467 Biochimica et Biophysics Acta, 575 (1979) o Elsvier/North-Holland Biomedical Press
467-470
BBA Report BBA 51263
MODULATION OF PURIFIED PHOSPHOLIPASE AZ ACTIVITY HUMAN PLATELETS BY CALCIUM AND INDOMETHACIN
ROBERT
L. JESSE and RICHARD
FROM
C. FRANSON
Department of Biophysics, Medical College of Virginia, Box 694, MCV Station, Richmond, VA 23298 (U.S.A.) (Received
September
6th, 1979)
Key words: Phospholipase A, ; Ca’+; Zndomethacin;
(Human platelets)
Summary A membrane bound phospholipase A2 (phosphatide 2acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase AZ activity was assayed using [ l-14C] oleate-labeled Escherichiu coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-l position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca”. Indomethacin, but not aspirin, inhibited phospholipase A2 activity.
Metabolytes of arachidonic acid, including prostaglandin endoperoxides and thromboxanes, are important in the function of human platelets, (for review see Ref. 1). Greater than 95% of this precursor fatty acid is esterified in the C-2 position of phospholipids; thus, for prostaglandin synthesis to occur, arachidonate is thought to be made available by the action of a phospholipase A2 [ 21. Despite its apparent role in regulating the availability of ‘free’ arachidonate, little is known about the cellular or pharmacologic control of this enzyme. We report that a preparation enriched 3500-fold in human platelet phospholipase A2 activity, is highly sensitive to Ca” and, as recently demonstrated for the rabbit leukocyte phospholipase A2 [ 31, is inhibited by indomethacin. Outdated platelet rich plasma, collected from normal donors in acid citrate dextrose with a Hemonetics Model 30 cell separator was centrifuged at 200 X g for 15 min, then at 2500 X g for 15 min. The platelet pellet was washed with 0.9% saline and resuspended in 1.15% KC1 to a concentration of 15 mg platelet protein/ml. By light microscopic examination, this preparation was essentially free of contaminating white or red blood cells. This
468
concentrate was diluted 1: 1 with ice-cold H,SO, to a final concentration of 0.18 N HZS04 and was homogenized with a Potter-Elvehjem homogenizer. After 1 hour at 4”C, the suspension was rehomogenized and centrifuged at 10 000 X g for 15 min. The resulting supernatant fraction was dialyzed against four changes of 50 mM citrate buffer pH 6.0, then centrifuged at 40 000 X g for 30 min. The pellet, enriched in phospholipase AZ activity, was resuspended in 100 mM citrate pH 3.0 and centrifuged as above; activity was found almost entirely in the supernatant fraction, designated the ‘pellet extract’. The pellet extract was applied to a sulfa propyl-Sephadex column (SPSephadex C25-120, Pharmacia) equilibrated with 100 mM NaCl - 20 mM citrate pH 3.0; washed first with 150 ml of buffer, then with 250 ml of buffered 0.75 M NaCl. Phospholipase AZ activity was subsequently eluted with 120 ml of a linear NaCl gradient (0.75 M-3.0 M, pH 3.0) collected in 2.0 ml fractions. Phospholipase AZ activity was measured by established methods using l-[ 1-14C] oleate labeled autoclaved E. coli [ 41 or 1-acyl 2-C1-14C] linoleoyl 3-sn-phosphatidylethanolamine as substrate [ 51. To confirm positional specificity, l-[ l-i4C] stearyl 2-acyl phosphatidylethanolamine was used as substrate. Unless indicated otherwise, incubation mixtures contained in a total volume of 0.5 ml, 5 mM Ca*‘, 100 mM Tris-HCl buffer pH 9.5 and 2.5*10* cells of autoclaved E. coli (corresponding to approximately 4 nmol of phospholipid and 6000 cpm). Using sonicated phospholipid, reaction mixtures in a total volume 1.0 ml were as described above except that 50 nmol of substrate were used. In both assay systems platelet protein and incubation times (at 37°C) were adjusted to maintain linear kinetics. Reactions were stopped and radioactive lipids extracted and isolated as previously described [4,5]. Phospholipase A activity is expressed as nmol product formed (free fatty acid) per h. Protein was measured by the method of Bradford [6] modified by the addition of a 5 min incubation at 37°C prior to measuring absorption at 595 nm. Table I summarizes the purification and recovery of enzymatic activity and protein. These data represent one experiment; an extraction of 50 units TABLE
I
RECOVERY OF PROTEIN AND ENZYMATIC ACTIVITY IN VARIOUS PURIFICATION OF HUMAN PLATELET PHOSPHOLIPASE A,
FRACTIONS
DURING
Platelets were prepared and the fractions were isolated as described in methods. The data are derived kom a single experiment with an initial extraction of 50 units of platelets, and are representative of four other experiments. In aU experiments, unless indicated otherwise, phospholipase A, activity was assayed in duplicate or triplicate using the standard E. coli incubation, (see methods). The data were averaged and are expressed as nmol free fatty acid formed/h per mg. All values are corrected for nonenzymatic hydrolysis, which was less than 3.0% in alI experiments. ___Fraction
Protein -
Homogenate Pellet extract SP-Sephadex
column
PhosphoIipase activity
A,
Specific activity
mg
% Recovery
nmol/b
% Recovery
(nmoIF/mg)
2390.0 67.9 1.1
100.0 2.3 .04
1718.0 3163.2 2253.2
100.0 179.9 128.1
0.6 46.6 2110.0
Fold increase
77.6 3520.0
-
469
of platelets, and are representative of four similar experiments. Sulfuric acid extraction of whole platelets and selective pH precipitation resulted in a pellet extract enriched 77.6-fold in phospholipase AZ activity over the homogenate. The recovery of soluble activity was almost twice that of the homogenate. Further enrichment was achieved by SP-Sephadex ion exchange chromatography; a single symmetrical peak of enzyme activity, eluting between 1.0 and 2.0 M NaCl, contained approximately one mg of protein and was purified 3500-fold over the homogenate in a yield of 128%. Incubation of purified platelet phospholipase AZ and l-[ 1-14C] stearyl 2acyl 3-sn-phosphatidylethanolamine yielded quantitatively as product only radioactive lysophosphatidylethanolamine confirming the 2-acyl specificity of this enzyme. The isolated enzyme is active toward exogenous substrates over a wide pH range (pH 7.0-10.5) (not shown) in contrast to platelet membrane preparations which hydrolyze endogenous phospholipids maximally between pH 8.0 and 9.5 [2]. Figs. 1A and 1B show the requirement of the purified phospholipase Az for Ca2’ and the inhibition of this activity by indomethacin. While platelet membrane preparations have maximal activity toward exogenous substrate with approximately 5.0 mM Ca2+, the isolated enzyme was maximally active in the presence of only 0.5 to 0.75 mM Ca” (Fig. 1A) and was inhibited by EDTA (not shown). Recent studies with intact platelets and divalent cation ionophore A23187 suggest that mobilization of internal stores of Ca” rather than an influx of external Ca2+ may initiate platelet phospholipase A2 activity [ 7 ] . Both 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoa~ and dibutyryl cyclic AMP inhibited the A23187 induced activation of platelet phospholipase A?, presumably by “fixing” Ca2+ at membrane storage sites [ 81. The inhibition was significantly reduced by added Ca2+. These observations and our data demonstrating the dramatic increase in enzymic activity in response to micromolar concentrations of Ca2’ strongly support the concept that the
I
/
10.”
INDOMETHACIN
10-q
10-3
M
Fig. 1. Calcium requirement for isolated phospholipase activity and the inhibition of activity by indomethacin. Phospholipid hydrolysis was assayed using autoclaved E. coli as described in the methods with varying concentrations of calcium (A) and with varying concentrations of indomethacin in the presence of 5.0 mM calcium (B). Maximal activity (100%) equals 2.1 pmol free fatty acid/h per mg protein.
470
availability of free intracellular Ca*+ is a major regulatory step in the generation of free fatty acids from phospholipids in human platelets. At present little is known concerning the precise mechanism by which platelet phospholipase A2 is modulated by Ca*+. However, studies with both the isolated pancreatic and leukocyte enzymes [9,10] indicate that Ca*’ can interact with either the phospholipid substrate, the enzyme, or both to markedly influence catalysis. Although the inhibitory effects of indomethacin on prostaglandin biosynthesis have been attributed solely to its action on the cyclooxygenase enzyme that converts arachidonate into endoperoxide, it has recently been shown to inhibit the release of arachidonate in intact platelets [ 111. This drug also inhibited the isolated platelet phospholipase A,. Fig. 1B shows the dose-dependent inhibition of this activity by indomethacin. Inhibition was detectable at lo-’ M indomethacin and one-half maximal inhibition was noted at 7.5. lo-’ M. Aspirin on the other hand had no effect on platelet phospholipase A, activity (not shown). Kaplan et al. [ 31 have previously described the inhibition of leukocyte phospholipase A, by indomethacin in both disrupted leukocytes and purified protein preparations. Interestingly, in their study indomethacin inhibition (ID,, = 30 PM) was immediate and non-competitive, presumably due to specific interaction of the drug with enzyme. That four other soluble phospholipases were insensitive to 50 PM concentrations of the drug may indicate that this is not a result of some non-specific inhibition, (i.e., interaction with substrate). Thus, inhibition of the platelet and leukocyte phospholipase A, activities by non-steroidal anti-inflammatory agents such as indomethacin may contribute to the anti-inflammatory action of these drugs in vivo. This work has been funded in part by grants HL 21116 and HL 19148 from the National Institute of Health. References 1 2 3 4 5 6 7 8 9 10 11
Marcus, A. (1978) J. Lipid Res. 19, 793-826 Derksen. A. and Cohen, P. (1975) J. Biol. Chem. 250, 9342-9347 Kaplan, L., Weiss. J. and Elsbach. P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2955-2958 Franson, R., Patriarca, P. and Elsbach, P. (1974) J. Lipid Res. 15, 380-388 Waite, B.M. and van Deenen. L.L.M. (1967) Biochim. Biophys. Acta 137, 498-517 Bradford, M. (1976) Anal. Biochem. 72, 248-254 Pickett. W., Jesse, R. and Cohen, P. (1977) Biochim. Biophys. Acta 486. 209-213 Rittenhouse-Simmons, S. and Deykin. D. (1978) Biochim. Biophys. Acta 543. 409422 de Haas, G.H.. Bonsen, P.P.M., Peiterson, W.A. and van Deenen, L.L.M. (1971) Biochim. Acta 239. 252-266 Franson, R. and Waite, M. (1978) Biochemistry 17, 40294033 Deykin, D. and Russell, F. (1978) Circulation 57-58 Suppl. II. II-125 (Abstr.)
Biophys.
467-470
BBA Report BBA 51263
MODULATION OF PURIFIED PHOSPHOLIPASE AZ ACTIVITY HUMAN PLATELETS BY CALCIUM AND INDOMETHACIN
ROBERT
L. JESSE and RICHARD
FROM
C. FRANSON
Department of Biophysics, Medical College of Virginia, Box 694, MCV Station, Richmond, VA 23298 (U.S.A.) (Received
September
6th, 1979)
Key words: Phospholipase A, ; Ca’+; Zndomethacin;
(Human platelets)
Summary A membrane bound phospholipase A2 (phosphatide 2acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase AZ activity was assayed using [ l-14C] oleate-labeled Escherichiu coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-l position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca”. Indomethacin, but not aspirin, inhibited phospholipase A2 activity.
Metabolytes of arachidonic acid, including prostaglandin endoperoxides and thromboxanes, are important in the function of human platelets, (for review see Ref. 1). Greater than 95% of this precursor fatty acid is esterified in the C-2 position of phospholipids; thus, for prostaglandin synthesis to occur, arachidonate is thought to be made available by the action of a phospholipase A2 [ 21. Despite its apparent role in regulating the availability of ‘free’ arachidonate, little is known about the cellular or pharmacologic control of this enzyme. We report that a preparation enriched 3500-fold in human platelet phospholipase A2 activity, is highly sensitive to Ca” and, as recently demonstrated for the rabbit leukocyte phospholipase A2 [ 31, is inhibited by indomethacin. Outdated platelet rich plasma, collected from normal donors in acid citrate dextrose with a Hemonetics Model 30 cell separator was centrifuged at 200 X g for 15 min, then at 2500 X g for 15 min. The platelet pellet was washed with 0.9% saline and resuspended in 1.15% KC1 to a concentration of 15 mg platelet protein/ml. By light microscopic examination, this preparation was essentially free of contaminating white or red blood cells. This
468
concentrate was diluted 1: 1 with ice-cold H,SO, to a final concentration of 0.18 N HZS04 and was homogenized with a Potter-Elvehjem homogenizer. After 1 hour at 4”C, the suspension was rehomogenized and centrifuged at 10 000 X g for 15 min. The resulting supernatant fraction was dialyzed against four changes of 50 mM citrate buffer pH 6.0, then centrifuged at 40 000 X g for 30 min. The pellet, enriched in phospholipase AZ activity, was resuspended in 100 mM citrate pH 3.0 and centrifuged as above; activity was found almost entirely in the supernatant fraction, designated the ‘pellet extract’. The pellet extract was applied to a sulfa propyl-Sephadex column (SPSephadex C25-120, Pharmacia) equilibrated with 100 mM NaCl - 20 mM citrate pH 3.0; washed first with 150 ml of buffer, then with 250 ml of buffered 0.75 M NaCl. Phospholipase AZ activity was subsequently eluted with 120 ml of a linear NaCl gradient (0.75 M-3.0 M, pH 3.0) collected in 2.0 ml fractions. Phospholipase AZ activity was measured by established methods using l-[ 1-14C] oleate labeled autoclaved E. coli [ 41 or 1-acyl 2-C1-14C] linoleoyl 3-sn-phosphatidylethanolamine as substrate [ 51. To confirm positional specificity, l-[ l-i4C] stearyl 2-acyl phosphatidylethanolamine was used as substrate. Unless indicated otherwise, incubation mixtures contained in a total volume of 0.5 ml, 5 mM Ca*‘, 100 mM Tris-HCl buffer pH 9.5 and 2.5*10* cells of autoclaved E. coli (corresponding to approximately 4 nmol of phospholipid and 6000 cpm). Using sonicated phospholipid, reaction mixtures in a total volume 1.0 ml were as described above except that 50 nmol of substrate were used. In both assay systems platelet protein and incubation times (at 37°C) were adjusted to maintain linear kinetics. Reactions were stopped and radioactive lipids extracted and isolated as previously described [4,5]. Phospholipase A activity is expressed as nmol product formed (free fatty acid) per h. Protein was measured by the method of Bradford [6] modified by the addition of a 5 min incubation at 37°C prior to measuring absorption at 595 nm. Table I summarizes the purification and recovery of enzymatic activity and protein. These data represent one experiment; an extraction of 50 units TABLE
I
RECOVERY OF PROTEIN AND ENZYMATIC ACTIVITY IN VARIOUS PURIFICATION OF HUMAN PLATELET PHOSPHOLIPASE A,
FRACTIONS
DURING
Platelets were prepared and the fractions were isolated as described in methods. The data are derived kom a single experiment with an initial extraction of 50 units of platelets, and are representative of four other experiments. In aU experiments, unless indicated otherwise, phospholipase A, activity was assayed in duplicate or triplicate using the standard E. coli incubation, (see methods). The data were averaged and are expressed as nmol free fatty acid formed/h per mg. All values are corrected for nonenzymatic hydrolysis, which was less than 3.0% in alI experiments. ___Fraction
Protein -
Homogenate Pellet extract SP-Sephadex
column
PhosphoIipase activity
A,
Specific activity
mg
% Recovery
nmol/b
% Recovery
(nmoIF/mg)
2390.0 67.9 1.1
100.0 2.3 .04
1718.0 3163.2 2253.2
100.0 179.9 128.1
0.6 46.6 2110.0
Fold increase
77.6 3520.0
-
469
of platelets, and are representative of four similar experiments. Sulfuric acid extraction of whole platelets and selective pH precipitation resulted in a pellet extract enriched 77.6-fold in phospholipase AZ activity over the homogenate. The recovery of soluble activity was almost twice that of the homogenate. Further enrichment was achieved by SP-Sephadex ion exchange chromatography; a single symmetrical peak of enzyme activity, eluting between 1.0 and 2.0 M NaCl, contained approximately one mg of protein and was purified 3500-fold over the homogenate in a yield of 128%. Incubation of purified platelet phospholipase AZ and l-[ 1-14C] stearyl 2acyl 3-sn-phosphatidylethanolamine yielded quantitatively as product only radioactive lysophosphatidylethanolamine confirming the 2-acyl specificity of this enzyme. The isolated enzyme is active toward exogenous substrates over a wide pH range (pH 7.0-10.5) (not shown) in contrast to platelet membrane preparations which hydrolyze endogenous phospholipids maximally between pH 8.0 and 9.5 [2]. Figs. 1A and 1B show the requirement of the purified phospholipase Az for Ca2’ and the inhibition of this activity by indomethacin. While platelet membrane preparations have maximal activity toward exogenous substrate with approximately 5.0 mM Ca2+, the isolated enzyme was maximally active in the presence of only 0.5 to 0.75 mM Ca” (Fig. 1A) and was inhibited by EDTA (not shown). Recent studies with intact platelets and divalent cation ionophore A23187 suggest that mobilization of internal stores of Ca” rather than an influx of external Ca2+ may initiate platelet phospholipase A2 activity [ 7 ] . Both 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoa~ and dibutyryl cyclic AMP inhibited the A23187 induced activation of platelet phospholipase A?, presumably by “fixing” Ca2+ at membrane storage sites [ 81. The inhibition was significantly reduced by added Ca2+. These observations and our data demonstrating the dramatic increase in enzymic activity in response to micromolar concentrations of Ca2’ strongly support the concept that the
I
/
10.”
INDOMETHACIN
10-q
10-3
M
Fig. 1. Calcium requirement for isolated phospholipase activity and the inhibition of activity by indomethacin. Phospholipid hydrolysis was assayed using autoclaved E. coli as described in the methods with varying concentrations of calcium (A) and with varying concentrations of indomethacin in the presence of 5.0 mM calcium (B). Maximal activity (100%) equals 2.1 pmol free fatty acid/h per mg protein.
470
availability of free intracellular Ca*+ is a major regulatory step in the generation of free fatty acids from phospholipids in human platelets. At present little is known concerning the precise mechanism by which platelet phospholipase A2 is modulated by Ca*+. However, studies with both the isolated pancreatic and leukocyte enzymes [9,10] indicate that Ca*’ can interact with either the phospholipid substrate, the enzyme, or both to markedly influence catalysis. Although the inhibitory effects of indomethacin on prostaglandin biosynthesis have been attributed solely to its action on the cyclooxygenase enzyme that converts arachidonate into endoperoxide, it has recently been shown to inhibit the release of arachidonate in intact platelets [ 111. This drug also inhibited the isolated platelet phospholipase A,. Fig. 1B shows the dose-dependent inhibition of this activity by indomethacin. Inhibition was detectable at lo-’ M indomethacin and one-half maximal inhibition was noted at 7.5. lo-’ M. Aspirin on the other hand had no effect on platelet phospholipase A, activity (not shown). Kaplan et al. [ 31 have previously described the inhibition of leukocyte phospholipase A, by indomethacin in both disrupted leukocytes and purified protein preparations. Interestingly, in their study indomethacin inhibition (ID,, = 30 PM) was immediate and non-competitive, presumably due to specific interaction of the drug with enzyme. That four other soluble phospholipases were insensitive to 50 PM concentrations of the drug may indicate that this is not a result of some non-specific inhibition, (i.e., interaction with substrate). Thus, inhibition of the platelet and leukocyte phospholipase A, activities by non-steroidal anti-inflammatory agents such as indomethacin may contribute to the anti-inflammatory action of these drugs in vivo. This work has been funded in part by grants HL 21116 and HL 19148 from the National Institute of Health. References 1 2 3 4 5 6 7 8 9 10 11
Marcus, A. (1978) J. Lipid Res. 19, 793-826 Derksen. A. and Cohen, P. (1975) J. Biol. Chem. 250, 9342-9347 Kaplan, L., Weiss. J. and Elsbach. P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2955-2958 Franson, R., Patriarca, P. and Elsbach, P. (1974) J. Lipid Res. 15, 380-388 Waite, B.M. and van Deenen. L.L.M. (1967) Biochim. Biophys. Acta 137, 498-517 Bradford, M. (1976) Anal. Biochem. 72, 248-254 Pickett. W., Jesse, R. and Cohen, P. (1977) Biochim. Biophys. Acta 486. 209-213 Rittenhouse-Simmons, S. and Deykin. D. (1978) Biochim. Biophys. Acta 543. 409422 de Haas, G.H.. Bonsen, P.P.M., Peiterson, W.A. and van Deenen, L.L.M. (1971) Biochim. Acta 239. 252-266 Franson, R. and Waite, M. (1978) Biochemistry 17, 40294033 Deykin, D. and Russell, F. (1978) Circulation 57-58 Suppl. II. II-125 (Abstr.)
Biophys.
