189
Biochimicu et Biophysicu Acru, 1046 (1990) 189-194 Elsevier
BBALIP 53483
Purification and characterization of phospholipase A 2 from rat stomach Tadahide Yasuda I, Junko Hirohara *, Tadayoshi Okumura
*
and Kunihiko Saito
’
’ Department of Medical Chemistry and 2 Third Department of fnrernul Medicine, Kansai Medicui School, Osaka (Japan) (Received 4 March 1990)
Key words: Phospholipase A,; Arachidonic acid; (Rat stomach)
Phospholipase A,, which is kcalixed in the mucosal part of the corpus of rat stomach (Hirohara et al. (1987) Biochim. Biophys. Acta 919,2314&Q, was puritied 998-fold from the supernatant of a tissue homogenate by heat treatment at acidic PI-&ammo&m sulfate fractionation, ion-exchange chromatography, gel-filtration and reverse-phase highh-performance liquid chromato8raphy (reverse-phase HPLC). The puritied enzyme gave a sin& protein band on sodium dodecyl sulfate (SDS)-~y~l~i~ gel electrophoresis with a molecular mass of approx. 17 hDa. The enzyme had a pH optimum of 8.0 and hydrolyzed the 2-arachidonoyl residue of phosphatidylcholine preferentially to the 2-oleoyl residue, the V_ and K, values for the two being 227 and 29 flmol/min per mg protein and 0.037 and 0.019 mM, respectively. The activity was ~ci~~~~t and was markedly increased by SDS and dhnethyl sulfoxide @MS@. The enzyme showed typical product hthibition. Free unsaturated fatty acids (oleic, arachidonic and docosahexaenoic acids), which are supposedly the main enzymatic products in vivo, inhibited the activity. Arachidonic acid caused ~c~~titive ~bition and its c~~n~ti~ for its maximal i~biti~ (!!%% in~bition~ was 5 10 -’ M. Lysophosphatidylcholine, free saturated fatty acids (palmitic and steak acids) and arachidonic acid metabolites (leuhotrienes and prostaglandins) had no effect on the activity. l
Introduction Phospho~p~ A, (EC 3.1.1.4), which hydrolyzes fatty acids esterified at the C-2 position of glycerophospholipids, is thought to be a rate-limiting enzyme for biosynthesis of prosta~~d~s, leukot~enes and even a platelet-activating factor in various cells and tissues. In human and rat stomach, endogenous gastric prostaaxons and exogenous treatment with their analogues have been shown to have cytoprotective effects and cause healing of peptic ulcers [l-6]. Recently, we (71 reported that the level of platelet-activating factor in rat stomach rapidly decreased during water immersion stress, which might be associated with ulcer formation. Rat stomach contains several lipolytic enzymes, such as phospho~p~s A, and A,, ly~phospho~pase and transacylase [&lo]. In particular, we [12] found that the
stomach has a higher amount of phospholipase A, than other organs, and that its activity is mainly present in the mucosal part (mucosa and submucosa) of the corpus. Grataroli et al. Ill] and we [12] reported some properties of crude phospholipase A, from the stomach, but further e~~olo~c~ studies are necessary to clarify whether this phospholipase AZ is involved in regulation of active lipid metabolism in the stomach. Tojo et al. [13] purified a pancreatic type phospholipase A, from the supematant of a rat stomach homogenate, but did not characterize it well. In this study, we purified phospholipase A, from rat stomach to a homogeneous state with high recovery and determined its substrate specificity and the effects of various compounds, such as DMSO, SDS, fatty acids and arachidonic acid metabolites, on its activity. Materials and Methods
Abbreviations: HPLC, high performance liquid chromatography; DMSO dimethyl sulfoxide; EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecyl sulfate; Tris-HCl, t~~hydrox~ethyI~n~ methane-HCI. Correspondence: T. Okumura, Department of Medical Chemistry, Kansai Medical School, l-Fumizonocho, Mo~guc~, Osaka 570, Japan.
Chemicals l-~l-‘4C]Palmitoyl-2-lyso-~~-~ycero-3-phosph~h~ line (50 mCi/mmol) and l-p~~toyl-Z-[l-‘4C]~ac~donoyl-sn-glyccro-3-phosphocholine (55 mCi/ mmol) were purchased from New England Nuclear, Boston, U.S.A. l-Acyl-Z-[l-‘4C]~ac~donoyl-~~-~y~r~3-phosph~
0005-2760/90/$03.50 B 1990 Elsevier Science publishers B.V. (Biomedical Division)
190 ethanolamine (60 mCi/mol) was from Amersham International, U.K. l-Acyl-2-[l-‘4C]oleoyl-sn-glycero-3phosphocholine (0.86 mCi/mmol) was prepared by the method of Pugh and Kates [14] as described previously [12]. l-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was from Avanti Polar-Lipids, AL, U.S.A. Phosphatidylcholine and its lysophosphatidylcholine were prepared from egg yolk [15]. Phosphatidylethanolamine purified from porcine liver, fatty acids and prostaglandins were from Funakoshi, Tokyo, Japan. CM-Sephadex C-50 and Sephadex G-50 were from Pharmacia, Uppsala, Sweden. TSK gel phenyl 5-PW was from Tosoh, Tokyo, Japan.
Preparation of enzyme source Male Wistar strain rats (180-250 g) were starved for 24 h and decapitated under light diethylether anesthesia. The stomach was removed, opened along the greater curvature and rinsed thoroughly with cold 0.15 M NaCI. The forestomach, which is the nonsecreting part covered with squamous epithelium, was discarded. The glandular stomach (1 g wet weight), consisting of the corpus and the antrum, was homogenized with 25 ml of 50 mM Tris-HCl buffer (pH 7.4) in an Ultra Turrax (Ika Werk, F.R.G.) for 4 min X 2 and then sonicated (50 W output 5, Branson Sonic Power, U.S.A.) for 1 min X 3 at 4O C and used as the enzyme source. Unless otherwise stated, all subsequent procedures were carried out at 4°C. Measurement of phospholipase A, activity In the standard assay conditions, the reaction mixture (0.4 ml) consisted of 0.1 mM 1-palmitoyl-2-[1r4C]arachidonoyl-sn-glycero-3-phosphocholine (40 000 dpm) in 50 mM glycylglycine-NaOH buffer, containing 2 mM CaCl, .2H,O and 50% DMSO (pH 8.0) and the test enzyme preparation. After incubation for 15 min at 37 “C, the reaction was stopped and the radioactive fatty acid released was determined by the method of Katsumata et al. [16] with some modifications as follows. The reaction was terminated by adding 0.2 ml of 60 mM EDTA containing 3% Triton X-100. The reaction product was extracted with 6 ml of n-hexane/O.l% acetic acid following the additions of 1.5 ml of H,O and approx. 1.5 g of anhydrous Na,SO,. After centrifugation, 3 ml of the n-hexane layer was mixed with 10 ml of Scintillator 299 (Packard, U.S.A.) and counted (TrisCarb, Packard). For measuring lysophospholipase and transacylase activities, the conditions were the same except that 1 mM l-[l-‘4C]palmitoyl-2-lyso-sn-glycero-3-phosphocholine was used as substrate. The reaction was terminated by adding 1.5 ml of chloroform/methanol (1 : 2, v/v). Then the reaction products were extracted by the method of Bligh and Dyer [17], separated by thin-layer chromatography and detected with radioac-
tive autoscanner as described previously [12]. Protein concentration was determined by the method of Lowry et al. [18] with bovine serum albumin as standard. Results Purification of rat stomach phospholipase A, Table I shows the activities of three lipolytic enzymes in the homogenate of rat glandular stomach. Phospholipase A, activity in the homogenate was stable for several months during storage at -20°C. The homogenate was centrifuged (105 000 x g for 60 min) and approx. 50% of the phospholipase A, activity and protein were recovered in the supernatant. The supernatant was acidified to pH 3.5 for 15 min and centrifuged (12000 X g, 15 min). The acidic supernatant was heated at 95” C for 7 min and centrifuged. The activity was quite stable during these treatments, in contrast to a report by Grataroli et al. [ll] that phospholipase A, from rat stomach is heat- and acid-labile. The heattreated supernatant was neutralized and the fraction precipitated with 25-85% saturation of ammonium sulfate was separated. At this step, the yield of activity was high (70% of that of the extract) but the specific activity was not increased appreciably (4-fold). However, the lysophospholipase and transacylase activities were both completely removed (data not shown). The ammonium sulfate fraction was applied to a CM Sephadex C-50 column (Fig. 1A). On stepwise increase in the NaCl concentration, the activity was eluted with 75 mM NaCl. This fraction was then applied to a Sephadex G-50 column (Fig. 1B). The active fraction recovered from the column was dialyzed, lyophilized and applied to a reverse phase HPLC column at room temperature (Fig. 1C). On elution with a linear gradient of decreasing (NH4)*S04 concentration, the activity was eluted at 0.28 M (NH4)*S04 with a retention time of 55 min. The active fraction was rechromatographed under the same conditions. The final enzyme preparation gave a single protein band (approx. 17 kDa) on
TABLE
I
Phospholipase A2, lysophospholipase and transacylase activities in the homogenate of rat stomach Glandular stomachs of rats (190-370 g assays as described standard deviations. ses.
Phospholipase A2 Lysophospholipase Transacylase
(0.96-1.06 g wet weight, 113-131 mg as protein) body weight) were homogenized and used for in Materials and Methods. Values are means* Numbers of experiments are shown in parenthe-
Specific activity (~mol/min per mg protein)
Total activity (pmol/min per rat stomach)
0.230 f 0.06 (7) 0.054 (1) 0.019 (1)
27.6+6.5 6.3 2.2
(7) (1) (1)
191
kDd 94 67 43 30 IO
20
Fraction
20
number
14
20
40
Fraction
Fig. 2. SDS-polyacrylamide gel electrophoresis of purified phospholipase A, from rat stomach. The HPLC-purified sample (10 pg protein) was applied to the top of a lo-2046 gradient gel in the presence of &mercaptoe.thanol. Electrophoresis was carried out at 60 mA for 60 mm and the gel was stained with 0.25% Coomassie brilliant blue R-250. The standard proteins (STD) used were phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa).
40
number
-6
I -3
20
40
Retention
60
front
60
time (min)
Fig. 1. Purification of rat stomach phospholipase A, by column chromatograpbies. (A) Chromatography on CM Sephadex C-50. The ammonium sulfate fraction (25-85% saturation, 119 nmol/min per 175 mg protein) was dialyzed against 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and applied to a column (2.5 X6 cm) equilibrated with the same buffer. Material was eluted (50 ml/h, 20 ml/fraction) with buffer containing 0, 10, 75 and 500 mM NaCl. (B) Gel filtration on Sephadex G-50. The 75 mM NaCl fraction from the CM Sephadex C-50 column (92 pmol/min per 20 mg protein) was dialyzed, lyophilized and dissolved in 10 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and 0.1 mM EDTA and applied to a column (1.35 X86 cm) equilibrated and developed (20 ml/h, 2.4 ml/fraction) with the same buffer. (C) HPLC on phenyl 5-PW. The lyophilized post-Sephadex G-50 sample (74 pmol/min per 5.3 mg protein) was dissolved in 10 mM Tris-HCl buffer (pH 7.4) containing 1.7 M (NH&SO4 and applied to a column of TSK gel phenyl 5-PW (7.5 X 75 mm) equilibrated with the same buffer. Material was eluted (1 ml/mm) with a Tosoh CCPE dual-Pump HPLC system with the same buffer for 5 min, and then with a linear gradient of 1.7 to 0 M (NH4)2S04 in 60 mm at room temperature.
SDS-polyacrylamide gel electrophoresis (Fig. 2) and had a specific activity of 168 ~mol/rnin per mg protein. Table II summarizes results during the purification. The total recovery of activity was about 40% with 990-fold purification. Characteristics of purified rat stomach phospholipase A2
When l-palmitoyl-2-[l-‘4C]arachidonoyl-sn-glycero3-phosphocholine was used as substrate, the purified phospholipase A, had a pH optimum of 8.0 with a shoulder at pH 5 (Fig. 3, left). This pH profile was similar to that of the crude enzyme in the homogenate
2
4
6
PH
8
IO I2
&t2
(In&)
Fig. 3. Effects of pH and calcium on rat stomach phospholipase A,. The purified phospholipase A, was incubated under the conditions described in Material and Methods except that various pH values (A, acetate buffer; n, glycylglycine-NaOH buffer) and various concentrations of CaCl,.2H,O (0) or 2 mM CaCI,.2H,O in the presence of 2 mM EDTA (0) were used.
192 TABLE
II
Purification of rat stomach phospholipax A, The stomachs (19 g) from 23 male rats were homogenized and sonicated with 475 ml of 50 mM Tris-HC1 105000 x g for 60 min. The resulting supematant was used as starting material. Protein (mg)
Total activity (pmol/min)
1250 1190 210 28 10 0.48
210 (100) 180 (84) 150 (71) 150 (70) 140 (67) 80 (38)
Specific activity (pmol/min per mg protein)
buffer
(pH 7.4) and then centrifuged
at
Purification
@) Supematant Acid-heat treatment (NH4)2S04 fractionation CM-Sephadex C-50 Sephadex G-50 2nd HPLC (phenyl’5PW)
described previously [12]. The activity was calcium-dependent and was inhibited completely by addition of EDTA (Fig. 3, right); the calcium concentrations for half maximal and maximal activation were less than 0.1 mM and 1 mM, respectively. The release of radioactive fatty acid from the substrate increased linearly with increase in the reaction time and in the enzyme protein-concentration to approx. 6 nmol (15% of the total substrate) per reaction mixture and then continued with decreasing velocity (data not shown). This loss of linearity was presumably due to enzyme inhibition by released arachidonic acid as discussed later. Fig. 4 shows the effects of DMSO and SDS on the activity DMSO and SDS increased phospholipase A, activity in the assay system by 20- and 2-fold at concentrations of 40 and 0.025%, respectively. Furthermore, pretreatment of the enzyme protein with SDS stimulated the activity more efficiently: preincubation with 0.5-1.0% SDS for 15 min at room temperature increased the activity more than lo-fold and stabilized it for several days at room temperature (data not shown). Kinetic analyses of the purified phospholipase A, were made with three substrates, 1-palmitoyl-2-[l-
0.17 0.15 0.68 4.9 14 168
1 0.9 4.0 29 82 990
“C]arachidonoyl-sn-glycero-3-phosphocholine, l-acyl2-[l-‘4C]arachidonoyl-~~-glycero-3-phosphoethanolamine and l-acyl-2-[l-‘4C]oleoyl-sn-glycero-3-phosphocholine (Fig. 5). The K, values for these three substrates were similar (0.037, 0.018 and 0.019 mM, respectively), but the V,,, values with the former two phospholipids, with an arachidonoyl residue at position C-2, were higher (227 and 318 pmol/rnin per mg protein respectively) than that with the third substrate (29 pmol/rnin per mg protein). Fig. 6 shows the effects of various fatty acids and lysophosphatidylcholine, which are products of the phospholipase A, reaction, on the activity. Unsaturated fatty acids such as oleic acid (18 : l), arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6) inhibited the activity significantly, but saturated fatty acids such as palmitic acid (16 : 0) and stearic acid (18 : 0) and 2-lysophosphatidylcholine did not. The maximal inhibition by arachidonic acid was approx. 50% at 5. 1O-5 M and the concentration for half maximal inhibition was 1.2. lo-’ M (data not shown). Lineweaver-Burk plots for 1-palmitoyl-2-[114C]arachidonoy 1-sn- g 1y cero-3-phosphocholine in the presence of 4. lo-’ M arachidonic acid showed no change of the K, value (0.032 mM) but decrease of the per mg protein), indicating Vmax value (83 pmol/min that arachidonic acid acts as a noncompetitive inhibitor. The arachidonic acid metabolites leukotrienes (C, and D4) and prostaglandins (E2, D, and F,,) had no effect on the activity at concentrations of up to 10e5 M. Discussion
Fig. 4. Effects of DMSO and SDS on rat stomach phospholipase A,. The purified phospholipase A, was incubated under the standard conditions in the presence of various concentrations of SDS (A) or DMSO (0). In the case of DMSO, the enzyme was incubated with 0.5% SDS for 30 min at room temperature before the assay.
During the last 10 years, phospholipase A, has been purified from various sources, such as rat platelets [19], rat spleen [20], rat pancreas [21], rat stomach [13], rat liver [22], rabbit platelets [23], human platelets [24], human synovial fluid [25-281, mouse peritoneal macrophages [29] and macrophage-like cell line [30]. Characterizations of these enzymes have indicated the existence of two types of phospholipase A,: one is a
193 pancreatic type in various organs [13,20,21] besides the pancreas and pancreatic juice; and the other is a nonpancreatic type also in various organs [19,22-241 and in extracellular fluid [25-281. In this study we purified phospholipase A, from the supernatant of a homogenate of rat stomach by a simple procedure and obtained a substantial amount of enzyme protein with high recovery. After homogenization with an Ultra Turrax and sonication approx. half the activity was recovered in the supernatant, whereas by the methods used previously only 6 to 10% of the activity was recovered in the supernatant [12,13]. The residual activ-
0.0 5
0.1 5
0.1 0
Concentration
( mM)
I/KS1 (mM_‘)
/
I -50
0
50
100
I/ISl(mM”) Fig. 5. Kinetic analyses of rat stomach phospholipase A 2 with various phospholipid substrates. The effects of increasing phospholipid concentrations on phospholipase A, activities (top) and double reciprocal plots of the activities (middle and bottom) toward l-acyl-(l‘4C]arachidonoyl-rn-glycero-3-phosphoethanolamine (A, A), l-palmitoyl-2-[l-‘4C]arachidonoyl-sn-glycero-3-phosphocholine (0, l) and l-acyl-2-[l-‘4C]oleoyl-sn-glycero-3-phosphocholine (0, n) are shown.
Contmll6~0 80
184 204
2Z6 LPC
Fig. 6. Effects of fatty acids and lysophosphatidylcholine on rat stomach phospholipase A,. The purified phospholipase A, was incubated in the absence (Control) and presence of palmitic acid (16 : 0), stearic acid (18:0), oleic acid (18: l), arachidonic acid (20:4), deco sahexaenoic acid (22 : 6) or lysophosphatidylcholine (LPC) purified from egg yolk, each at 5.10-’ M.
ity in the insoluble fraction showed a similar pH optimum, and similar heat-resistance and calcium-dependence to the enzyme purified from the soluble fraction (data not shown), suggesting that it was that of the same enzyme. However, the possibility that the phospholipases A, in the insoluble fraction is a different enzyme cannot be ruled out, until it has been purified and characterized. Tojo et al. [13] purified phospholipase A r and its proenzyme from rat stomach and concluded from its N-terminal amino acid sequence that it was of the pancreatic type. The cellular fraction of human stomach also contains immunoreactive pancreatic type phospholipase A, [31]. Pancreatic type phospholipase A, mRNA has been detected in rat gastric mucosa and lung, and shown to have the same nucleotide sequence as that of mRNA from rat pancreas [32]. Recently, Okamoto’s group [33,34] purified both pancreatic and non-pancreatic type phospholipases A,, which have different characteristics, from the supematant and pellet fractions of a homogenate of rat spleen, respectively, indicating the presence of at least wo phospholipases A, with different functions in the same cells or tissues. The molecular mass of our enzyme, determined by SDSpolyacrylamide gel electrophoresis, was approx. 17 kDa (Fig. 2), which is slightly higher than that (14 kDa) of the enzyme from rat pancreas [21]. Aarsman et al. [22] found that the molecular mass of phospholipase A, purified from rat liver mitochondria was 17 kDa under similar conditions to those used here, but they concluded that it was actually 14 kDa, because its migration was identical with those of porcine pancreatic and Crotalus adamanteus phospholipases A,, The N-terminal amino acid sequence of this enzyme showed only 25% homology with that of rat pancreatic phospholipase A, [21] but 96% identity with those of phospholipases A, from rat platelets [19] and rat spleen membranes [34]. Judging from these reports, our purified phospholipase AZ seems likely to be the pancreatic type, but we do not yet know whether it is the same enzyme as that reported by Tojo et al. [13].
194 The phospholipase A 2 activity increased with increase in DMSO concentration at lower concentrations, but decreased at higher DMSO concentrations (more than 50% (Fig. 4). Similar results were reported with skin phospholipase A, [35], which shows maximum activity with 30% DMSO. Our enzyme was also activated and stabilized by pretreatment with SDS. The mechanisms of these activations are unknown. Probably, DMSO and SDS influence the enzyme-substrate interaction by modifying the physiochemical state of the enzyme and/or the substrate. Purified phospholipase A, preferentially hydrolyzed 2-arachidonoyl-phosphatidylcholine and -phosphatidylethanolamine (Fig. 5). Similar high selectivity for the arachidonoyl residue was also reported for phospholipase A, in human platelets [36]. The cytosolic fraction of human platelets contains phospholipase A, activity showing high selectivity for phospholipid containing arachidonoyl residues. Unsaturated fatty acids inhibited the activity, whereas saturated fatty acids, lysophosphatidylcholine and arachidonic acid metabolites had not effect on the activity (Fig. 6). These properties are very similar to those of macrophage phospholipase A, [30]. Macrophage phospholipase A 2 showed no preference for arachidonoyl-containing phospholipid, but arachidonic acid was found to be a competitive inhibitor, suggesting a possible regulatory role of arachidonic acid in vivo [30]. The selectivity of our enzyme for arachidonoyl residues and its inhibition by arachidonic acid indicate that rat stomach phospholipase A, is involved in phospholipid metabolism especially related to arachidonic acid release and participates in the production of arachidonic acid metabolites. To elucidate the role of this enzyme in regulation of phospholipid metabolism, we are now studying further details of its molecular and catalytic properties. Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and in part by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation (1989). References 1 Arakawa, T., Kobayashi, K., Nakamura, H., Chono, S., Yamada, H., Ono, T. and Yamamoto, S. (1981) Gastroenterol. Jpn. 16, 236-241. 2 Robert, A., Nezamis, J.E., Lancaster, C. and Hanchar, A.J. (1979) Gastroenterology 77, 433-443. 3 Vantrappen, G., Janssens, J., Popiela, T., Kulig, J., Tytgat, G.N.J., Huibregtse, K., Lambert, R., Pauchard, J.P. and Robert, A. (1982) Gastroenterology 83, 357-363. 4 Rachmilewitz, D., Ligumsky, M., Fich, A., Goldin, E., Eliakim, A. and Karmeli, F. (1986) Gastroenterology 90, 963-969. 5 Jentjens, T. and Strous, G.J. (1987) ProstagIand. Leuk. Med. 27, l-8.
6 Garrick, T., Kolve, E. and Kauffman, G.L.. Jr.. (1986) Digest. Dia Sci. 31, 40-405. 7 Sugatani, J., Fujimura, K.. Miwa, M., Mizuno. T., Sameshima. Y. and Saito, K. (1989) FASEB J. 3, 65-70. 8 Wassef, M.K., Lin, Y.N. and Horowitz, M.I. (1978) Biochim. Biophys. Acta 528, 318-320. 9 Lin, Y.N., Wassef, M.K. and Horowitz, M.I. (1979) Arch. Biothem. Biophys. 193, 213-220. 10 Wassef, M.K., Lin, Y.N. and Horowitz, M.I. (1980) in Membrane Fluidity: Biophysical Techniques and Cellular Regulation (Kates, M. and Kuksis, A., eds.), pp. 283-296, Humana, Clifton, NJ. 11 Grataroli, R., Charbonnier, M., Ltonardi, J., Grimaud, J-C., Lafont, H. and Nalbone, G. (1987) Arch. B&hem. Biophys. 258, 77-84. 12 Hirohara, J., Sugatani, J., Okumura, T., Sameshima. Y. and Saito. K. (1987) Biochim. Biophys. Acta 919, 231-238. 13 Tojo, H., Ono, T. and Okamoto, M. (1988) Biochem. Biophys. Res. Commun. 151, 1188-1193. 14 Pugh, E.L. and Kates, M. (1975) B&him. Biophys. Acta 380, 442-453. 15 Saito, K. and Sato, K. (1968) Biochim. Biophys. Acta 151,706-708. 16 Katsumata, M., Gupta, C. and Goldman, A.S. (1986) Anal. Biothem. 154, 676-681. 17 Bligh, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 37, 911-917. 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. BioI. Chem. 193, 265-275. 19 Hayakawa, M., Kudo, I., Tomita, M., Nojima, S. and Inoue, K. (1988) J. B&hem. (Tokyo) 104, 767-772. T., Yamano, T. and Okamoto, M. (1984) 20 ToJo, H., Teramoto, Anal. B&hem. 137, 533-537. H., Yamano, T. and 21 Ono,T., Tojo, H., Inoue, K., Kagamiyama, Okamoto, M. (1984) J. B&hem. (Tokyo) 96, 785-792. 22 Aarsman, A.J., de Jong, J.G.N., Amoldussen, E., Neys, F.W.. van Wassenaar, P.D. and Van den Bosch, H. (1989) J. Biol. Chem. 264, looO8-10014. 23 Mizushima, H., Kudo, I., Horigome, K., Murakami, M., Hayakawa, M., Kim, D-K., Kondo, E., Tomita, M. and Inoue, K. (1989) J. B&hem. (Tokyo) 105, 520-525. 24 Kramer, R.M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E.P., Tizard, R. and Pepinsky, R.B. (1989) J. Biol. Chem. 264, 5768-5775. 25 Hara, S., Kudo, I., Chang, H.W., Matsuta, K., Miyamoto, T. and lnoue, K. (1989) J. Biochem. (Tokyo) 105, 395-399. K., Miyamoto, T. and Inoue, K. 26 Hara, S., Kudo, I., Matsuta, (1988) J. Biochem. (Tokyo) 104, 326-328. Biophys. Res. Commun. 27 Lai, C-Y. and Wada, K. (1988) B&hem. 157, 488-493. W., Vadas, P., Plant, S., Miller, J.A., 28 Seilhamer, J.J., Pruzanski, KIoss, J. and Johnson, L.K. (1989) J. Biol. Chem. 264, 5335-5338. 29 Wijkander, J. and Sundler, R. (1989) FEBS Lett. 244, 51-56. 30 Lister, M.D., Deems, R.A., Watanabe, Y., Ulevitch, R.J. and Dennis, E.A. (1988) J. Biol. Chem. 263, 7506-7513. 31 Matsuda, Y., Ogawa, M., Shibata, T., Nakaguchi, K., Nishijima, J-I., Wakasugi, C. and Mori, T. (1987) Res. Commun. Chem. Pathol. Pharmacol. 58, 281-284. 32 Sakata, T., Nakamura, E., Tsuruta, Y., Tamaki, M., Teraoka, H., Tojo, H., Ono, T. and Okamoto, M. (1989) B&him. Biophys. Acta 1007, 124-126. 33 Tojo, H., Ono, T., Kuramitsu, S., Kagamiyama, H. and Okamoto, M. (1988) J. Biol. Chem. 263, 5724-5731. 34 Ono, T., Tojo, H., Kuramitsu, S., Kagamiyama, H. and Okamoto, M. (1988) J. Biol. Chem. 263, 5732-5738. 35 Bergers, M., Verhagen, A.R., Jongerius, M. and Mier, P.D. (1986) Biochim. Biophys. Acta 876, 327-332. 36 Kim, D.K., Kudo, 1. and Inoue, K. (1988) J. B&hem. (Tokyo) 104, 492-494.
Biochimicu et Biophysicu Acru, 1046 (1990) 189-194 Elsevier
BBALIP 53483
Purification and characterization of phospholipase A 2 from rat stomach Tadahide Yasuda I, Junko Hirohara *, Tadayoshi Okumura
*
and Kunihiko Saito
’
’ Department of Medical Chemistry and 2 Third Department of fnrernul Medicine, Kansai Medicui School, Osaka (Japan) (Received 4 March 1990)
Key words: Phospholipase A,; Arachidonic acid; (Rat stomach)
Phospholipase A,, which is kcalixed in the mucosal part of the corpus of rat stomach (Hirohara et al. (1987) Biochim. Biophys. Acta 919,2314&Q, was puritied 998-fold from the supernatant of a tissue homogenate by heat treatment at acidic PI-&ammo&m sulfate fractionation, ion-exchange chromatography, gel-filtration and reverse-phase highh-performance liquid chromato8raphy (reverse-phase HPLC). The puritied enzyme gave a sin& protein band on sodium dodecyl sulfate (SDS)-~y~l~i~ gel electrophoresis with a molecular mass of approx. 17 hDa. The enzyme had a pH optimum of 8.0 and hydrolyzed the 2-arachidonoyl residue of phosphatidylcholine preferentially to the 2-oleoyl residue, the V_ and K, values for the two being 227 and 29 flmol/min per mg protein and 0.037 and 0.019 mM, respectively. The activity was ~ci~~~~t and was markedly increased by SDS and dhnethyl sulfoxide @MS@. The enzyme showed typical product hthibition. Free unsaturated fatty acids (oleic, arachidonic and docosahexaenoic acids), which are supposedly the main enzymatic products in vivo, inhibited the activity. Arachidonic acid caused ~c~~titive ~bition and its c~~n~ti~ for its maximal i~biti~ (!!%% in~bition~ was 5 10 -’ M. Lysophosphatidylcholine, free saturated fatty acids (palmitic and steak acids) and arachidonic acid metabolites (leuhotrienes and prostaglandins) had no effect on the activity. l
Introduction Phospho~p~ A, (EC 3.1.1.4), which hydrolyzes fatty acids esterified at the C-2 position of glycerophospholipids, is thought to be a rate-limiting enzyme for biosynthesis of prosta~~d~s, leukot~enes and even a platelet-activating factor in various cells and tissues. In human and rat stomach, endogenous gastric prostaaxons and exogenous treatment with their analogues have been shown to have cytoprotective effects and cause healing of peptic ulcers [l-6]. Recently, we (71 reported that the level of platelet-activating factor in rat stomach rapidly decreased during water immersion stress, which might be associated with ulcer formation. Rat stomach contains several lipolytic enzymes, such as phospho~p~s A, and A,, ly~phospho~pase and transacylase [&lo]. In particular, we [12] found that the
stomach has a higher amount of phospholipase A, than other organs, and that its activity is mainly present in the mucosal part (mucosa and submucosa) of the corpus. Grataroli et al. Ill] and we [12] reported some properties of crude phospholipase A, from the stomach, but further e~~olo~c~ studies are necessary to clarify whether this phospholipase AZ is involved in regulation of active lipid metabolism in the stomach. Tojo et al. [13] purified a pancreatic type phospholipase A, from the supematant of a rat stomach homogenate, but did not characterize it well. In this study, we purified phospholipase A, from rat stomach to a homogeneous state with high recovery and determined its substrate specificity and the effects of various compounds, such as DMSO, SDS, fatty acids and arachidonic acid metabolites, on its activity. Materials and Methods
Abbreviations: HPLC, high performance liquid chromatography; DMSO dimethyl sulfoxide; EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecyl sulfate; Tris-HCl, t~~hydrox~ethyI~n~ methane-HCI. Correspondence: T. Okumura, Department of Medical Chemistry, Kansai Medical School, l-Fumizonocho, Mo~guc~, Osaka 570, Japan.
Chemicals l-~l-‘4C]Palmitoyl-2-lyso-~~-~ycero-3-phosph~h~ line (50 mCi/mmol) and l-p~~toyl-Z-[l-‘4C]~ac~donoyl-sn-glyccro-3-phosphocholine (55 mCi/ mmol) were purchased from New England Nuclear, Boston, U.S.A. l-Acyl-Z-[l-‘4C]~ac~donoyl-~~-~y~r~3-phosph~
0005-2760/90/$03.50 B 1990 Elsevier Science publishers B.V. (Biomedical Division)
190 ethanolamine (60 mCi/mol) was from Amersham International, U.K. l-Acyl-2-[l-‘4C]oleoyl-sn-glycero-3phosphocholine (0.86 mCi/mmol) was prepared by the method of Pugh and Kates [14] as described previously [12]. l-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was from Avanti Polar-Lipids, AL, U.S.A. Phosphatidylcholine and its lysophosphatidylcholine were prepared from egg yolk [15]. Phosphatidylethanolamine purified from porcine liver, fatty acids and prostaglandins were from Funakoshi, Tokyo, Japan. CM-Sephadex C-50 and Sephadex G-50 were from Pharmacia, Uppsala, Sweden. TSK gel phenyl 5-PW was from Tosoh, Tokyo, Japan.
Preparation of enzyme source Male Wistar strain rats (180-250 g) were starved for 24 h and decapitated under light diethylether anesthesia. The stomach was removed, opened along the greater curvature and rinsed thoroughly with cold 0.15 M NaCI. The forestomach, which is the nonsecreting part covered with squamous epithelium, was discarded. The glandular stomach (1 g wet weight), consisting of the corpus and the antrum, was homogenized with 25 ml of 50 mM Tris-HCl buffer (pH 7.4) in an Ultra Turrax (Ika Werk, F.R.G.) for 4 min X 2 and then sonicated (50 W output 5, Branson Sonic Power, U.S.A.) for 1 min X 3 at 4O C and used as the enzyme source. Unless otherwise stated, all subsequent procedures were carried out at 4°C. Measurement of phospholipase A, activity In the standard assay conditions, the reaction mixture (0.4 ml) consisted of 0.1 mM 1-palmitoyl-2-[1r4C]arachidonoyl-sn-glycero-3-phosphocholine (40 000 dpm) in 50 mM glycylglycine-NaOH buffer, containing 2 mM CaCl, .2H,O and 50% DMSO (pH 8.0) and the test enzyme preparation. After incubation for 15 min at 37 “C, the reaction was stopped and the radioactive fatty acid released was determined by the method of Katsumata et al. [16] with some modifications as follows. The reaction was terminated by adding 0.2 ml of 60 mM EDTA containing 3% Triton X-100. The reaction product was extracted with 6 ml of n-hexane/O.l% acetic acid following the additions of 1.5 ml of H,O and approx. 1.5 g of anhydrous Na,SO,. After centrifugation, 3 ml of the n-hexane layer was mixed with 10 ml of Scintillator 299 (Packard, U.S.A.) and counted (TrisCarb, Packard). For measuring lysophospholipase and transacylase activities, the conditions were the same except that 1 mM l-[l-‘4C]palmitoyl-2-lyso-sn-glycero-3-phosphocholine was used as substrate. The reaction was terminated by adding 1.5 ml of chloroform/methanol (1 : 2, v/v). Then the reaction products were extracted by the method of Bligh and Dyer [17], separated by thin-layer chromatography and detected with radioac-
tive autoscanner as described previously [12]. Protein concentration was determined by the method of Lowry et al. [18] with bovine serum albumin as standard. Results Purification of rat stomach phospholipase A, Table I shows the activities of three lipolytic enzymes in the homogenate of rat glandular stomach. Phospholipase A, activity in the homogenate was stable for several months during storage at -20°C. The homogenate was centrifuged (105 000 x g for 60 min) and approx. 50% of the phospholipase A, activity and protein were recovered in the supernatant. The supernatant was acidified to pH 3.5 for 15 min and centrifuged (12000 X g, 15 min). The acidic supernatant was heated at 95” C for 7 min and centrifuged. The activity was quite stable during these treatments, in contrast to a report by Grataroli et al. [ll] that phospholipase A, from rat stomach is heat- and acid-labile. The heattreated supernatant was neutralized and the fraction precipitated with 25-85% saturation of ammonium sulfate was separated. At this step, the yield of activity was high (70% of that of the extract) but the specific activity was not increased appreciably (4-fold). However, the lysophospholipase and transacylase activities were both completely removed (data not shown). The ammonium sulfate fraction was applied to a CM Sephadex C-50 column (Fig. 1A). On stepwise increase in the NaCl concentration, the activity was eluted with 75 mM NaCl. This fraction was then applied to a Sephadex G-50 column (Fig. 1B). The active fraction recovered from the column was dialyzed, lyophilized and applied to a reverse phase HPLC column at room temperature (Fig. 1C). On elution with a linear gradient of decreasing (NH4)*S04 concentration, the activity was eluted at 0.28 M (NH4)*S04 with a retention time of 55 min. The active fraction was rechromatographed under the same conditions. The final enzyme preparation gave a single protein band (approx. 17 kDa) on
TABLE
I
Phospholipase A2, lysophospholipase and transacylase activities in the homogenate of rat stomach Glandular stomachs of rats (190-370 g assays as described standard deviations. ses.
Phospholipase A2 Lysophospholipase Transacylase
(0.96-1.06 g wet weight, 113-131 mg as protein) body weight) were homogenized and used for in Materials and Methods. Values are means* Numbers of experiments are shown in parenthe-
Specific activity (~mol/min per mg protein)
Total activity (pmol/min per rat stomach)
0.230 f 0.06 (7) 0.054 (1) 0.019 (1)
27.6+6.5 6.3 2.2
(7) (1) (1)
191
kDd 94 67 43 30 IO
20
Fraction
20
number
14
20
40
Fraction
Fig. 2. SDS-polyacrylamide gel electrophoresis of purified phospholipase A, from rat stomach. The HPLC-purified sample (10 pg protein) was applied to the top of a lo-2046 gradient gel in the presence of &mercaptoe.thanol. Electrophoresis was carried out at 60 mA for 60 mm and the gel was stained with 0.25% Coomassie brilliant blue R-250. The standard proteins (STD) used were phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa).
40
number
-6
I -3
20
40
Retention
60
front
60
time (min)
Fig. 1. Purification of rat stomach phospholipase A, by column chromatograpbies. (A) Chromatography on CM Sephadex C-50. The ammonium sulfate fraction (25-85% saturation, 119 nmol/min per 175 mg protein) was dialyzed against 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and applied to a column (2.5 X6 cm) equilibrated with the same buffer. Material was eluted (50 ml/h, 20 ml/fraction) with buffer containing 0, 10, 75 and 500 mM NaCl. (B) Gel filtration on Sephadex G-50. The 75 mM NaCl fraction from the CM Sephadex C-50 column (92 pmol/min per 20 mg protein) was dialyzed, lyophilized and dissolved in 10 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and 0.1 mM EDTA and applied to a column (1.35 X86 cm) equilibrated and developed (20 ml/h, 2.4 ml/fraction) with the same buffer. (C) HPLC on phenyl 5-PW. The lyophilized post-Sephadex G-50 sample (74 pmol/min per 5.3 mg protein) was dissolved in 10 mM Tris-HCl buffer (pH 7.4) containing 1.7 M (NH&SO4 and applied to a column of TSK gel phenyl 5-PW (7.5 X 75 mm) equilibrated with the same buffer. Material was eluted (1 ml/mm) with a Tosoh CCPE dual-Pump HPLC system with the same buffer for 5 min, and then with a linear gradient of 1.7 to 0 M (NH4)2S04 in 60 mm at room temperature.
SDS-polyacrylamide gel electrophoresis (Fig. 2) and had a specific activity of 168 ~mol/rnin per mg protein. Table II summarizes results during the purification. The total recovery of activity was about 40% with 990-fold purification. Characteristics of purified rat stomach phospholipase A2
When l-palmitoyl-2-[l-‘4C]arachidonoyl-sn-glycero3-phosphocholine was used as substrate, the purified phospholipase A, had a pH optimum of 8.0 with a shoulder at pH 5 (Fig. 3, left). This pH profile was similar to that of the crude enzyme in the homogenate
2
4
6
PH
8
IO I2
&t2
(In&)
Fig. 3. Effects of pH and calcium on rat stomach phospholipase A,. The purified phospholipase A, was incubated under the conditions described in Material and Methods except that various pH values (A, acetate buffer; n, glycylglycine-NaOH buffer) and various concentrations of CaCl,.2H,O (0) or 2 mM CaCI,.2H,O in the presence of 2 mM EDTA (0) were used.
192 TABLE
II
Purification of rat stomach phospholipax A, The stomachs (19 g) from 23 male rats were homogenized and sonicated with 475 ml of 50 mM Tris-HC1 105000 x g for 60 min. The resulting supematant was used as starting material. Protein (mg)
Total activity (pmol/min)
1250 1190 210 28 10 0.48
210 (100) 180 (84) 150 (71) 150 (70) 140 (67) 80 (38)
Specific activity (pmol/min per mg protein)
buffer
(pH 7.4) and then centrifuged
at
Purification
@) Supematant Acid-heat treatment (NH4)2S04 fractionation CM-Sephadex C-50 Sephadex G-50 2nd HPLC (phenyl’5PW)
described previously [12]. The activity was calcium-dependent and was inhibited completely by addition of EDTA (Fig. 3, right); the calcium concentrations for half maximal and maximal activation were less than 0.1 mM and 1 mM, respectively. The release of radioactive fatty acid from the substrate increased linearly with increase in the reaction time and in the enzyme protein-concentration to approx. 6 nmol (15% of the total substrate) per reaction mixture and then continued with decreasing velocity (data not shown). This loss of linearity was presumably due to enzyme inhibition by released arachidonic acid as discussed later. Fig. 4 shows the effects of DMSO and SDS on the activity DMSO and SDS increased phospholipase A, activity in the assay system by 20- and 2-fold at concentrations of 40 and 0.025%, respectively. Furthermore, pretreatment of the enzyme protein with SDS stimulated the activity more efficiently: preincubation with 0.5-1.0% SDS for 15 min at room temperature increased the activity more than lo-fold and stabilized it for several days at room temperature (data not shown). Kinetic analyses of the purified phospholipase A, were made with three substrates, 1-palmitoyl-2-[l-
0.17 0.15 0.68 4.9 14 168
1 0.9 4.0 29 82 990
“C]arachidonoyl-sn-glycero-3-phosphocholine, l-acyl2-[l-‘4C]arachidonoyl-~~-glycero-3-phosphoethanolamine and l-acyl-2-[l-‘4C]oleoyl-sn-glycero-3-phosphocholine (Fig. 5). The K, values for these three substrates were similar (0.037, 0.018 and 0.019 mM, respectively), but the V,,, values with the former two phospholipids, with an arachidonoyl residue at position C-2, were higher (227 and 318 pmol/rnin per mg protein respectively) than that with the third substrate (29 pmol/rnin per mg protein). Fig. 6 shows the effects of various fatty acids and lysophosphatidylcholine, which are products of the phospholipase A, reaction, on the activity. Unsaturated fatty acids such as oleic acid (18 : l), arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6) inhibited the activity significantly, but saturated fatty acids such as palmitic acid (16 : 0) and stearic acid (18 : 0) and 2-lysophosphatidylcholine did not. The maximal inhibition by arachidonic acid was approx. 50% at 5. 1O-5 M and the concentration for half maximal inhibition was 1.2. lo-’ M (data not shown). Lineweaver-Burk plots for 1-palmitoyl-2-[114C]arachidonoy 1-sn- g 1y cero-3-phosphocholine in the presence of 4. lo-’ M arachidonic acid showed no change of the K, value (0.032 mM) but decrease of the per mg protein), indicating Vmax value (83 pmol/min that arachidonic acid acts as a noncompetitive inhibitor. The arachidonic acid metabolites leukotrienes (C, and D4) and prostaglandins (E2, D, and F,,) had no effect on the activity at concentrations of up to 10e5 M. Discussion
Fig. 4. Effects of DMSO and SDS on rat stomach phospholipase A,. The purified phospholipase A, was incubated under the standard conditions in the presence of various concentrations of SDS (A) or DMSO (0). In the case of DMSO, the enzyme was incubated with 0.5% SDS for 30 min at room temperature before the assay.
During the last 10 years, phospholipase A, has been purified from various sources, such as rat platelets [19], rat spleen [20], rat pancreas [21], rat stomach [13], rat liver [22], rabbit platelets [23], human platelets [24], human synovial fluid [25-281, mouse peritoneal macrophages [29] and macrophage-like cell line [30]. Characterizations of these enzymes have indicated the existence of two types of phospholipase A,: one is a
193 pancreatic type in various organs [13,20,21] besides the pancreas and pancreatic juice; and the other is a nonpancreatic type also in various organs [19,22-241 and in extracellular fluid [25-281. In this study we purified phospholipase A, from the supernatant of a homogenate of rat stomach by a simple procedure and obtained a substantial amount of enzyme protein with high recovery. After homogenization with an Ultra Turrax and sonication approx. half the activity was recovered in the supernatant, whereas by the methods used previously only 6 to 10% of the activity was recovered in the supernatant [12,13]. The residual activ-
0.0 5
0.1 5
0.1 0
Concentration
( mM)
I/KS1 (mM_‘)
/
I -50
0
50
100
I/ISl(mM”) Fig. 5. Kinetic analyses of rat stomach phospholipase A 2 with various phospholipid substrates. The effects of increasing phospholipid concentrations on phospholipase A, activities (top) and double reciprocal plots of the activities (middle and bottom) toward l-acyl-(l‘4C]arachidonoyl-rn-glycero-3-phosphoethanolamine (A, A), l-palmitoyl-2-[l-‘4C]arachidonoyl-sn-glycero-3-phosphocholine (0, l) and l-acyl-2-[l-‘4C]oleoyl-sn-glycero-3-phosphocholine (0, n) are shown.
Contmll6~0 80
184 204
2Z6 LPC
Fig. 6. Effects of fatty acids and lysophosphatidylcholine on rat stomach phospholipase A,. The purified phospholipase A, was incubated in the absence (Control) and presence of palmitic acid (16 : 0), stearic acid (18:0), oleic acid (18: l), arachidonic acid (20:4), deco sahexaenoic acid (22 : 6) or lysophosphatidylcholine (LPC) purified from egg yolk, each at 5.10-’ M.
ity in the insoluble fraction showed a similar pH optimum, and similar heat-resistance and calcium-dependence to the enzyme purified from the soluble fraction (data not shown), suggesting that it was that of the same enzyme. However, the possibility that the phospholipases A, in the insoluble fraction is a different enzyme cannot be ruled out, until it has been purified and characterized. Tojo et al. [13] purified phospholipase A r and its proenzyme from rat stomach and concluded from its N-terminal amino acid sequence that it was of the pancreatic type. The cellular fraction of human stomach also contains immunoreactive pancreatic type phospholipase A, [31]. Pancreatic type phospholipase A, mRNA has been detected in rat gastric mucosa and lung, and shown to have the same nucleotide sequence as that of mRNA from rat pancreas [32]. Recently, Okamoto’s group [33,34] purified both pancreatic and non-pancreatic type phospholipases A,, which have different characteristics, from the supematant and pellet fractions of a homogenate of rat spleen, respectively, indicating the presence of at least wo phospholipases A, with different functions in the same cells or tissues. The molecular mass of our enzyme, determined by SDSpolyacrylamide gel electrophoresis, was approx. 17 kDa (Fig. 2), which is slightly higher than that (14 kDa) of the enzyme from rat pancreas [21]. Aarsman et al. [22] found that the molecular mass of phospholipase A, purified from rat liver mitochondria was 17 kDa under similar conditions to those used here, but they concluded that it was actually 14 kDa, because its migration was identical with those of porcine pancreatic and Crotalus adamanteus phospholipases A,, The N-terminal amino acid sequence of this enzyme showed only 25% homology with that of rat pancreatic phospholipase A, [21] but 96% identity with those of phospholipases A, from rat platelets [19] and rat spleen membranes [34]. Judging from these reports, our purified phospholipase AZ seems likely to be the pancreatic type, but we do not yet know whether it is the same enzyme as that reported by Tojo et al. [13].
194 The phospholipase A 2 activity increased with increase in DMSO concentration at lower concentrations, but decreased at higher DMSO concentrations (more than 50% (Fig. 4). Similar results were reported with skin phospholipase A, [35], which shows maximum activity with 30% DMSO. Our enzyme was also activated and stabilized by pretreatment with SDS. The mechanisms of these activations are unknown. Probably, DMSO and SDS influence the enzyme-substrate interaction by modifying the physiochemical state of the enzyme and/or the substrate. Purified phospholipase A, preferentially hydrolyzed 2-arachidonoyl-phosphatidylcholine and -phosphatidylethanolamine (Fig. 5). Similar high selectivity for the arachidonoyl residue was also reported for phospholipase A, in human platelets [36]. The cytosolic fraction of human platelets contains phospholipase A, activity showing high selectivity for phospholipid containing arachidonoyl residues. Unsaturated fatty acids inhibited the activity, whereas saturated fatty acids, lysophosphatidylcholine and arachidonic acid metabolites had not effect on the activity (Fig. 6). These properties are very similar to those of macrophage phospholipase A, [30]. Macrophage phospholipase A 2 showed no preference for arachidonoyl-containing phospholipid, but arachidonic acid was found to be a competitive inhibitor, suggesting a possible regulatory role of arachidonic acid in vivo [30]. The selectivity of our enzyme for arachidonoyl residues and its inhibition by arachidonic acid indicate that rat stomach phospholipase A, is involved in phospholipid metabolism especially related to arachidonic acid release and participates in the production of arachidonic acid metabolites. To elucidate the role of this enzyme in regulation of phospholipid metabolism, we are now studying further details of its molecular and catalytic properties. Acknowledgments
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