Preferential hydrolysis of oxidized phospholipids by peritoneal fluid of rats treated with casein

192

Biochimica et 3joph.v~lcu Actu, 963 (1988) 192-200 Elsevier

BBA 52969

Preferential hydrolysis of oxidized phospholipids by peritoneal fluid of rats treated with casein Hiroyuki

Itabe, Ichiro Kudo and Keizo Inoue

Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Tok.yo (Japan)

(Revised

Key words:

Phospholipase

A,;

(Received 9 May 1988) manuscript received 16 August

Rat peritoneal fluid; Casein induced 2-Azelaoylphosphatidylchoiine

1988)

inflammation:

Oxidized

phospholipids;

l-Palmitoyl-2-azelaoyl-PC, which is one of the possible cytotoxic products generated by the oxyhemoglobin-induced lipid peroxidation of 1-palmitoyl-2-linoleoyl-PC, was found to be efficiently hydrolyzed by the peritoneal fluid of rats treated with casein. The rate of hydrolysis of l-palmitoyl-2-azelaoyl-PC was approx. E-fold higher than that observed with l-palmitoyl-2-linoleoyl-PC. When l-palmitoyl-%-linoleoyl-PC pretreated with oxyhemoglobin was incubated with the ~ritone~ fluid, oxidized products of PC were hydrolyzed more efficiently than the intact I-palmitoyl-2-linoleoyl-PC. When 1-I1-‘4C]paimitoyl-2azelaoyl-PC was incubated with the peritoneal fluid, radiolabeled IysoPC was formed, whereas radiolabeled neutral lipids were not formed, indicating that the hydrolytic activity was of the ‘phospholipase A 2’ type. We previously found and purified an ex~acellufar phosphoiip~e A, (Chang, H.W. et al. (1987) J. Biochem. 102, 147-154) in the peritoneal fluid of rats injected intraperitoneally with casein. Hydrolysis of I-palmitoyl-Z azelaoyl-PC by this purified phospholipase A, was as low as that of l-palmitoyl-2-linoleoyl-PC. These two phospholipase A, activities showed different pH optima and Ca*+ requirements. The present phospholipase A, activity, which preferentially hydrolyzes oxidized products of PC, may play an important role in detoxification or repair of damaged membrane in infl~ed sites. Introduction Although high levels of extracellular phospholipase A, activity have often been found in inflamed sites 111, such as glycogen-induced ascites in rabbits [2,3], peritoneal fluid of casein-treated rats [4,5], gram-negative septic shock in human [6] and synovial fluid in patients with rheumatoid

Abbreviations: ethanolamine; mance liquid ane.

PC, phosphatidylcholine: PS. phosphatidyl~~ne; chromatography; ADAM,

Correspondence: K. Inoue, Faculty of Pharmaceutical Hongo, Tokyo 113, Japan. 0005-2760/88/$03.50

Department Sciences,

PE, phosphatidylHPLC, high-perfor9-anth~ldi~ometh-

of Health University

Chemistry, of Tokyo,

0 1988 Elsevier Science Publishers

arthritis [7,8], their physiological or pathological function has not yet been clarified. In inflamed sites, various serial events, such as an increase of blood permeability, accumulation of leukocytes, generation and release of chemical mediators, generation of oxygen free radicals, and peroxidation of lipids have often been observed. The involvement of lipid peroxidation in tissue damage is becoming obvious: accumulation of peroxidized lipid products was observed in the synovial fluid of patients with rheumatoid arthritis [9], the synovial fluid of rats with adjuvant arthritis [lo], ischemic rat brain 1111, rat liver administered with carbon tetrachloride [12], advanced atherosclerotic plaque of human aorta [13], and in disordered erythrocytes [14]. Some of the products generated during lipid peroxidation showed cyto-

B.V. (Biomedical

Division)

193 0

CH,O-&CH,),,CH, HOOCtCH,),

:: ’ C-OCH I F CH,O-~OCH,CH,ihCH,,,

bFig. 1. Structure

of l-palmitoyl-2-azelaoyl-PC.

toxic activities [15-171 and were suggested to be involved in pathological conditions. Cytotoxic phospholipids are generated during oxyhemoglobin-induced peroxidation of PC-containing polyunsaturated fatty acyl chains [l&19]. These products are separated from non-cytotoxic products by a straight-phase HPLC. We have identified 1-O-octadecyl-2-azelaoyl-PC as one of these cytotoxic products generated from 1-O-octadecyl-2-linoleoyl-PC [17]. It is reasonable to assume that 1-palmitoyl-2-azelaoyl-PC might be generated under the same conditions as when lpalmitoyl-2-linoleoyl-PC is oxidized instead of lO-octadecyl-2-linoleoyl-PC (Fig. 1). In fact, when 1-palmitoyl-2-linoleoyl-PC was treated with oxyhemoglobin and then coupled with a fluorescent reagent, we detected a fluorescent derivative corresponding to a compound generated from l-palmitoyl-2-azelaoyl-PC. Oxidized cytotoxic products of PC, including 1-palmitoyl-2-azelaoyl-PC, were hydrolyzed efficiently by the peritoneal fluid of rats treated with casein. Materials and Methods Chemicals. [ l-l4 C]Acetate, [ 3HImethyl iodide and l-[l-‘4C]palmitoyl-2-lysoPC were purchased from Amersham, Buckinghamshire, U.K. Linoleic acid and dipalmitoyl-PC were donations from Japan Fat and Oil CO., Tokyo, Japan. N,N-dimethylaminopyridine was purchased from Aldrich, Milwaukee, WI, U.S.A. Sodium caseinate and snake venom (Trimeresus flavovilidis) were purchased from Wako Pure Chemical Co., Osaka, Japan. Phospholipase A, (porcine pancreas, Naja naja and Crotarus adamanteus) were purchased from Sigma, St. Louis, MO, U.S.A. Synthesis of I-palmitoyl-2-azelaoyl-PC. 1-Palmitoyl-ZlysoPC was obtained from 1,2-dipalmitoylPC by treatment with snake venom phospholipase

AZ followed by a silica gel column chromatography separation on Bio-sil A (Bio-Rad, Richmond, MO, U.S.A.). Azelaoyl chloride (2.50 g, 10 mmol) dissolved in 10 ml of dry diethyl ether was added slowly to a solution of benzylalcohol (1.08 g, 10 mmol) and pyridine (0.79 g, 10 mmol) in 30 ml of dry diethyl ether. The reaction mixture was stirred and refluxed for 2 h. Distilled water (40 ml) was then added to the reaction mixture and the organic phase was separated. Azelaoylmonobenzyl ester was purified by column chromatography on Kiesel gel 60 (Merck, Darmstadt, F.R.G.) using chloroform as an eluant. A pure product (0.72 g, 2.5 mmol) was obtained having the following ‘H-NMR spectrum: 6 = 1.2-1.4, multiplet, 6H, methylenes; S = 1.5-1.9, multiplet, 4H, acyl y-methylenes; 6 = 2.3, triplet, 4H, acyl P-methylenes; 6 = 5.04, singlet, 2H, benzylmethylene; 6 = 7.23, singlet, 5H, benzyl protons. Acylation of 1ysoPC was performed by the method of Gupta et al. [20] with a slight modification. Azelaoylmonobenzyl anhydride was prepared by the reaction of azelaoylmonobenzyl ester (0.54 g, 1.9 mmol) with dicyclohexylcarbodiimide (0.31 g, 1.5 mmol) in 10 ml dry carbon tetrachloride for 2 h. The precipitate was removed by filtration. The azelaoylmonobenzyl anhydride fraction dissolved in dry chloroform was added to the solution of 1-palrnitoyl-2-1ysoPC (150 mg, 0.3 mmol) and N, N-dimethylaminopyridine (37 mg, 0.3 mmol) in 3 ml of chloroform. The reaction mixture was stirred at 60°C for 2 h. After addition of 0.1 M HCl, 1-palmitoyl-2-(9-benzyloxy) azelaoyl-PC was extracted by the method of Bligh and Dyer [21]. 1-Palmitoyl-2-azelaoyl-PC was obtained by reductive debenzylation using Pd(OH), as a catalyst [22]. I-Palmitoyl-2-azelaoyl-PC generated was further purified by silica gel column chromatography on Bio-sil A. A pure product (100 mg, 0.15 mmol) was obtained. TLC showed a single spot, R, 0.3 with a chloroform/methanol/ water (65 : 35 : 8) solvent system, and an R, 0.27 with a chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2) solvent system. The ‘H-NMR spectrum was as follows: 6 = 0.88, triplet, 3H, methyl; S = 1.2-1.4, multiplet, 30H, methylenes; S = 1.6, multiple& 6H, acyl y-methylenes; 6-2.3, multiplet, 6H, acyl fl-methylenes; S = 3.3. singlet, 9H, Nmethyls; S = 3.8, multiplet, 2H, choline N-methyl-

194

ene; a-3.95, multiplet, 2H, glycerol sn-3-methylene; 6 = 4.1, multiplet, 2H, glycerol sn-1 methylene; S = 4.35, multiplet, 2H, choline O-methylene; S = 5.2, singlet, lH, glycerol sn-2 proton. Preparation of radiolabeled substrates. Dipalmitoyl-[ N-Me-3H]PC was synthesized by the method of Stoffel [23]. l-Palmitoyl-2-lyso[ N-Me-3H]PC was obtained by treatment of dipalmitoyl-[ N-Me3H]PC with snake venom phospholipase A,. lPalmitoyl-2-linoleoyl-[ N-Me- 3H]PC was synthesized by the method of Gupta et al. [20]. lPalmitoyl-2-azelaoyl-[ N-Me-3H]PC and 1-[1-14C] palmitoyl-2-azelaoyl-PC were synthesized as described above. All synthetic PCs were purified by silica gel column chromatography. [14C]PE was prepared from cells of E. coli SN17 [23] grown in the presence of [l-‘4C]acetate and purified by a column chromatography on DEAE-cellulose [24]. Preparation of oxidized PC. Oxidized phospholipids were prepared by incubation of lpalmitoyl-2-linoleoyl-[ N-Me-3H]PC with freshly prepared human oxyhemoglobin at 37’C for 2 h. Oxidized 1-palmitoyl-2-linoleoyl-[ N-Me- 3H]PC was extracted by the method of Bligh and Dyer [21]. Determination of hydrolysis of oxidized [NMe-3H]PC was performed by fractionation of the mixtures with a straight-phase HPLC on a Zorbax-SIL column (4.6 x 250 mm, Shimadzu Co., Kyoto, Japan) eluted with n-hexane/ isopropanal/water (46 : 46 : 8) at 0.7 ml/mm. In some experiments, products fractionated as described above were used as substrates for the phospholipase. Preparation of rat peritoneal fluid. SpragueDawley rats (male, 250-300 g, obtained from Nippon Bio-Supp. Center, Tokyo, Japan), which had received an intraperitoneal injection of 15 ml of saline-containing 5% sodium caseinate under light anesthesia, were killed by bleeding at the indicated times. The peritoneal cavity was washed with 15 ml of saline. Cells were removed by centrifugation at 160 X g for 7 min and cell-free peritoneal fluid was obtained. Assay of phospholipase AI activities. When lpalmitoyl-2-azelaoyl-PC or 1-palmitoyl-2-linoleoyl-PC was used as a substrate, the standard incubation system (100 ~1) containing 50 mM Tris-HCl (pH 7.0) 1 mM CaCl,, 200 PM of radiolabeled PCs ([3H]: 100000 dpm; [14C]: 20000

dpm) and the peritoneal fluid was incubated at 37°C for 15 min. The reaction was stopped by adding 0.6 ml of chloroform/methanol (1 : 2). Lipids were extracted immediately by the method of Bligh and Dyer [21] and separated on thin-layer chromatography (Merck) using a chloroform/ methanol/ acetic acid/ water (25 : 15 : 4 : 2) solvent system. The plastic plates of the thin-layer chromatography were cut and the radioactivity of each spot was measured by a liquid scintillation counter (Packard 3255, Downers Grove, IL, U.S.A.). When E. coli [14C]CPE was used as a substrate, the reaction mixture (250 ~1) containing 50 mM Tris-HCl (pH 9.0) 4 mM CaCl,, 200 PM radiolabeled PE and peritoneal fluid was incubated at 37°C for 15 min [4,5]. The reaction was stopped by adding 1.25 ml of Dole’s reagent and the radiolabeled free fatty acid was extracted by the method previously described [26,27]. Derivation of peroxidized PC products by 9anthryldiazomethane (ADAM). PCs which have carboxyl groups were labeled with ADAM (Funakoshi, Tokyo, Japan), a carboxyl groupspecific fluorescent reagent [28]. Phospholipids were dried under nitrogen and dissolved in 50 ~1 methanol. ADAM was dissolved in one drop of acetone followed by an addition of methanol (1 mg/ml). 200 ~1 of the ADAM solution was added to the sample. The reaction mixture was kept at room temperature in the dark for at least 4 h. Before analysis by HPLC, the sample was passed through a silica gel column (0.1 ml) to remove the excess fluorescent dye. Conditions for HPLC were the same as described above. The ADAM derivatives were detected with the fluorescent detector (excitation at 365 nm and emission at 412 nm). Results Detection of the ADAM derivative of I-palmitoyl2-azelaoyl-PC in oxyhemoglobin-treated 1 -palmitoyl-2-linoleoyl-PC When l-palmitoyl-2-linoleoyl-PC was incubated with human oxyhemoglobin and then treated with ADAM, a sharp peak monitored by fluorescent detector (excitation at 265 nm and emission at 412 nm) was detected after a retention time of 16.5 min (Fig. 2B). The peak might be due to an ADAM derivative of 1-palmitoyl-2-azelaoyl-

195

A

B

Peaks appearing around 5 min in every sample might be ADAM contaminated in the final preparations. It is noteworthy that synthetic 1-palmitoyl-2azelaoyl-PC eluted at 26 min with a tailing peak when the sample was loaded on a straight-phase HPLC (Fig. 2D). Tailing of the peak may be due to the interaction of the carboxyl group of the compound with silica gel. The present findings are consistent with our previous observations that lO-octadecyl-2-azelaoyl-PC, a related compound, was formed from 1-O-octadecyl-2-linoleoyl-PC by treatment with oxyhemoglobin [19]. Hydrolysis of I -palmitoyl-2-azelaoyl-PC by the peritoneal fluid of rats treated with casein Accumulation of leukocytes in the peritoneal cavity of rats was observed 4-24 h after treatment with casein. An increase of acid phosphatase, a lysosomal enzyme, in the peritoneal fluid was also observed (data not shown). The total phospholipase activity in the cell-free peritoneal fluid of casein-treated rats, which was measured using lpalmitoyl-2-azelaoyl-PC as a substrate, was much higher than that of non-treated rats (Fig. 3). The highest phospholipase activity (about 25-times the control level) was observed in the peritoneal fluid

Retention

time

(min)

Fig. 2. Detection of a fluorescent derivative of 1-palmitoyl-2azelaoyl-PC in the reaction mixture of oxyhemoglobin-treated 1-palmitoyl-2-linoleoyl-PC and ADAM. Solvent, isopropanol/n-hexane/water (46 : 46 : 8); flow rate, 0.7 ml/min; column, Zorbax-SIL (4.6 X250 mm). (A) Elution profile of a synthetic I-palmitoyl-2-azelaoyl-PC (200 pmol) treated with ADAM, which was monitored by a fluorescent detector (excitation at 365 nm, emission at 412 nm). (B) Elution profile of ‘ADAM-reactive’ product(s) of oxyhemoglobin-induced peroxidation of l-palmitoyl-2-linoleoyl-PC. After l-palmitoyl-2linoleoyl-PC had been incubated with oxyhemoglobin at 37 ’ C for 2 h. the oxidized mixture (10 nmol) was treated with ADAM as described in Materials and Methods. (C) Elution profiles of 1,2-dipalmitoyl-PC (10 nmol) treated with ADAM. (D) Elution profile of a synthetic I-palmitoyl-2-azelaoyl-PC (100 nmol) monitored by an ultraviolet detector at 206 nm.

PC, since exactly the same peak was detected when a synthetic 1-palmitoyl-2-azelaoyl-PC was treated with ADAM (Fig. 2A). No appreciable fluorescent peak was detected in the reaction mixture of dipalmitoyl-PC and ADAM (Fig. 2C).

046

16 Time

24 after casein

46

injection

72 th)

Fig. 3. Phospholipase activity of cell-free peritoneal fluid of rats treated with casein. Rats (250-300 g) were injected intraperitoneally with 15 ml of saline containing 5% casein. After indicated periods, rats were killed and the peritoneal cavity was washed with 15 ml of saline. Phospholipase A, activity was determined using I-palmitoyl-2-azelaoyl-PC as a substrate. Specific activity (nmol/min per mg. 0): total activity (X 100 nmol/min. 0); number of peritoneal exudate cells (X10* cells), shaded bar.

196

of rats 8 h after injection, followed by a gradual decrease. The change of specific enzyme activity was different from the change of the total activity. The difference may be due to the increase of cell-free proteins exuded to the sites. When l-palmitoyl-2-azelaoyl-[N-Me-3H]PC was incubated with the peritoneal fluid, a decrease of the starting material and a concomitant increase of radioactive 1ysoPC was observed (data not shown). It is noteworthy that l-palmitoyl-2linoleoyl-PC was not appreciably hydrolyzed under the same conditions. Specific activity of the enzyme in the peritoneal fluid, which was obtained using 1-palmitoyl-2-azelaoyl-PC as a substrate, was 15fold higher than that obtained with I-palmitoyl-2-linoleoyl-PC (Table I). Generation of radioactive 1ysoPC was again observed when l-[l-‘4C]palmitoyl-2-azelaoyl-PC was incubated with the peritoneal fluid. However, no appreciable formation of radiolabeled neutral lipids was observed, indicating that the 2-azelaoylPC was hydrolyzed by the ‘phospholipase A,‘-type enzyme (data not shown). Hydrolysis of 1 -palmitoyl-2-linoleoyl-PC pretreated with oxyhemoglobin When l-palmitoyl-2-linoleoyl-[N-Me-3H]PC was treated with oxyhemoglobin at 37°C for 2 h, it was partially oxidized (Fig. 4A). The reaction mixture was separated into three fractions. Fraction I (retention time, 9-12 min) is an intact 1-palmitoyl-2-linoleoyl-PC, judging from its retention time. Products in fraction II (12-20 min) are supposed to contain conjugated dienes because of significant absorbance at 233 nm. Pure lpalmitoyl-2-azelaoyl-PC was eluted at 26 min (Fig. 2D) and the cytotoxicity such as that seen in the hemolytic activity was mostly recovered from fraction III (20-30 min) (data not shown). Fraction III may contain cytotoxic phospholipids and lpalmitoyl-2-azelaoyl-PC seems to be one of those products, since the amount of the 2-azelaoyl-PC formed during peroxidation of the 2-linoleoyl-PC was estimated at l-2% of them (Fig. 2). When the oxidized mixture was incubated with the peritoneal fluid and then separated by HPLC, a new radioactive peak appeared at 38 min (Fig. 4B). The peak is due to 1-palmitoyl-2-lysoPC, since exactly the same peak was observed with pure

(a) A

+

(b)

$

I

0

Fig. 4. Hydrolysis of the oxidized l-palmitoyl-2-linoleoyl-PC by the cell-free peritoneal fluid of rats treated with casein. The conditions for HPLC were as described in the Fig. 2 legend. The absorbances at 206 () and 233 nm (- - - - - -) were monitored. Eluates were collected and the radioactivity of each fraction was measured (0). (A) Elution profile of the oxidized 1-palmitoyl-2-linoleoyl-PC. l-[ 3H]Palmitoyl-2-linoleoyl-PC was incubated with oxyhemoglobin at 37’ C for 2 h. The incubation mixture was further fractionated into three fractions, fractions I, II and III. These fractions were used for the next experiment as substrates (see Table I). (B) Elution profile of oxidized l-[ 3H]palmitoyl-2-linoIeoyl-PC (20 nmol) treated with the peritoneal fluid (10 ~1) for 2 h. Arrows indicate the retention times of pure l-palmitoyl-2-linoleoyl-PC (a) and lpalmitoyl-2-IysoPC (b).

l-palmitoyl-2-lyso[N-Me-3H]PC. Most of 1ysoPC may be derived from ‘oxidized’ PC, since the radioactivity recovered from fractions II and III decreased 13 and 38%, respectively, whereas that from fraction I did not change appreciably. Products generated upon oxidation of lpalmitoyl-2-linoleoyl-PC were separated next and their sensitivity to the peritoneal enzymes was examined. 35, 50 and 15% of the total radioactivity in the whole reaction mixture was recovered from fractions I, II and III, respectively, which are designated as shown in Fig. 4A. When these fractions were incubated with the peritoneal fluid, fraction III was most efficiently hydrolyzed, fol-

197 TABLE

I

HYDROLYSIS OF OXIDIZED I-PALMITOYL-2-LINOLEOYL-PC BY THE PERITONEAL FLUID OF RATS TREATED WITH CASEIN Radiolabeled substrates, fractions I, II and III. were obtained from the reaction mixture of 1-palmitoyl-2-linoleoyl-PC and oxyhemoglobin as described in Fig. 4 legend. Fraction I is an intact I-palmitoyl-2-Iinoleoyl-PC. Fraction II contains hydroperoxides and alcohols with a conjugated diene because of appreciable absorbance at 233 nm. Fraction III, showing cytotoxic activity, contains 1-palmitoyl-2-azelaoyl-PC. These lipids and two other pure phospholipids (20 nmol as phosphorus) were incubated with the peritoneal fluid of rats treated with casein (40 pg protein), as described in Materials and Methods. Values are expressed as means f S.D. (n > 3). Substrates

Specific activity (nmol/min per mg)

I II III

0.37 f 0.23 1.61 f 0.54 4.26 f 0.64

I-Pahnitoyl-2-linoleoyl-PC l-Palmitoyl-2-azelaoyl-PC

0.26 f 0.08 3.95 f 0.26

lowed by fraction II. The sensitivity observed with fraction III was close to that observed with a pure phospholipid, l-palmitoyl-2-azelaoyl-PC (Table I). Factors affecting the phospholipase activity. The effect of pH on hydrolysis of I-palmitoyl-2azelaoyl-PC by the peritoneal fluid was examined (Fig. 5A). The activity was detected in the acidic to neutral pH range and was optimum at pH 7. The activity was affected by the presence of Ca2+ ions (Fig. 5B). Addition of EDTA to the reaction mixture partially suppressed the activity. Maximum activity was obtained in the presence of 1 mM CaCl, and the activity was suppressed by higher Ca2 + concentration.

ber of phospholipases A, [29], resulted in the loss of the present activity. The activity was also inhibited by dithiothreitol, which reduces the disulfide bonds and which destabilizes the conformation. No inhibition was observed by adding iodoacetoamide, p-methylsulfonyl fluoride, or pchloromercuribenzenesulfonic acid.

A

1

J

4

6

7

a

9

10

PH

EDTA

5

0

10

Concentration

20

C&I,

CmM)

C

u__L 0

Inhibition of enzyme activity More than 50% of the activity remained during incubation at 55 “C for 10 min. The activity was almost completely lost after boiling for 10 min (Fig. 5C). The effects of various reagents on the activity of the peritoneal fluid were examined next (Table II). Incubation of the peritoneal fluid with 5 mM p-bromophenacyl bromide, which is known to be an active site-directed histidine reagent for a num-

5

25

50

Temperature

100

Co0

Fig. 5. Effects of pH (A). concentration of Ca2+ (B) and heat treatment (C) on phospholipase A, activity of the peritoneal fluid of rats treated with casein. I-Palmitoyl-2-azelaoyl[ N-Me‘H]PC was incubated with the peritoneal fluid at 37O C for 15 min. The buffers used were 50 mM sodium acetate in the pH 3.5-6 range, 50 mM Tris-malate, pH 6-7. 50 mM Tris-HCI. pH 7-9, and 50 mM glycine-NaOH, pH 9-10. (B) Different concentrations of Ca2+ or EDTA were added to the standard reaction mixture. (C) Cell-free peritoneal fluid was preincubated for 10 min at various temperatures.

198 TABLE

II

TABLE

IV

EFFECTS OF VARIOUS REAGENTS ON THE PHOSACTIVITY OF THE PERITONEAL PHOLIPASE A, FLUID OF RATS TREATED WITH CASEIN

HYDROLYSIS OF I-PALMITOYL-2-AZELAOYL-PC I-PALMITOYL-2-LINOLEOYL-PC BY SNAKE OR PANCREATIC PHOSPHOLIPASE A,

The peritoneal fluid (1 mg/ml) was preincubated with or without various reagent (5 mM) at 37OC. After a 30 min and a 120 min preincubation, an aliquot (20 ~1) was taken for measurement of phospholipase A, activity using I-palmitoyl2-azelaoyl-[ N-Me-3H]PC as a substrate.

The reaction mixture containing phospholipase A, (0.8 nmol PC/min), radiolabeled substrates (200 pl), 50 mM Tris-HCI (pH 7.4) and 10 mM CaCl a was incubated at 37 o C for 15 min. Percentages of 1ysoPC formed are indicated. Enzyme source

Reagent

added

None p-Bromophenacyl bromide Dithiothreitol Iodoacetoamide Phenylmethylsulfonyl fluoride p-Chloromercuribenzensulfinic

acid

Remaining

activity (%)

30 mm

120 min

79 9 58 89 85 85

79 9 18 90 92 72

Lack of ‘oxidized phospholipid-specific’ phospholipase activity in the purified alkaline phospholipase A2 We had previously purified a 13.5K phospholipase A, from the peritoneal fluid of rats treated with casein [5]. This enzyme showed optimum activity at pH 8.5-9.0 when E. coli [14C]CPE was used as a substrate. The question arose as to whether or not this purified enzyme showed ‘oxidized phospholipid-specific’ hydrolytic activity. Specific activities of the purified enzyme were determined using 1-palmitoyl-2-azelaoyl-[ N-Me-

TABLE

III

HYDROLYSIS OF OXIDIZED PHOSPHOLIPIDS BY THE PURIFIED FROM THE PERIPHOSPHOLIPASE A, TONEAL FLUID OF RATS TREATED WITH CASEIN The hydrolysis of I-palmitoyl-2-azelaoyl-[ N-J%I~-~H]PC, lpalmitoyl-2-linoleoylj N-Me-3H]PC, or E. co/i [14C]PE by the phospholipase A, previously purified from peritoneal fluid of rats treated with casein [5] was determined as described in Materials and Methods under two different experimental conditions. Substrates

Specific activity ( X lo4 nmol/min

I-Palmitoyl-2-azelaoyl-PC I-Palmitoyl-2-linoleoyl-PC E. CO/I PE

per mg)

pH 9.0, 4 mM CaCl ,

pH 7.0, 1 mM CaCl,

0.2 0.8 9.0

0.7 0.9 3.0

Porcine pancreas N. naja C. adamanteur

AND VENOM

Substrate l-palmitoyl2-azelaoyl-PC

l-palmitoyl2-linoleoyl-PC

0.5 0.0 2.5

4.0 4.5 4.2

3H]PC, 1-palmitoyl-2-linoleoyl-[ N-Me- 3H]PC and E. coli [14C]PE as substrates under two different pHs (9.0 and 7.0). The purified enzyme hydrolyzed 1-palmitoyl-2-linoleoyl-PC more efficiently than l-palmitoyl-2-azelaoyl-PC under both conditions. Hydrolysis of 1 -palmitoyl-2-azelaoyl-PC by the other phospholipases A2 The same units of phospholipase A, obtained from porcine pancreas or snake venom (N. naja, C. adamanteus) were incubated with l-palmitoyl2-azelaoyl-PC or l-palmitoyl-2-linoleoyl-PC (Table IV). Phospholipases A, from either porcine pancreas or N. naja hardly hydrolyzed lpalmitoyl-2-azelaoyl-PC. The enzyme of C. adamanteus hydrolyzed 2-azelaoyl-PC, although the sensitivity was again poorer than that observed with 2-linoleoyl-PC. Discussion In the cell-free peritoneal fluid of rats treated with casein, we found a novel enzyme activity that preferentially hydrolyzes 1-palmitoyl-2-azelaoylPC, one of the possible products formed during peroxidation of phospholipids. The hydrolysis of 1-palmitoyl-2-azelaoyl-PC was due to the phospholipase A z activity, since radiolabeled 1ysoPC was exclusively formed when the PC with radiolabeled palmititic acid at the C-l position was incubated with the peritoneal fluid. This phospholipase A, may hydrolyze not only 2-azelaoyl-PC but also various oxidized PC. Frac-

199

tion II separated from the reaction mixture of I-palmitoyl-2-linoleoyl-PC and oxyhemoglobin, which probably contains phospholipids with hydroperoxide, was also susceptible to the present although the sensitivity of phospholipase A,, Fraction II was lower than that observed with fraction III, in which 2-azelaoyl-PC may be involved. Although 2-azelaoyl-PC is a rather minor product of peroxidation, it may be one of the useful analogs to detect the phospholipase A, activity which hydrolyzes oxidized phospholipids. It is noteworthy here that 2-azelaoyl-PC is one of the most cytotoxic products generated during oxyhemoglobin-induced peroxidation of 2-linoleoyl-PC [19]. The present phospholipase A, activity detected in the inflamed sites may play an important role either in detoxification of cytotoxic phospholipids generated locally or in repair in damaged membranes. are observed in some Phospholipases A, inflamed peritoneal fluid [2-51. The enzyme previously purified showed a pH optimum of 8.5-9 and required Ca ions (completely inhibited in the presence of EDTA) [5]. The optimum pH and the Ca2+ requirement of the purified enzyme were quite different from those obtained in the peritoneal fluid with l-palmitoyl-2-azelaoyl-PC as a substrate. The alkaline phospholipase A, purified from the peritoneal cavity showed no preference for 2-azelaoyl-PC. The extracellular phospholipase A 2, which hydrolyzes oxidized PCs preferentially, may be different from the alkaline phospholipase A, described above. This extracellular phospholipase A, may have originated from lysosomes of some inflammatory cells, since properties such as the pH optimum and the Ca2+ requirement are rather similar to those previously observed with lysosomal phospholipases [30]. Another possibility which cannot be excluded is that the substrate specificity of the phospholipase A, is affected by a constituent in the crude peritoneal fluid. The enzyme which hydrolyzes the 2-azelaoyl-PC may not recognize the structure of the 2-azelaoylPC molecule, since a variety of products formed during peroxidation of PC, fraction II as well as fraction III, were hydrolyzed. Phospholipases A, are known to recognize lipid/ water interfaces [28]. Recently, however, it was hypothesized that relatively hydrophilic phospholipids are susceptible to

phospholipases A, because hydrophilic acyl chains are in contact with aqueous phase [31]. The enzyme in the peritoneal fluid may recognize hydrophilic phospholipids which are loosely bound to the membranes. It has been reported that lipid peroxidation in lysosomes or mitochondria induced formation of lysophospholipids [32,33]. Accumulation of lysophospholipids was observed also in hepatocytes treated with carbon tetrachloride [34]. These findings suggest the possible relationship between activity of cellular phospholipases and lipid peroxidation. Sevanian et al. [35] reported that hydroperoxides of bovine liver PC were hydrolyzed by from snake venom (C. phospholipase A, adumanteus). The release of polyunsaturated fatty acids by the same enzyme increased when liposomes composed of bovine liver PC and PE were peroxidized by Fe2+ plus ascorbate [36]. Porcine pancreatic phospholipase A, could also hydrolyze hydroperoxides of 1-palmitoyl-2-linoleoyl-PC [37,38]. It was proposed that phospholipase A, might play a protective role against lipid peroxidation by coupling with glutatione peroxidase. Snake venom or pancreatic phospholipase A, have, however, different properties from phospholipase A, detected in inflamed sites [29.30]. In fact, we demonstrate here that, unlike the enzyme in the peritoneal fluid, neither snake venom (N. nuju) nor porcine pancreatic phospholipase A, could hydrolyze 1-palmitoyl-2-azelaoyl-PC significantly. Recently, Franson and his colleagues [39] reported that the cardiac lysosome fraction hydrolyzed auto-oxidized PE preferentially. Production of radiolabeled monoacylglycerol and diacylglycerol was observed with a loss of auto-oxidized l-acyl-2-[i4C]linoleoyl-PE, indicating that autooxidized PE was sequentially hydrolyzed by phospholipase C and diglyceride lipase. In the present study, we demonstrated that a phospholipase A, activity which hydrolyzes oxidized phospholipids preferentially was induced in inflamed sites. The findings may suggest that degradation of peroxidized lipids may be of crucial importance during the inflammation process. It is of interest to elucidate the physiological or pathophysiological roles of phospholipase A, present in inflamed sites.

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Acknowledgements We are grateful to Dr. Tetsuo Nagano of University of Tokyo for his crucial advice in the synthesis of 1-palmitoyl-2-azelaoyl-PC. This work was supported in part by Grant-in-Aid for Scientific Research (Nos. 62624504 and 62870093) from the Ministry of Education, Science and Culture of Japan). References 1 Vadas, P. and Pruzanski, W. (1986) Lab. Invest. 55, 391-404. 2 Franson, R., Dobrow, R., Weiss, J., Elsbach, P. and WegIicki, W.B. (1978) J. Lipid Res. 19, 18-23. 3 Frost, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, I. and Heimikson, R.L. (1986) Biochemistry 25, 8381-8385. 4 Chang, H.W., Kudo, I., Hara, S., Karasawa, K. and Inoue, K. (1986) J. B&hem. 100, 1099-1101. 5 Chang, H.W., Kudo, I., Tomita, M. and Inoue, K. (1987) J. B&hem. 102, 147-154. 6 Weiss, J., Elsbach, P., Olsson, 1. and Odeberg, H. (1978) J. Biol. Chem. 253, 2664-2672. 7 Stefanski, E., Pruzanski, W., Stemby, B. and Vadas, P. (1986) J. B&hem. 100, 1297-1303. 8 Punzi, L., Todesco, S., Toffano, G., Catana, R., Bigon, E. and Bnmi, A. (1986) Rheumatol. Int. 6, 7-11. T., Yokoe, N., Takemura, S., Kato, M., 9 Yoshikawa, Hosokawa, K. and Kondo, M. (1979) Jpn. J. Med. 18, 199-204. T., Tamura, M. and Kondo, M. (1985) Bio10 Yoshikawa, them. Med. 33, 320-326. 11 Kogure, K., Watson, B.D., Busto, R. and Abe, K. (1982) Neurochem. Res. 7, 437-454. A.M. 12 Recknagel, R.O., Glende, Jr., E.A. and Hruszkewycz, (1977) in Free Radicals in Biology (Pryor, W.A., ed.), vol. 3, pp. 97-132, Academic Press, New York. W.A., Gilbert, J.D. and Procks, C.J.W. (1973) 13 Harland, B&him. Biophys. Acta 316, 378-385. 14 Chiu, D., Lubin, B. and Shohet, S.B. (1982) in Free Radicals in Biology (Pryor, W.A., ed.), vol. 5, pp. 115-160, Academic Press, New York. A., Comporti, M. and Esterbauer, H. (1980) 15 Benedetti, B&him. Biophys. Acta 620, 281-296.

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