©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Macrophage Ca-independent Phospholipase A by Bromoenol Lactone and Trifluoromethyl Ketones (*)

(Received for publication, September 23, 1994; and in revised form, October 28, 1994)

Elizabeth J. Ackermann (§) Kilian Conde-Frieboes (¶) Edward A. Dennis (**)

From the Department of Chemistry and Biochemistry, Revelle College and School of Medicine, University of California at San Diego, La Jolla, California 92093-0601

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel Ca-independent phospholipase A(2) (PLA(2)) has recently been purified from the murine macrophage-like cell line P388D(1) (Ackermann, E. J., Kempner, E. S., and Dennis, E. A.(1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF(3)), arachidonyl trifluoromethyl ketone (AACOCF(3)), and a bromoenol lactone (BEL). Both PACOCF(3) and AACOCF(3) were found to inhibit the macrophage PLA(2) in a concentration-dependent manner. PACOCF(3) was found to be 4-fold more potent than AACOCF(3), with IC values of 3.8 µM (0.0075 mol fraction) and 15 µM (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF(3)bulletPLA(2) complex rapidly dissociated upon dilution.

BEL was also found to inhibit the macrophage PLA(2) in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40 °C. Inhibition was not reversed after extensive dilution of the enzyme into assay buffer. Treatment of the PLA(2) with BEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF(3). In addition, PLA(2) treated with [^3H]BEL resulted in the covalent labeling of a major band at M(r) 80,000. Inactivation of the PLA(2) by 5,5`-dithiobis(2-nitrobenzoic acid) prior to treatment with [^3H]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a M(r) 80,000 band rather than a M(r) 40,000 band upon treatment with [^3H]BEL distinguishes the macrophage Ca-independent PLA(2) from a previously identified myocardial Ca-independent PLA(2) and provides strong evidence that the M(r) 80,000 protein is the catalytic subunit.


INTRODUCTION

Phospholipase A(2) (PLA(2)) (^1)has been the focus of considerable research over the years due to its potential involvement in the release of arachidonic acid from membrane phospholipids and the subsequent production of prostaglandins and leukotrienes (for review, see (1) ). PLA(2) are also thought to play key roles in phospholipid metabolism, digestion, and various disease states. Even though it is now evident that they represent a very large and diverse family of enzymes (for review, see (2) ), the vast majority of the structural and mechanistic information available is from the Ca-dependent Group I, II, and III secreted PLA(2) (sPLA(2)). These enzymes are characterized by their low molecular weight, high disulfide bond content, conserved three-dimensional structures, and a requirement for calcium during hydrolysis (for reviews, see (3) and (4) ).

Recently, a number of unique intracellular cytosolic PLA(2) have been identified and purified that are distinct from the sPLA(2). Two well studied examples are the 85-kDa Group IV cytosolic PLA(2) (cPLA(2)) (5, 6) and the M(r) 40,000 myocardial Ca-independent PLA(2) (iPLA(2))(7) . In addition, we have also reported the purification of an apparent M(r) 80,000 cytosolic Ca-independent PLA(2) from the macrophage-like cell line P388D(1)(8) . Unlike the sPLA(2), very little is known about the catalytic mechanisms of these enzymes, their intracellular roles, or their relationships to one another. This is especially true in the case of the myocardial iPLA(2) and the macrophage iPLA(2). These two enzymes are unique among the known PLA(2) in that they are both modulated by ATP and they both form high molecular weight complexes of 400,000(8, 9) . Because of these similarities, there has been some uncertainty as to whether they represent similar enzymes modulated by the same regulatory protein (10) or whether they are truly distinct enzymes. Unfortunately, sequences have not been available for either of these two iPLA(2).

One advantageous method for studying and comparing kinetic and chemical mechanisms between enzymes is through the use of inhibitors. Recently, two active site-directed inhibitors have been reported in the literature, arachidonyl trifluoromethyl ketone (AACOCF(3)), which reportedly displays specificity for the Group IV cPLA(2)versus the Group II sPLA(2)(11) , and a bromoenol lactone (BEL), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, which reportedly displays specificity for the myocardial iPLA(2)versus both Group I and III sPLA(2)(12) . In this study, we have investigated the action of each of these inhibitors as well as several additional compounds including a new potent inhibitor, palmitoyl trifluoromethyl ketone (PACOCF(3)), toward the purified macrophage iPLA(2), and we have utilized [^3H]BEL to help identify the catalytic subunit of the macrophage iPLA(2).


EXPERIMENTAL PROCEDURES

Materials

1-Palmitoyl-2-[1-^14C]palmitoyl-sn-glycero-3-phosphorylcholine was purchased from Amersham Corp., 1-[1-^14C]palmitoyl-2[1-^14C]palmitoyl-sn-glycero-3-phosphorylcholine was purchased from DuPont NEN, and unlabeled phospholipids were purchased from Avanti Polar Lipids, Inc. All other reagents were analytical grade or better. (E)-6-(Bromomethylene)tetrahydro-3-(1-[4-^3H]naphthalenyl)-2H-pyran-2-one ([^3H]BEL) was the generous gift of Randy H. Weiss, and Philip Needleman (Monsanto Co., St. Louis, MO)(13) . Anandamide (arachidonylethanolamide) (14) was kindly provided by Ester Fride and Raphael Mechoulam (Hebrew University, Jerusalem, Israel). The two palmitoyl anandamide analogs (N-(2-hydroxyethyl)hexadecanesulfonamide and 3-(hexadecanesulfonyl)-2-methoxy-1,3-oxazolidine) were synthesized and will be described elsewhere. (^2)Synthesis of the phospholipid substrate analogs 1-(hexylthio)-2-(hexanoylamine)-1,2-dideoxy-sn-3-phosphocholine and 1-(hexylthio)-2-(hexanoylamine)-1,2-dideoxy-sn-3-phosphoethanolamine is described elsewhere(15) , and they were provided by Scott Boegeman.

AACOCF(3)

AACOCF(3) was prepared according to the general procedure of Boivin et al.(16) with the following modifications. Instead of using an acylchloride, a mixed anhydride was formed in the reaction mixture by the following procedure. 0.50 g of arachidonic acid (1.6 mmol; Aldrich) was dissolved in 25 ml of dichloromethane under a nitrogen atmosphere. 1.3 ml of pyridine (16.4 mmol) and 1.9 ml of trifluoroacetic anhydride (13.1 mmol) were added at room temperature, and the mixture was stirred for 1 h. The solution was cooled in a ice bath followed by the addition of 10 ml of water. Workup with 100 ml of water, extraction of the aqueous phase with 3 times 30 ml of dichloromethane, and evaporation of the organic phase after drying with sodium sulfate gave 0.35 g (60%) of AACOCF(3) after chromatography with ether/hexane (1:2) on silica gel (R(F) = 0.67). Mass spectroscopy (fast atom bombardment) M - 1: 355.2248 (theoretical), 355.2260 (found). ^1H and C NMR data were consistent with previously published results(11) .

PACOCF(3)

PACOCF(3) was synthesized in the same manner as described above for the arachidonoyl analog. Using 0.95 g (3.7 mmol) of palmitic acid as starting material yielded 0.68 g (60%) of PACOCF(3) (R(F) = 51; ether/hexane (1:2)). ^1H NMR (CDCl(3), 300.0 MHz) 0.88 (t, 3H), 1.2-1.4 (m, 24H), 1.6-1.7 (m, 2H), and 2.69 (t, 2H); C NMR (CDCl(3), 75.48 MHz) 14.1, 22.4, 22.8, 28.8, 29.3, 29.5, 29.6, 29.7, 29.8, 32.0, 36.4, 115.7 (q, J = 292 Hz), and 191.5 (q, J = 35 Hz).

BEL and 6-Bromo-2-(1-naphthyl)-5-oxohexanoic Acid (Bromomethyl Ketone)

BEL was synthesized as described previously by Daniels et al.(17) . For the bromomethyl ketone, 0.10 g (0.3 mmol) of BEL was dissolved in 5 ml of tetrahydrofuran, and 1 ml of 5 M HCl was added. After 1 h of stirring at room temperature, 50 ml of water was added to the reaction mixture, and the aqueous phase was extracted three times with 50 ml of dichloromethane. Drying the organic phase over sodium sulfate, removing the solvent, and chromatography on silica gel gave 14 mg of a clear oil. ^1H NMR (CDCl(3), 300.0 MHz) 2.2-2.7 (m, 4H), 3.9 (s, 2H), 4.4 (t, 1H), 7.3-7.6 (m, 4H), 7.7-7.9 (m, 2H), and 8.1 (m, 1H).

Preparation of P388D(1) Ca-independent Phospholipase A(2)

The P388D(1) Ca-independent PLA(2) was purified utilizing an ammonium sulfate precipitation of the whole cell homogenate followed by sequential column chromatography as described previously(8) . Mono Q eluents (purified 100,000-fold with a specific activity of 1.3 µM/min/mg(8) ) were utilized for most experiments. ATP-agarose eluents (purified 26,000-fold with a specific activity of 0.32 µM/min/mg(8) ) were utilized for the time-dependent inactivation experiments because the Tris buffer present in the Mono Q eluents catalyzed the hydrolysis of BEL(17) .

Phospholipase A(2) Assay

Each PLA(2) assay contained 400 µM Triton X-100, 100 µM dipalmitoylphosphatidylcholine (DPPC) (containing 200,000 cpm 1-palmitoyl-2-[1-^14C]palmitoyl-sn-glycero-3-phosphorylcholine), 100 mM Hepes, pH 7.5, 5 mM EDTA, and 0.1 mM ATP in a final volume of 500 µl. The mixed micellar substrate was prepared as described previously(8) . Assays were incubated at 40 °C for 30 min with agitation (unless otherwise indicated), and the reaction was stopped by the addition of 2.5 ml of Dole reagent (2-propanol, heptane, 0.5 M H(2)SO(4) (400:100:20, v/v/v))(18) . The product mixture was subsequently processed according to the modified (19) Dole extraction system (18) as described previously(20) . Control reactions lacking enzyme were routinely carried out and subtracted from the reported activities.

Inhibition with Trifluoromethyl Ketones

Stock solutions and serial dilutions of inhibitors and fatty acids were prepared in Me(2)SO. Each assay tube was prepared by the addition of 5 µl of the appropriate inhibitor to 445 µl of the mixed micellar substrate followed by vortexing, bath sonication, and vortexing (30 s each). Assays were initiated by the addition of 50 µl of the P388D(1) Ca-independent PLA(2) (sufficient to produce 3000 counts in 30 min) to the substrate mixture and were incubated at 40 °C for 30 min. Time course experiments were carried out in a similar fashion with variable incubation times. Inhibitor concentrations expressed as mole fractions were calculated utilizing the total concentration of lipid present in the assay (i.e. 100 µM DPPC and 400 µM Triton X-100 plus the inhibitor concentration). Because under our assay conditions the monomeric concentration of Triton X-100 is not known, we did not take it into account in calculating the mole fractions reported herein(21) .

Inhibition with BEL

Stock solutions and serial dilutions of BEL were prepared in Me(2)SO. The P388D(1) Ca-independent PLA(2) (sufficient to produce 3000 cpm in 30 min) was preincubated with the indicated amounts of BEL in 10 mM Tris (or 10 mM Hepes), pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mM ATP, 1 mM Triton X-100, and 10% glycerol. Preincubations were carried out at 40 °C for 5 min with agitation in a volume of 55 µl. The remaining enzyme activity was assayed by the addition of 445 µl of substrate mixture followed by incubation at 40 °C for 30 min. The reactions were stopped and processed as described above. The time course of inactivation was carried out in the same manner, except that preincubation times were varied as indicated.

For the competition experiments, PACOCF(3) and BEL were added simultaneously to the enzyme and incubated as described above. Upon dilution into the assay mixture, the concentration of PACOCF(3) was 1.1 µM, a concentration that results in 16% inhibition of the PLA(2) activity (see Fig. 1). For a control, the PLA(2) was routinely preincubated in the presence of PACOCF(3) alone, and the resulting activity (measured after dilution into the assay mixture) was defined as 100% of control for all assays in which PACOCF(3) was present.


Figure 1: Concentration-dependent inhibition of the P388D(1) Ca-independent PLA(2) by PACOCF(3) and AACOCF(3). The P388D(1) PLA(2) was assayed in the presence of increasing concentrations of PACOCF(3) (), AACOCF(3) (bullet), palmitic acid ([), or arachidonic acid ([) utilizing a mixed micelle assay containing Triton X-100 and DPPC. The enzyme activity is plotted as the percentage of the control enzyme assayed in the absence of inhibitor. Each point represents the average of duplicates.



For dilution experiments, the PLA(2) was concentrated 6-fold using a Centricon 10 apparatus (Amicon, Inc.). The resulting enzyme preparation was preincubated at 40 °C for 5 min with either Me(2)SO alone or 10 µM BEL and Me(2)SO. After preincubation, a 2-µl aliquot was removed and diluted 1500-fold into 3 ml of assay buffer containing 400 µM Triton X-100, 100 µM DPPC (with 200,000 cpm 1-palmitoyl-2-[1-^14C]palmitoyl-sn-glycero-3-phosphorylcholine/50 µl of assay buffer), 100 mM Hepes, pH 7.5, 0.8 mM ATP, 1.0 mM dithiothreitol, and 5 mM EDTA. At the indicated time intervals, a 50-µl aliquot was removed, and the released radiolabeled fatty acid was measured as described above.

Covalent Modification of P388D(1) Ca-independent PLA(2) by [^3H]BEL

2-3 µg of the P388D(1) Ca-independent PLA(2) was incubated with 2 µM [^3H]BEL for 30 min at 40 °C. Excess unreacted [^3H]BEL along with the Triton X-100 was removed through four cycles of concentration and dilution utilizing a Centricon 10 apparatus. Samples were subsequently lyophilized, resuspended in sample buffer, and separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a prepoured 10% gel (Novex). Gels were fixed with 50% methanol and visualized by autoradiography. Experiments utilizing the DTNB-inactivated enzyme (P388D(1) PLA(2) preincubated with 1.0 mM DTNB at 40 °C for 15 min) were carried out in a similar fashion, expect that 1-2 µg of enzyme was utilized and the fixed gels were soaked for 30 min in Amplify (Amersham Corp.) prior to visualization.

Control experiments were also carried out that demonstrated that under these conditions, [^3H]BEL inhibited 83% of the PLA(2) activity. For these experiments, the PLA(2) activity was assayed utilizing a double-labeled phospholipid (1-[1-^14C]palmitoyl-2-[1-^14C]palmitoyl-sn-glycero-3-phosphorylcholine), and the activity was measured by following the release of 1-[1-^14C]palmitoyl-2-lyso-sn-glycero-3-phosphorylcholine separated by TLC as described previously(8) . This method was used because the presence of [^3H]BEL would have interfered with the detection of released radiolabeled fatty acid in the Dole extraction method.


RESULTS

Inhibition by Trifluoromethyl Ketones

Fig. 1shows the concentration-dependent inhibition of the P388D(1) Ca-independent PLA(2) by trifluoromethyl ketone analogs of palmitic acid (PACOCF(3)) and arachidonic acid (AACOCF(3)) using a mixed micelle assay system with 400 µM Triton X-100 and 100 µM DPPC (4:1 molar ratio of Triton to phospholipid). The saturated analog of AACOCF(3) was also synthesized, but it could not be utilized in these experiments due to its low solubility. Also shown in Fig. 1are the results obtained with the free fatty acids palmitate and arachidonate, which were not inhibitory. Interestingly, PACOCF(3) was found to be 4-fold more potent than AACOCF(3), with an IC of 3.8 µM (0.0075 mol fraction) compared with 15 µM (0.028 mol fraction) for AACOCF(3). This selectivity is consistent with the substrate preference found earlier utilizing mixed micellar substrates in which the rate of hydrolysis for dipalmitoylphosphatidylcholine was 4-fold faster than that for 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphorylcholine (8) .

Because AACOCF(3) has recently been shown to be a slow, tight-binding inhibitor of the Group IV cPLA(2)(11) , we examined the time course and reversibility of inhibition with the P388D(1) Ca-independent PLA(2). The P388D(1) PLA(2) was assayed in the presence of either 8 µM PACOCF(3) or 12 µM AACOCF(3) or in the absence of inhibitor. As shown in Fig. 2, linear progress curves were observed under all three conditions, with each curve passing through the origin, indicating that these are not slow binding inhibitors. In addition, preincubation of the P388D(1) Ca-independent PLA(2) with 300 µM PACOCF(3) for 5 min at 40 °C followed by a 1500-fold dilution into assay buffer resulted in near complete recovery of the PLA(2) activity at all time points measured (15-240 min). This is in contrast to the slow dissociation rate observed for the CabulletAACOCF(3)bulletcPLA(2) complex, where under similar conditions, only 14% of the complex dissociated over a 5-h period(11) . Thus, the association/dissociation rates observed between these inhibitors and the P388D(1) PLA(2) appear to be much faster than the rates observed between the Group IV cPLA(2) and AACOCF(3).


Figure 2: Reaction progress curves of the P388D(1) Ca-independent PLA(2) in the presence of trifluoromethyl ketone inhibitors. The P388D(1) PLA(2) was assayed under standard assay conditions in the absence of inhibitor (bullet) or in the presence of either 12 µM AACOCF(3) () or 8 µM PACOCF(3) ([). Each point represents the average of duplicates.



Concentration-dependent Inhibition of P388D(1) Ca-independent PLA(2) by BEL

BEL was found to be a potent inhibitor of the purified P388D(1) Ca-independent PLA(2). As shown in Fig. 3, preincubation of the P388D(1) PLA(2) for 5 min at 40 °C with increasing amounts of BEL resulted in a concentration-dependent inhibition of activity. Half-maximal activity was found at 60 nM BEL. In contrast, similar experiments carried out utilizing the hydrolyzed form of the inhibitor (bromomethyl ketone) did not result in any appreciable inhibition of the PLA(2) activity at concentrations up to 700 nM (see ``Discussion''). Because BEL has been shown to be a suicide inhibitor of the myocardial iPLA(2), we investigated the mechanism of inhibition with the P388D(1) iPLA(2).


Figure 3: Concentration-dependent inhibition of the P388D(1) Ca-independent PLA(2) by BEL. The P388D(1) PLA(2) was incubated with the indicated concentrations of BEL for 5 min at 40 °C in buffer containing 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mM Triton X-100, 10% glycerol, and 1 mM ATP. After this preincubation, the enzyme was diluted 10-fold into the assay mixture, and the remaining activity was measured. Each point represents the average of duplicates and is plotted as the percent of control (enzyme preincubated in the absence of inhibitor).



Irreversible Inhibition

The reversibility of inhibition could not be assessed using either gel filtration or dialysis techniques due to the presence of Triton X-100 with the enzyme, which was necessary for stabilization of the PLA(2) activity(8) . Instead, the P388D(1) PLA(2) was preincubated in the presence or absence of BEL and subsequently diluted 1500-fold into assay buffer. Aliquots were removed at various time intervals, and the enzyme activity was determined. As shown in Fig. 4, enzyme not treated with inhibitor resulted a linear time course over the entire assay period, while enzyme pretreated with BEL did not regain any measurable activity in the 4 h following dilution, indicating tight or covalent inhibition of the enzyme by BEL. It should be noted that the enzyme used for the dilution studies was initially concentrated 6-fold, and therefore, the Triton X-100 present with the enzyme was also concentrated. We have found that in the presence of high concentrations of Triton X-100, higher concentrations of BEL were required in order to achieve full inhibition of the P388D(1) PLA(2) activity. Consequently, 10 µM BEL was utilized in these experiments to ensure complete inactivation of the enzyme. Whether this phenomenon is due to a protection of the enzyme by Triton X-100 or a partitioning effect is unknown at this time.


Figure 4: Irreversible inhibition of the P388D(1) Ca-independent PLA(2) by BEL. The P388D(1) PLA(2) was preincubated at 40 °C for 5 min with either 10 µM BEL in Me(2)SO () or Me(2)SO alone (bullet). After preincubation, a 2-µl aliquot was removed and diluted 1500-fold into 3 ml of assay buffer. At the indicated time intervals, a 50-µl aliquot was removed, and the amount of released radiolabeled fatty acid was determined. Each point represents the average of duplicates.



Time-dependent Inactivation and Protection by PACOCF(3)

To explore the characteristics of this inhibition, the time course of inactivation was examined. The P388D(1) PLA(2) was preincubated with BEL for periods of 1-30 min, followed by dilution into the assay mixture and quantification of remaining activity. A semilogarithmic plot of the remaining activity versus preincubation time resulted in a linear inactivation time course (Fig. 5) for each concentration tested up to at least 7 min, indicating pseudo first-order kinetics. This time-dependent inactivation is indicative of a direct binding of the inhibitor to the enzyme. At longer preincubation times (7-30 min), first-order kinetics were no longer observed. This is most likely due to a depletion of the inhibitor concentration to a point below or equal to the enzyme concentration (22) . In addition, the initial steady-state rate was apparently preceded by a burst of inhibition as evidenced by the lack of intersection at 100% activity for any of the concentrations tested. Suicide inhibition of both the myocardial PLA(2) and chymotrypsin by BEL also resulted in a similar burst of inhibition(12, 23) .


Figure 5: Time-dependent inactivation of the P388D(1) Ca-independent PLA(2) by BEL. The P388D(1) PLA(2) was preincubated with 30 nM BEL (bullet), 60 nM BEL (), or 100 nM BEL () for the indicated time periods at 40 °C, followed by dilution into assay buffer and quantification of remaining activity. Each point represents the average of duplicates and is plotted on a semilogarithmic plot as the percent of control enzyme incubated in the absence of inhibitor.



Time-dependent experiments were also carried out in the presence of the reversible inhibitor PACOCF(3). The P388D(1) PLA(2) was preincubated with BEL alone or with BEL and 10 µM PACOCF(3). As shown in Fig. 6, the rate of inactivation in the presence of PACOCF(3) was significantly less than that observed in its absence. This protection afforded by PACOCF(3) suggests that the binding sites on the enzyme for these two inhibitors are at least partially overlapping.


Figure 6: Protection of the P388D(1) Ca-independent PLA(2) from inhibition by BEL. The P388D(1) PLA(2) was preincubated with 100 nM BEL in the presence (bullet) or absence () of 10 µM PACOCF(3) for the indicated time periods at 40 °C, followed by dilution into the assay mixture and quantification of remaining activity. These results are expressed relative to the control rate measured in the presence of Me(2)SO (for ) or Me(2)SO plus PACOCF(3) (for bullet) and are plotted on a semilogarithmic plot. Each point represents the average of duplicates.



Covalent Labeling of P388D(1) Ca-independent PLA(2) with [^3H]BEL

Finally, we examined the ability of [^3H]BEL to covalently label the P388D(1) PLA(2). The P388D(1) PLA(2) was preincubated with [^3H]BEL, the excess inhibitor was removed, and the preparation was separated by SDS-PAGE. As shown in Fig. 7A, a single major band was visualized upon autoradiography at M(r) 80,000. This molecular weight correlated with that observed previously in purified preparations of the P388D(1) PLA(2) after SDS-PAGE and silver staining(8) . This result is in contrast to that obtained with the myocardial PLA(2) in which [^3H]BEL labeled the myocardial M(r) 40,000 catalytic subunit(12) . In addition to the M(r) 80,000 band, we also observed a diffuse band near the top of the autoradiogram. This band was located near the junction between the stacking and running gels and therefore is most likely due to aggregated protein that did not enter the gel.


Figure 7: Covalent modification of the P388D(1) Ca-independent PLA(2) by [^3H]BEL and its attenuation with DTNB-inactivated enzyme. A, 2-3 µg of the P388D(1) PLA(2) was incubated with 2 µM [^3H]BEL for 30 min at 40 °C, followed by separation by SDS-PAGE on a 10% gel and visualization by autoradiography. B, control(-) or DTNB-inactivated (+) P388D(1) PLA(2) (1-2 µg) was incubated with 2 µM [^3H]BEL for 30 min at 40 °C. Samples were subsequently separated by SDS-PAGE on a 10% gel and visualized by fluorography.



A similar experiment was also carried out utilizing enzyme that had been treated with DTNB prior to inhibition with [^3H]BEL. Treatment of the P388D(1) Ca-independent PLA(2) with 1 mM DTNB resulted in the complete loss of PLA(2) activity. As shown in Fig. 7B, utilization of this DTNB-inactivated enzyme resulted in the near complete lack of covalent binding of [^3H]BEL to the M(r) 80,000 protein, as well as the complete lack of the label at the top of the gel, suggesting the necessity for a catalytically competent enzyme for incorporation of label. It should be noted that visualization for the experiment shown in Fig. 7B was carried out utilizing fluorography and therefore resulted in a much higher degree of sensitivity than that observed in Fig. 7A. As can be seen, under these more sensitive conditions, the M(r) 80,000 band appears much darker and even overloaded, and there is also a previously unobserved very faint band near the bottom of the gel at M(r) 36,000. Because this band is only a very small fraction of the total label and was only detected under these more sensitive conditions, it is most likely the result of a degradation product or contamination with another protein that reacts with BEL.

Other Inhibitors

During the course of our studies, we have also investigated the action of several other potential inhibitors on the P388D(1) Ca-independent PLA(2). Each of these compounds gave an estimated IC value of >50 µM (10 mol %) and therefore were not pursued further. They include nordihydroguaiaretic acid, anandamide (arachidonylethanolamide), two analogs of anandamide (N-(2-hydroxyethyl)hexadecanesulfonamide and 3-(hexadecanesulfonyl)-2-methoxy-1,3-oxazolidine), and two phospholipid substrate analogs (1-(hexylthio)-2-(hexanoylamine)-1,2-dideoxy-sn-3-phosphocholine and the corresponding phosphoethanolamine), which are potent inhibitors of the Group I PLA(2)(15) .


DISCUSSION

Trifluoromethyl Ketones: P388D(1) iPLA(2) Versus Group IV cPLA(2) Specificity

Recently, Street et al. (11) have reported the inhibition of the 85-kDa cytosolic Group IV cPLA(2) by a trifluoromethyl ketone analog of arachidonic acid (AACOCF(3)). AACOCF(3), which presumably binds directly to the active site of the cPLA(2), was found to be a slow and tight-binding inhibitor as demonstrated by nonlinear progress curves and a very slow dissociation rate. From these experiments, an upper estimate for the K(i) value was estimated at 5 times 10 mol fraction. When compared with the 0.1 mol fraction estimated for the sPLA(2), it was apparent that AACOCF(3) is a selective inhibitor for the cPLA(2)versus the sPLA(2).

Results reported herein demonstrate that the P388D(1) PLA(2) is also inhibited by AACOCF(3) as well as a new analog, PACOCF(3). Inhibition was found to be concentration-dependent, with IC values of 0.028 and 0.0075 mol fractions, respectively. Competition experiments carried out in the presence of BEL demonstrate that this inhibition is mediated through a direct binding of the inhibitor to the enzyme, most likely at the active site, consistent with results obtained with the cPLA(2). However, in contrast to the Group IV cPLA(2), kinetic experiments reveal linear progress curves in the presence of both inhibitors, and the PACOCF(3)bulletPLA(2) complex was found to rapidly dissociate upon dilution. Taken together, these data are consistent with a classical mechanism of reversible inhibition.

These data represent the first report of inhibition of a Ca-independent PLA(2) by trifluoromethyl ketone inhibitors. It is difficult to directly compare the exact potency of inhibition between the macrophage iPLA(2) and the cPLA(2) due to the different types of inhibition observed. In addition, it should be kept in mind that the IC values reported for the macrophage iPLA(2) are a function of the assay conditions utilized and should only be taken as an upper limit estimate of the true K(i) value, which may be much lower. In any case, the ability of these compounds to inhibit multiple intracellular PLA(2) in the low micromolar range indicates that their use as specific inhibitors for in vivo studies should be carried out with some caution. For example, the P388D(1) macrophages contain a Group IV cPLA(2), a Group II sPLA(2), and the Ca-independent iPLA(2)(24) , and therefore, these inhibitors cannot be used indiscriminately in this cell type to inhibit specifically either the Group IV cPLA(2) or the Ca-independent iPLA(2). On the other hand, AACOCF(3) has been used recently in calcium ionophore- and thrombin-stimulated platelets to implicate the involvement of the cPLA(2) rather than the sPLA(2) in arachidonic acid release(25, 26) . Structure/function studies were also carried out with several different compounds besides AACOCF(3) to help eliminate nonspecific inhibition effects(25, 26) .

BEL: P388D(1) PLA(2) Versus Myocardial PLA(2)

The macrophage iPLA(2) and the myocardial iPLA(2) are unique among the known PLA(2) in that they are both modulated by ATP, and they both form oligomeric complexes of M(r) 400,000(8, 9) . Despite these similarities, there has been some uncertainty as to whether they are similar enzymes modulated by the same regulatory protein or whether they are truly distinct enzymes. For example, in the case of the myocardial PLA(2), these characteristics are thought to be due to the regulation of the M(r) 40,000 catalytic PLA(2) by phosphofructokinase(10) , a tetrameric enzyme composed of M(r) 85,000 subunits. Together, the myocardial PLA(2) and the phosphofructokinase are thought to form an ATP-sensitive regulatory complex, composed of one M(r) 40,000 PLA(2) subunit and four M(r) 85,000 phosphofructokinase regulatory subunits. The P388D(1) PLA(2), on the other hand, is thought to be a M(r) 80,000 protein that is active as a tetramer (radiation inactivation experiments indicated a catalytically active complex of 337 ± 25 kDa), and it is thought to bind ATP directly(8) . However, in our previous studies(8) , we were unable to completely rule out a model in which the M(r) 80,000 P388D(1) protein is actually phosphofructokinase that was purified along with an undetected M(r) 40,000 catalytic subunit, in analogy with the myocardial PLA(2).

Recently, BEL has been shown to be a potent suicide inhibitor of the myocardial PLA(2)(12, 27) . This inhibition was found to be at least 1000-fold more potent toward the myocardial PLA(2) than toward the Group I or III sPLA(2)(12) . Because of the similarities between the myocardial PLA(2) and the macrophage PLA(2), we examined the ability of BEL to inhibit the P388D(1) PLA(2) in the hope of gaining insight into the relationship between these two enzymes. We have found that (a) BEL is a potent inhibitor of the P388D(1) iPLA(2), with half-maximal activity found at 60 nM after a 5-min preincubation at 40 °C; (b) inhibition is irreversible when subjected to a 1500-fold dilution and covalent as demonstrated by the incorporation of [^3H]BEL, which persisted through SDS-PAGE treatment; (c) inhibition is time-dependent and shows pseudo first-order kinetics; (d) this time-dependent inactivation is slowed in the presence of the reversible inhibitor PACOCF(3); and (e) a catalytically active enzyme is necessary for covalent modification as DTNB-inactivated PLA(2) had a greatly diminished capacity for incorporation of label.

Taken together, these data indicate that BEL is a covalent irreversible inhibitor of the P388D(1) PLA(2), with inactivation proceeding through an enzyme-mediated process. These results are consistent with the documented cases of suicide inhibition utilizing BEL with both the myocardial PLA(2)(12) and chymotrypsin (17) . In addition, we have found that the hydrolyzed form of BEL (bromomethyl ketone) is not inhibitory. This indicates that the bromomethyl ketone (which is proposed to be the reactive species responsible for irreversible modification of both the myocardial PLA(2) and chymotrypsin) is not released from the enzyme prior to irreversible inactivation, i.e. ruling out a metabolically activated mechanism(22) . Furthermore, the sensitivities and characterizations of BEL inhibition observed with the P388D(1) PLA(2) were strikingly similar to those observed with the myocardial PLA(2). Both enzymes were inhibited by BEL in the nanomolar range, both showed an initial burst of inhibition followed by a first-order time-dependent inactivation, and both enzymes lost their ability to incorporate [^3H]BEL label after inactivation with DTNB. These data are intriguing in that they suggest that these two enzymes may share very similar active-site environments.

However, despite these similar sensitivities to BEL, treatment of the macrophage PLA(2) with [^3H]BEL resulted in the labeling of a single major M(r) 80,000 protein, as opposed to the M(r) 40,000 band documented with the myocardial PLA(2). These data distinguish the macrophage PLA(2) from the myocardial PLA(2) and provide strong evidence that the M(r) 80,000 protein is the catalytic subunit, and not phosphofructokinase. Thus, the ATP activation and oligomerization observed with both of these enzymes appear to be a function of distinct regulatory mechanisms.

In conclusion, we have demonstrated that PACOCF(3), AACOCF(3), and BEL inhibit the macrophage Ca-independent PLA(2) activity. Based on the data presented herein and in analogy with results obtained with the cPLA(2) and the myocardial iPLA(2), we propose that the trifluoromethyl ketones are classical reversible inhibitors of the macrophage iPLA(2) and that BEL is a suicide inhibitor. More important, these data demonstrate that the myocardial iPLA(2) and the macrophage iPLA(2) are distinct enzymes and provide new evidence that the M(r) 80,000 protein is the macrophage iPLA(2) catalytic subunit.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM-20501, GM-51606, and HD-26171. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Department of Education Graduate Assistance in Areas of National Need fellowship.

Recipient of a Deutsche Forschungsgemeinschaft postdoctoral fellowship.

**
To whom correspondence should be addressed.

(^1)
The abbreviations used are: PLA(2), phospholipase(s) A(2); sPLA(2), secreted Ca-dependent PLA(2); cPLA(2), cytosolic PLA(2); iPLA(2), Ca-independent PLA(2); AACOCF(3), arachidonyl trifluoromethyl ketone; PACOCF(3), palmitoyl trifluoromethyl ketone; BEL, bromoenol lactone; DPPC, dipalmitoylphosphatidylcholine; PAGE, polyacrylamide gel electrophoresis; DTNB, 5,5`-dithiobis(2-nitrobenzoic acid).

(^2)
K. Conde-Frieboes and E. A. Dennis, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Laure Reynolds and Raymond Deems for critical suggestions and discussions during the course of this study. We also thank Dr. Richard Ulevitch and Lois Kline (Research Institute of Scripps Clinic) for generously growing the P388D(1) cells.


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