secondly, a number of recent papers have reported evidence for the occurrence of arachidonate-selective phospholipases A 2 in cell-free systems such as those prepared from bone marrow-derived macrophages [14], human neutrophils [15], human platelets [16] and mouse peritoneal macrophages [17]. With the exception of the latter case the in vitro acyl-chain selectivity of these phospholipases A, was assessed by using separate vesicles of phosphatidylcholine species differing in their fatty acyl constituent at the sn-2-position. With this approach the results may be influenced by the physical-chemical properties and structural arrangements of the substrates. For example, the arachidonoyl species may form a less densely packed bilayer, thus allowing better penetration of the enzyme and giving rise to a higher hydrolysis rate that is governed by the properties of the substrate rather than by a true acyl-chain selectivity of the enzyme. In this paper we have used mixed and differentially labeled phospholipid substrates to investigate whether arachidonate specific phospholipases A, are lost during preparation of enzyme extracts from human PMN. In addition, the acyl-chain selectivity of phospholipases AZ in preparations from human PMN and human and rat platelets was investigated using separate substrates and doubly-labeled substrates, in which the two species were mixed before preparation of the phospholipids vesicles in order to present the substrates to the enzyme in a single bilayer. Materials and Methods Materials
[9,10-3H]Oleic acid, [l-i4C]oleic acid, [l-‘4C]linoleic acid, l-palmitoyl-2-[l-‘4C]oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-[5,6,8,9,11,12,14,15-3H]arachidonoyl-sn-glycero-3-phosphocholine and l-acyl-2-[l-i4C]arachidonoyl-sn-glycero-3-phosphoethanolamine were all obtained from Amersham (Buckinghamshire, U.K.). lStearoyl-2-arachidonoylphosphatidylcholine was bought from Sigma (St. Louis, MO, U.S.A.). 1-Palmitoyl-2oleoylphosphatidic acid, disodium salt, was purchased from Avanti Polar Lipids (Birmingham, AL, U.S.A.).
Methods Preparation
of substrates. I-Acyl-2-[ 3H]oleoyland 1-acyl-2-[‘4C]linoleoylphosphatidylethanolamine were biosynthetically prepared using rat liver microsomes as previously described [ 181. Phospholipase A 2 degradation of the purified compounds indicated that 86% of [3H]oleate and 97% of [‘4C]linoleate was located at the sn-Zposition. Escherichia coli membrane substrate labeled with either [3H]oleate or [‘4C]oleate was prepared as described [19,20]. Attempts to doubly label the membrane phospholipids by growing the cells in the presence of [ 3H]oleate and [i4C]arachidonate were unsuccessful due to low incorporation of arachidonic acid. Satisfactory results were obtained by growing cells in the presence of 10 PCi of [3H]oleate and 2.5 PCi of 1-acyl-2-[1i4C]arachidonoy 1p hosphatidylethanolamine in 50 ml medium. Washing the cells two times with saline containing 0.5% bovine serum albumin and autoclaving them to inactivate endogenous phospholipases yielded a double-labeled E. coli membrane substrate with a 3H/‘4C ratio of 0.86 from which pancreatic phospholipase A, released fatty acids with an isotopic ratio of 2.6. Specific radioactivities of the substrate are indicated in the legends to the tables. For the acyl-chain selectivity studies with phosphatidylcholines, l-palmitoyl-2-[l-‘4C]oleoyl-GPC was used at its commercially available specific radioactivity of 52 mCi/mmol. I-Stearoyl-2-[ 3H]arachidonoyl-GPC (120 Ci/mmol) was diluted with the unlabeled species to give a final specific radioactivity of 52 mCi/mmol. Doubly labeled substrates were prepared by mixing equimolar amounts of the two species in organic solvents. After addition of 5 mol% phosphatidic acid the phospholipids were dried under nitrogen and dispersed in distilled water at a concentration of 19 PM by sonication for three times 20 s in an ice bath under nitrogen. Enzyme preparations. Isolation of human polymorphonuclear leukocytes and preparation of cell homogenates and acid extracts were done as previously described [20], except that the stirring after addition of ice-cold 0.18 M H,S0,/2 M NaCl at pH 1.6 was done for 4 h at 0” C rather than overnight. A 1 M KC1 extract from PMN homogenate was prepared by mixing equal volumes of homogenate and 2 M KCl, stirring at 4” C overnight and centrifugation for 30 min at 100 000
x g. Isolation of rat and human platelets and preparation of a 1 M KC1 extract from rat platelets, containing over 90% of the phospholipase A, activity measurable in platelet lysates, was done as described [21,22]. The preparation of human platelet extract was done as described by Kramer et al. [23]. It has previously been shown [16,20,21,23] that release of sn-Zfatty acids from
141 phospholipids in these enzyme preparations is caused by phospholipase A, activity. Phospholipase A, assays. Enzyme activity was measured at 37 o C in a total volume of 0.5 ml containing 5 mM Ca2+ and 0.1 M Tris-HCl buffer and substrates as specified in the legends of tables and figures. E. coli substrates were assayed at the optimal pH of 7.0, whereas phosphatidylethanolamine and phosphatidylcholine assays were run at the optimal pH of 8.5. Where specific activities are given it was ensured that the reactions were linear with time and amount of enzyme preparation. Typically, an amount of enzyme equivalent to (20-40). lo6 PMN cell equivalents was used and the reaction was linear up to at least 80. lo6 cell equivalents. Reactions were stopped after 1 h by a modified Dole extraction procedure [18] and radioactivities in released fatty acids were determined by liquid scintillation spectrometry according to established procedures, Analytical procedures. Protein was determined by the dye binding assay described by Bradford [24]. E. coli membrane phospholipids were extracted according to Bligh and Dyer [25] and quantitated by lipid phosphorus analysis as described by Rouser et al. [26]. Results Previous results have shown [20] that phospholipase A, activity in homogenates of human polymorphonuclear leukocytes is suppressed by endogenous inhibitors that can be separated from the enzyme by acid extraction. Studies on the acyl-chain selectivity of the phospholipase(s) A, in such an extract should preferably include experiments with the original homogenate to exclude the possibility that a putative enzyme with selectivity for a certain fatty acid, e.g., arachidonate, was lost during the acid extraction procedure. In initial experiments we compared the phospholipase A, activities of homogenates with those in extracts and pellets obtained after a harsh extraction procedure using sulfuric acid at pH 1.6 and a mild extraction procedure using 1 M KC1 at pH 7.4. In all cases, phospholipase A, activity was measured both with l-acyl-2-[‘4C]linoleoylphosphatidylethanolamine at the optimal pH of 8.5 and with [14C]oleate-labeled E. coli membranes at the optimal pH of 7.0. Table I shows that the use of E. cofi membranes represents a much more sensitive assay system than pure phosphatidylethanolamine, especially for fractions other than the acid extracts. The total activity with phosphatidylethanolamine as substrate increased 9-fold in the acid extract when compared to homogenate, presumably due to removal of inhibiting substances. Such inhibitors are apparently not separated from the enzyme upon extraction of the homogenate with 1 M KCl. The endogenous suppressors of phospholipase A, activity in PMN homogenates have been identified as unsaturated fatty acids and derivatives
TABLE
I
Phospholipase A, activities in human PMN fractions Enzyme activities with the indicated substrates were determined as described under Materials and Methods. Mean values of two experiments differing by less than 15% are expressed in pmol/min per IO9 cell equivalents. Data in brackets represent specific activities in pmol/min per mg protein. PE substrate, l-acyl-2-[i4C]linoleoylphosphatidylethanolamine (100 yM, 250000 dpm); E. coli substrate, [“‘Cloleate-labeled E. coli membranes (80 PM, 12000 dpm). Fraction
Phospholipase
Homogenate Acid pellet Acid extract KC1 pellet KC1 extract
A 2 activity
PE substrate
E. coli substrate
36 (2) 20 (2) 315 (57)
1250 (55) 425 (32) 1675 (310) 425 (SO) 755 (58)
IO (2) 2 (0.2)
thereof [20]. Attempts to increase the phospholipase A, activity by extraction of the homogenate with diethyl ether prior to assay with phosphatidylethanolamine or by including bovine serum albumin or octyl glucoside yielded activities ranging from 15 to 40% of the original activity. For unknown reasons the effect of removal of endogenous suppressors during acid extraction was much less using E. coli substrate. The specific phospholipase A, activity of 55 pmol. min-’ 1mg protein-’ with E. coli substrate in homogenates is in good agreement with the value of 31 pmol *rnin-’ . mg protein-’ reported by Victor et al. [27]. The activities measured with E. coli substrate in homogenates, acid extract and KC1 extract allowed a comparison of the acyl-chain selectivity of the enzyme(s) in these fractions using double-labeled E. coli membranes containing [ 3H]oleate and [‘4C]arachidonate. The results (Table II), indicate that the enzyme(s) in both acid extract and KC1 extract release the two fatty acids in a ratio that is actually
TABLE
II
Acyl-chain selectivity of human PMN phospholipase A, Enzyme activities with the indicated double-labeled substrates containing [3H]oleate and [‘4C]arachidonate at the sn-2-position were determined as described under Materials and Methods. 3H/‘4C ratios at the sn-2-position of the substrates were determined after complete hydrolysis by porcine pancreatic phospholipase A, and amounted to 1.3 fO.l for PE substrate and 2.6kO.2 for E. cofi substrate. Mean values+S.D. (n = 3) are given. n.c., not conclusive. PE substrate, I-acyl-2-[ 3H]oleoyl/[14C]arachidonoylphosphatidylethanolamine (100 PM, 325000 dpm 3H and 217500 dpm 14C). E. coli substrate, [3H]oleate/[‘4C]arachidonate-labeled E. coli membranes (80 FM, 13 300 dpm ‘H and 15 300 dpm 14C). PMN fraction
3H/‘4C
PE substrate
E. coli substrate
Homogenate Acid extract KC1 extract
n.c. 1.4*0.1
3.4f0.4 1.1*0.1 1.9+0.4
XC.
in released
fatty acids
142 300
200
100
0
0 Time
(min)
Fig. 1. Acyl-chain selectivity of human PMN phospholipase A,. The reaction mixture with a total volume of 0.5 ml contained 0.1 M Tris-HCl buffer (pH 8.5) 10 mM CaCl,, 1.9 nmol of sonicated substrate including 5 mol% phosphatidic acid and an amount of immunopurified human PMN phospholipase A, equivalent to 42.106 cells. (A), l-Stearoyl-2-[3H]arachidonoyl-GPC (o) or l-palmitoyl-2-[‘4C]oleoyl-GPC (+). (B), Cosonicate of 1.9 nmol each of the substrates used in A. After incubation at 37°C for the indicated time periods the reaction was stopped and radioactivities in the released fatty acids were determined as described under Materials and Methods.
somewhat lower than that in which they are present at the sn-2-position of the phospholipids as determined by hydrolysis of the substrate with pancreatic phospholipase A,. Thus, the extracted enzyme(s), while hydrolyzing less than 5% of labeled substrates, certainly show no loss of preference for arachidonate release. In addition, a comparison with the [3H]oleate/[‘4C]arachidonate ratio released by the homogenate enzyme(s) clearly shows no evidence for a specific loss of an arachidonate releasing enzyme (in which case the ‘H/14C ratio should have increased) upon preparation of the acid extract by a harsh procedure or the KC1 extract by a mild procedure. The enzymatic activities in homogenates and KC1 extract with doubly labeled phosphatidylethanolamine as substrate were too low (compare Table I) to give conclusive results. As can be seen in Table II, the acid extract released [ 3H]oleate and [‘4C]arachidonate in a ratio identical to that present at the sn-2-position of this substrate and thus confirmed the lack of acyl-chain selectivity of the phospholipase A,. Further experiments were done with the acid extract and aimed to study acyl-chain selectivities with phosphatidylcholine substrates. Pure rat liver phosphatidylethanolamine adopts a hexagonal H,, organization at the assay conditions at 37 o C in the presence of remain 10 mM Ca 2+ [28], whereas p hosphatidylcholines organized in extended bilayers [28,29]. Because of this difference in lipid polymorphism we decided to perform acyl-chain selectivity experiments with phosphatidylcholine, a substrate representing the natural bilayer membrane as much as possible with respect to lipid organization. Since the phospholipase A, activity in the acid extract towards phosphatidylcholine was lo-fold lower
than that towards phosphatidylethanolamine, a further purification of the enzyme(s) was attempted. This was achieved by immunoprecipitation of the enzyme. As can be seen in Table III, essentially all of the enzyme was bound to Sepharose particles to which a monoclonal antibody against rat liver mitochondrial phospholipase A, had been coupled [22,30]. The enzyme could be recovered in good yield by washing the precipitate with glycine buffer at pH 2.5. Enzyme thus obtained was used to study acyl-chain selectivities with l-palmitoyl2-[i4C]oleoyl-GPC and l-stearoyl-2-[ 3H]arachidonoylGPC. In Fig. 1A the release of oleate and arachidonate from the corresponding phosphatidylcholine membranes in separate tubes is plotted as function of time. Clearly, there is no preferential release of arachidonate TABLE
III
Binding of human PMN phospholipuse A2 to anii-rat liver phospholipase AZ monoclonal antibodies Acid extracts of human PMN were incubated with Sepharose carrying antibodies (spec. either anti-rat liver phospholipase A, monoclonal McAb) or non-related monoclonal antibodies (aspec. McAb) as previously described [22,30]. Phospholipase A z activities were measured in the supernatant remaining after centrifugation and in the supernatant obtained after desorbing the enzyme from the Sepharose pellet with glycine buffer (pH 2.5) and are expressed in pmol phosphatidylethanolamine hydrolyzed per min. Results of two experiments are given. Enzyme
fraction
Acid extract Supernatant Desorbed Bound (8) Total recovery
(W)
Spec. McAb
Aspec. McAb
112; 101 17; 8 73; 64
112; 101 52; 48 4; 1
81; 80;
89 71
7; 50;
2 49
143 chains. This was confirmed by the experiments depicted in Fig. 1B in which the differentially labeled phosphatidylcholines were mixed in organic solvents prior to vesicle formation. Since the reported [14] selectivity for arachidonate release from single-labeled phosphatidylcholine species by a macrophage phospholipase A, was no longer expressed at substrate concentrations above 10 PM, we investigated the acyl-chain selectivity of the PMN phospholipase A, at lower substrate concentrations. As can be seen in Fig. 2 lowering the concentration of either substrate did not result in an apparent selectivity for arachidonate. A similar lack of acyl-chain selectivity as observed for human PMN phospholipase A, (Fig. 1) was also found in studies with an enzyme preparation from rat platelets (Fig. 3). This is in agreement with the lack of acyl-chain selectivity of this enzyme as recently
6-
1
0
4
3
2 Substrate
(JAM1
Fig. 2. Effect of substrate concentration on acyl-chain selectivity of human PMN phospholipase A,. Conditions and symbols as in the legend of Fig. lA, except that substrate concentrations were varied as indicated.
5
600
600
A
B 1
01
I
I
I
I
I
I
I
I
0
2
4
6
0
0
2
4
6
Protein Fig. 3. Acyl-chain
selectivity of rat platelet phospholipase of rat platelets prepared as described
IO 8
lpg )
A,. Conditions and symbols as in the legend of Fig. l.A, except that a 1 M KC1 extract [22] was used as enzyme source and incubation was for 1 h at 37 o C.
Protein
t pg 1
Fig. 4. Acyl-chain selectivity of human platelet phospholipase A,. Conditions and symbols as in the legend of Fig. lA, except that a human platelet supernatant fraction prepared as described [23] was used as enzyme source and incubation was for 1 h at 37 o C. In panel A and B the substrates were as in Fig. 1A and lB, respectively. In panel C the substrate consisted of vesicles composed of 1.9 nmol each of the labeled substrates used in A and B together with 9.5 nmol I-palmitoyl-2-linoleoyl-GPC and 0.5 nmol phosphatidic acid.
144 deduced from experiments utilizing separate vesicles prepared from either sn-2-arachidonoyl-GPC or sn-2linoleoyl-GPC, respectively [16]. In contrast to the rat platelet enzyme, the phospholipase A, in the cytosolic fraction of human platelets showed a lo-fold selectivity for arachidonate versus linoleate release from these substrates [16]. As can be seen in Fig. 4A a similar selectivity was observed for arachidonate versus oleate release when vesicles composed of single phosphatidylcholine species were compared. However, this apparent selectivity for arachidonoyl chains became much reduced when the substrates were presented in a mixed bilayer with a constant quality of the lipid/water interface (Fig. 4B) and was completely lost when the oleateand arachidonate-containing substrates were embedded at a concentration of 14 mol% each in a matrix otherwise composed of 68 mol% of l-palmitoyl-2-linoleoylGPC and 4 mol% phosphatidic acid (Fig. 4C). Exactly the same results as described in Figs. 1 to 4 were obtained when phosphatidic acid, used to introduce a negative charge in the membranes, was omitted from the vesicles, except that the phospholipase activities were about 20% less (data not shown). Discussion As briefly reviewed in the Introduction, the initial indications for the possible existence of arachidonatespecific phospholipases A, were provided by the selective release of this fatty acid after stimulation of prelabeled intact cells. In these cases, other factors such as specific labeling of certain phospholipid pools and compartmentalization of the activated enzyme can contribute to the observed selectivity [5]. The ultimate proof for the existence of an arachidonate-specific phospholipase A, should eventually come from studies with homogeneous enzymes under well-defined conditions. The purification of such an enzyme requires a cell-free system in which the selectivity for arachidonate is maintained in order to trace the enzyme during purification. Only a few cell-free systems have been reported to possibly contain arachidonate-selective phospholipases A *. Flesch et al. [14] described the acyl-chain specificity and kinetic properties of a phospholipase A, in homogenate and supernatant of bone marrow-derived macrophages. The soluble phospholipase A, preparation showed a marked specificity for arachidonate release when incubated with low concentrations of either l-palmitoyl-2-oleoyl-GPC, 1-palmitoyl-2-linoleoyl-GPC or 1 -palmitoyl-2-arachidonoyl-GPC. Thus, at substrate concentrations of 0.76 PM, values of 9.0, 4.0 and 84 pmol/min per mg were measured for oleate, linoleate and arachidonate release, respectively. However, when each substrate was used at its saturating concentration, these differences had largely disappeared due to the fact that the enzyme showed by far the highest affinity for
arachidonate-containing phosphatidylcholine species. Thus, the apparent specificity for arachidonate appears to be mainly caused by a K, effect, which may be influenced by substrate organization rather than being a true acyl-chain specificity due to specific recognition of the arachidonate structure in the active site of the enzyme involved. An arachidonoylphosphatidylcholine-hydrolyzing phospholipase A, has also been described in the cytosolic fraction of human neutrophils [15]. Competitive experiments with unlabeled substrates suggested that the phospholipase showed specificity for arachidonate compared to oleate and linoleate. In these experiments, preformed vesicles of unlabeled phosphatidylcholine species were added to vesicles composed of l-palmitoyl-2-[‘4C]arachidonoyl-GPC. In principle, the results of such an approach to study acyl-chain specificities might also be influenced by differential binding of the enzyme to the different vesicles. The fact that the addition of an equimolar amount of unlabeled arachidonoylphosphatidylcholine caused only 5% inhibition of [i4C]arachidonate release, rather than the expected 50%, stresses this point. As emphasized previously [31], in vitro studies on the acyl-chain selectivity of phospholipases A, should preferably use the phospholipids under investigation in a mixed membrane, rather than as separate lipid aggregates, to minimize the influence of physical-chemical factors and quality of the interfaces on the outcome of the experiments. When this approach was taken utilizing double labeled substrates in a single lipid aggregate, no indication for the existence of an arachidonate-specific phospholipase A, in homogenates or extracts of human PMN was obtained. It can not be excluded that neutrophils may contain several phospholipases A, and that the enzyme isolated by acid treatment is involved in other processes than arachidonate release, such as degradation of ingested bacterial phospholipids and thus might not be expected to exhibit arachidonate specificity. It should be mentioned, however, that no evidence for the selective loss of such an arachidonate-selective enzyme during extract preparation was found (Table II). The immunopurified enzyme hydrolyzed sn-2-oleoyl phosphatidylcholine equally well as the sn-2-arachidonoyl species, irrespective of whether the substrates were presented as pure species or in mixed bilayers (Fig. 1) and independent of the substrate concentration (Fig. 2). Similar results were obtained (Fig. 3) for a rat platelet phospholipase A, preparation that contains at least 90% of the measurable phospholipase A, activity in rat platelet lysates [22]. This confirms previous results comparing pure phospholipid species [9,16] and extends the lack of arachidonate specificity to the use of substrates in a mixed bilayer. An interesting phenomenon was observed with human platelet cytosol. In full agreement with results recently reported by Kim et al. [16] the data obtained
145 with single phosphatidylcholine species (Fig. 4A) were highly suggestive for the occurrence of an arachidonate-selective phospholipase A, in human platelets. This interpretation was strengthened by the fact that a selective release of arachidonate using the same substrates and conditions was not found with rat platelets and, therefore, seemed to exclude substrate effects. Yet, when we extended the studies with human platelet phospholipase A, to substrates in mixed bilayers (Fig. 4B and C), the apparent selectivity for arachidonate was nearly completely lost. This prompted a possible alternative explanation for the initially observed acyl-chain selectivity. This is schematically indicated in Fig. 5. It is quite reasonable to assume that the lipid packing in vesicles composed of 1-saturated-2-oleoyl-GPC is different from that in vesicles composed of 1-saturated-2arachidonoyl-GPC. Monolayer studies [32] have shown that the area per molecule at a constant surface pressure of 12 dynes/cm amounted to 77, 84 and 88 A2 for phosphatidylcholines having a saturated fatty acid at the l-position and oleate, linoleate or arachidonate, respectively, at the 2-position. It can thus be expected that the molecules in an arachidonoylphosphatidylcholine vesicle are more loosely packed than those in an oleoylphosphatidylcholine vesicle and allow for a better penetration of the phospholipases A,. Large differences in the membrane-penetrating capacity of various phospholipases A, have been reported [33]. Porcine pancreatic phospholipase A, hydrolyzed monolayers of lpalmitoyl-2-oleoyl-GPC only below initial surface pressures of 16.5 dynes/cm, whereas these cut-off points were 23.0, 34.8 and 35.3 dynes/cm for Crotalus adamanteus, Naja naja and bee venom phospholipases A,, respectively. It could thus be envisaged that phospholipase A, from human PMN and rat platelet penetrate as efficiently in 1-saturated-2-oleoyl-GPC vesicles as in 1-saturated-2-arachidonoyl-GPC vesicles
and hence show no apparent acyl-chain selectivity. Human platelet phospholipase A, might penetrate much more efficiently in the more loosely packed l-saturated2-arachidonoyl-GPC vesicles resulting in a lower K, value and an apparent selectivity for arachidonate when vesicles composed of single phosphatidylcholine species are compared. Mixing of the phosphatidylcholine species in vesicles may result in less efficient penetration (compare total hydrolyses rates in Fig. 4A and B) and a loss of most of the acyl-chain selectivity. Although such an interpretation could explain the different acyl-chain selectivities observed with single versus mixed-species substrates, it cannot be validated until more insight is obtained in the penetrating capacity of purified cellular phospholipases A,. In the absence of such data it is still felt that these possibilities should be kept in mind before definitive statements on the existence of arachidonate-specific phospholipases A, can be made. On the other hand the model, although emphasizing some of the pitfalls that can be encountered in acyl-chain selectivity studies, is not meant to deny that arachidonate specific phospholipases A, might exist. For example, a recently described 70 kDa phospholipase A, from mouse peritoneal macrophages [17] exhibited a 3.5-fold preference for arachidonate versus oleate release, even though the substrate consisted of an equimolar mixture of the parent phosphatidylcholines. As so often, however, a definitive answer can probably only be obtained by the complete purification of such a putative enzyme or, at least, its physical separation during chromatographic steps from other phospholipases A,. References 1 Bills, T.K., Smith, J.B. and Silver, M.J. (1977) J. Clin. Invest. 60, 1-6. 2 Mahadevappa, V.G. and Holub, B.J. (1984) J. Biol. Chem. 259, 9369-9373.
1 -sat. - 2 - oleoyl - GPC
1 - sat. - 2 - arachidonoyl - GPC
MIXED SPECIES
Fig. 5. Model to explain
the different
acyl-chain
selectivities
of various
phospholipases
A,. Compare
text for details
146 3 Holub, B.J. (1984) Can. _I. Biochem. Cell Biol. 62, 341-351. 4 Chilton, F.H. and Connel, T.R. (1988) J. Biol. Chem. 263, 52605265. 5 Purdon, A.D., Patelunas, D. and Smith, J.B. (1987) Biochim. Biophys. Acta 920, 205-214. 6 Kannagi, R. and Koizumi, K. (1979) Arch. Biochem. Biophys. 196, 534-542. 7 Derksen, A. and Cohen, P. (1975) J. Biol. Chem. 250, 9342-9347. 8 Kannagi, R., Koizumi, K. and Masuda, T. (1981) J. Biol. Chem. 256, 1177-1184. 9 Colard, O., Breton, M. and Bereziat, G. (1987) Biochim. Biophys. Acta 921, 333-334. 10 Ramesha, C.S. and Pickett, W.C. (1986) J. Biol. Chem. 261, 7592-7595. 11 Chilton, F.H., O’Flaherty, J.T., Ellis, J.M., Swendsen, C.L. and Wykle, R.L. (1983) J. Biol. Chem. 258, 726X-7271. 12 Chilton, F.H., Ellis, J.M., Olson, S.C. and Wykle, R.L. (1984) J. Biol. Chem. 259, 12014-12019. 13 Kramer, R.M., Patton, G.M., Pritzker, C.R. and Deykin, D. (1984) J. Biol. Chem. 259, 13316-13320. 14 Flesch. I., Schmidt, B. and Ferber, E. (1985) Z. Naturforsch. 4OC, 356-363. 15 Alonso, F., Henson, P.M. and Leslie. C.C. (1986) Biochim. Biophys. Acta 878, 273-280. 16 Kim, D.K., Kudo, I. and moue, K. (1988) J. Biochem. 104, 492-494. 17 Wijkander. J. and Sundler, R. (1989) FEBS Lett. 244, 51-56. 1X Van den Bosch, H., Aarsman, A.J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 348, 197-207. 19 Franson, R., Patriarca, P. and Elsbach, P. (1974) J. Lipid Res. 15, 3X0-388.
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