34

Biochimrcu et Bwphysica

Actu. 1043 (1990) 34-42 Elsevier

BB.ALIP 53333

Immunochemical Makoto Department

detection of ‘ platelet type’ phospholipase A 2 in the rat

Murakami,

Ichiro I&do,

Yasuhiro

Natori



and Keizo Inoue

of Wealth Chemrstry, Faculty of Pharmaceutical Sciences, The Unroersit,v oJ Tokyo and .~uttonui Medical Centre, Tokyo (Japan) (Received

Key words:

Phospholipase

7 August

AZ; Antibody;

’ Clinical Reseurch Institute,

1989)

ELISA;

Sandwich

ELISA: (Rat platelet)

Polyclonal antibodies were raised against rat platelet phospholipase A *. One of them, designated as R377 was prepared by immunizing a rabbit with the intact enzyme. The other antibody, designated as R385, was prepared by immunizing with the enzyme treated with 2-mercaptoethanol. The antibody R377 bound to rat platelet phospholipase A, almost exclusively, while the antibody R385 reacted not only with rat platelet phospholipase A, but with the enzymes obtained from snake venom or mammalian pancreas. The antibody R377 bound to the non-reduced rat platelet phospholipase A, bearing intact intramolecular disulfide bonds, but not to the reduced enzyme. In contrast, the antibody R385 reacted with both non-reduced and reduced enzymes. R377 may recognize the conformational structure of the enzyme. Both antibodies inhibited the enzyme activity. The antibody R377, but not R385, interfered with the interaction of the enzyme with heparin, which is one of the characteristic properties of rat platelet phospholipase A,. The antibody R377 reacted with phospholipases A, of bone-marrow cells and of peritoneal exudated cells prepared from caseinate-treated rats, indicating that some myeloid cells other than platelets also contain ‘platelet type’ phospholipase A,. An immunochemical method for measurement of rat ‘platelet type’ phosphoiipase AZ was developed. Tbe sensitivity of this method was 10 ng/ ml of ph~pholip~e A 2 in the preparation. One of the advantages of the present immun~hemica1 method is that the measurement was not affected by the presence of an endogenous inhibitor(s) of enzymatic activity.

Correspondence: K. Inoue, Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.

due. Much is known about the structures, functions and molecular mechanisms of the enzymatic action of mammalian pancreas or snake venom phospholipases A, [8]. In contrast, phospholipases A, of mammalian non-pancreatic sources have been characterized to a lesser extent than these exocrine enzymes. We have purified [9] and determined the primary structure [lo] of the phospholipase A 2 from rat platelets, which contain relatively high amounts of the enzyme [ll]. The analysis of the whole amino acid sequence of the enzyme revealed that it belongs to the ‘Group II’ family. Mammalian ‘nonpancreatic, Group II’ phospholipases A, have recently been detected in spleen [12], liver [13], intestine [14] or fluid exudated into inflamed sites of various species [15-191. In understanding the physiological functions, distribution, dynamics and structural characteristics, application of antibodies to these phospholipases A, may provide useful information. In our previous paper 1201, we have reported the establishment and characterization of monoclonal antibodies against rat platelet phospholipase A,. In the present paper, the distribution and the quantitation of rat ‘platelet type’ phospholipase A 2 was

~5-2760/9~/~03.50

Diviston)

Phospholipase A, plays important roles in several biological phenomena such as membrane phospholipid turnover, signal transduction through liberation of arachidonate from membrane phospholipids [I] and the process of inflammation [2-41. Phospholipases A, have been classified into two groups in terms of their primary structures [5-71. The ‘Group I’ family include the enzymes from mammalian pancreatic juices or snake venoms of Elapidae and Hydrophidue, and the ‘Group II’ family include the enzymes from snake venoms of Crotalidae and Viperi-

Abbreviations: 2-ME, 2-mercaptoethanol; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TBS, 10 mM Tris-HCI-buffered saline; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

“z 1990 Elsevier Science Publishers

B.V. (Biomedical

35 studied platelet Materials

using polyclonal rabbit antibodies phospholipase A, as a probe.

against

rat

and Methods

Animals Wistar rats (male, 400-500 g) and Japanese albino rabbits (male, 2-3 kg) were purchased from Nippon Bio-Supply Center (Tokyo). Materials Sepharose CL-4B conjugated with antibody MD7.1 was prepared as described previously [20]. 1-Acyl-2-[‘4C]linoleoyl-sn-3-glycerophospho-L-serine was prepared and used as a substrate in the phospholipase A, assay procedure as previously described [9]. Freund’s complete and incomplete adjuvants were purchased from Difco Laboratories (Detroit, MI). Porcine pancreas phospholipase A, was obtained from Boehringer-Mannheim (F.R.G.). Naja naja and Crotalus adamanteus phospholipases A, were from Sigma (St. Louis, MO). Rabbit platelet phospholipase A, was purified as described [21]. Poly(viny1 chloride) microtiter plates (Immulon 2), Millipore GVHP filters, ortho-phenylenediamine and HRP color-developing reagent were obtained as described [20]. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was obtained from Cappel Laboratories (Downingtown, PA). Diaminobenzidine and 2-mercaptoethanol (2-ME) were from Wako (Osaka). Biotinylated goat anti-rabbit immunoglobulins (Igs) and HRP-streptavidin were from Seikagaku Kogyo (Tokyo). HRP-avidin was purchased from Vector Laboratories (Burlingame, CA). IODOGEN was from Pierce Chemical (Rockford, IL). Nalz51 was obtained from Amersham International (U.K.). Purification of rat platelet phospholipase A, Phospholipase A, was purified from KC1 extracts of the whole lysate of rat platelets by immuno-affinity antibody MD7.1-conjugated chromatography, using Sepharose as described [21]. In brief, washed rat platelets were sonicated three times for 30 s each. The lysate was incubated for 12 h at 4°C in 10 mM Tris-HCl (pH 7.4) containing 1 M KCl, centrifuged at 100000 X g for 1 h, then the supernatant was applied onto an anti-rat platelet phospholipase AZ monoclonal antibody MD7.1-conjugated Sepharose affinity column. To avoid nonspecific adsorption, a precolumn of Sepharose CL4B (Pharmacia, Uppsala, Sweden) was attached in front of the affinity column. The column was washed extensively with 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl (TBS) and the bound enzyme was subsequently eluted with 50 mM glycine-HCl buffer (pH 2.2). The eluate at low pH gave a single protein band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of which was estimated to be 14

kDa, corresponding to phospholipase A *. The recovery of the enzyme activity was approx. 90%. 1.2 mg of phospholipase A, was obtained from platelets of 60 rats in a single preparation. Preparation of antiserum against rat platelet phospholipase A2 Immunoaffinity-purified phospholipase A 2 was used as an immunogen for raising antibodies in rabbits. Intact or denatured (treated with 2-ME for 10 min at 100 o C) phospholipase A z was injected subcutaneously into rabbits according to the following schedule; 250 pg of phospholipase A, in Freund’s complete adjuvant on day 0, 150 pg in Freund’s incomplete adjuvant every 2 to 3 weeks for 2 months. Animals were bled 1 week after each booster injection. Each serum was applied onto the protein A-Sepharose column (Pharmacia) to obtain IgG fractions [22]. The purified polyclonal antibodies gave homogeneous protein bands whose molecular weights were approx. 50 000 (heavy-chain) and 25 000 (light-chain) on SDS-PAGE. They were dialyzed against TBS and stored at - 20 o C before use. Assay of phospholipase A2 Phospholipase A 2 activity measured using 14C-labelled substrate as described [9].

of the test samples was phosphatidylserine as a

Iodination of phospholipase A2 Rat platelet phospholipase A, was iodinated with IODO-GEN [23]. Purified enzyme (100 pg in 100 ~1 of TBS) was added to an IODO-GEN-coated glass tube (10 pg/tube). Na “‘1 (0 .5 mCi) was then added and the mixture incubated for 15 min at room temperature. At the end of the incubation, the reaction mixture was loaded on antibody MD7.1-Sepharose affinity column to remove free 12’I. The specific activity of ‘251phospholipase A, was 0.5 pCi/pg. The iodinated enzyme showed the same affinity for heparin as an intact enzyme. Preparation of tissue and cell homogenates of rat Tissue and cell homogenates were prepared as described [18,24]. Peritoneal exudated fluid and cells were obtained from rat peritoneal cavity 8 h after injection of caseinate as described [25]. Immunoblotting Immunoblot analysis was performed as described [20]. Briefly, test samples were resolved by SDS-PAGE, transferred to a Millipore GVHP filter, then treated sequentially with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for blocking, antiserum (1 : 500 dilution), and HRP-conjugated goat anti-rabbit IgG (1 : 1000 dilution). Color development was performed with a HRP-color developing reagent as a substrate.

36 ~mmunoh~stochemica~ staining of rat hone marrow ceils Cells plated on slide glasses were washed twice with PBS, air-dried, and fixed for 30 min at room temperature in 3.7% formaldehyde in PBS. Then, the samples were treated sequentially as follows: (A) 0.3% H,O, in methanol for 40 min (to inactivate endogenous peroxidase); (B) 3% BSA in PBS for 30 min; (C) antiserum (1 : 500 dilution) for 1 h; (D) biotinylated goat anti-rabbit Igs (1 : 1000 dilution) for 40 min; (E) HRP-streptavidin (1 : 1000 dilution) for 20 min; (F) finally stained with diaminobenzidine. Results Characterization of antibodies Antisera raised in rabbits against rat platelet phospholipase A, were tested for specificity by either ELISA or immunoblotting. Antibody R377 obtained from a rabbit immunized with the intact enzyme as well as antibody R385 obtained from a rabbit immunized with the 2-ME-denatured enzyme bound to rat platelet phospholipase A z (Fig. 1). When immunoblotting was carried out on the whole lysate of rat platelets, R377 reacted exclusively with a single protein band, whose molecular mass was estimated to be about 14 kDa (Fig. 2A, lanes 3 and 4). On the other hand, R385 reacted with several other proteins present in the platelet lysate, though the extent of the reactivity was weaker than that observed with the band corresponding to the phospholipase A, (Fig. 2B, lanes 1 and 3). Reduced phospholipase A, did not react with R377 (Fig. 2A, lanes 1 and 2). indicating that the conformational structure of the enzyme with intramolecular disulfide bonds may be necessary for the enzyme to be recognized by R377. On

the other hand, R385 bound to both reduced and nonreduced forms of the phospholipase A, (Fig. 2B). Fig. 3 shows the crossreactivity of the antibodies. R385 reacted with all phospholipases A, so far tested (porcine pancreas, Naja naja, Crotalus adamanteus and rabbit platelet), though the affinity for them was rather weak compared with that for rat platelet phospholipase A,. It should be emphasized here that R377 obtained from rabbit immunized with an intact enzyme reacted with none of these enzymes, except for the rat platelet enzyme. When R377 was added to the phospholipase A, assay mixture, the activity of rat platelet phospholipase A 2 was effectively inhibited in a dose-dependent manner (Fig. 4A). Similar inhibitory activity was observed with R385 (Fig. 4B). Rat pancreatic phospholipase AZ was inhibited when incubated with R385 but not with R377, being consistent with their binding properties. It should be emphasized that phospholipase A, activity detected in the exudated cells recovered from peritoneal cavity of caseinate-treated rats was inhibited by R377. indicating that peritoneal cells may contain the ‘platelet type’ enzyme. Fig. 5 depicts the effect of antibodies on the binding of rat platelet phospholipase A, to heparin-Sepharose (Pharmacia). R377 interfered with the interaction of phospholipase A, with heparin, whereas antibody R385 did not. Distribution of ‘platelet fype phosphoiipa~e A, in rat We next examined the distribution of ‘platelet type’ phospholipase A, in rat tissues or cells using R377 as a probe. When the tissue or cell homogenates were incubated with R377, phospholipase A, activities of

Dilution of Serum Fig. 1. Titration of antisera. Polyfvinyl chloride) microtiter plates were coated with 25 ng/well of rat platelet phospholipase A, purified by monoclonal antibody-Sepharose column as described in Materials and Methods. After blocking with 1% BSA in PBS, it was incubated with and developed with HRP-conjugated goat antiserum R377 (0). R385 (A), and pre-immune serum of R377 (0) and R385 (A), respectively, anti-rabbit IgG and ~ri~#-phenylenediamine.

37 TABLE

I

Inhibition

of phospholipase

A, activity in various sources

Homogenates containing approx. 4 units of phospholipase A, were mixed with 100 gg of antibody R377 and the mixtures were added to enzyme assay mixtures. The remaining activities were measured. Specific activity (rim01 fatty acid released per min per mg)

Enzyme source

Platelets Bone marrow cells Megakaryocytes Spleen Lung Liver Kidney Pancreas Inflamed sites peritoneal exudated peritoneal exudated

fluids cells

Inhibition by R377 (S)

17.1 0.12 6.30 0.46 0.32 0.067 0.093 99.0

97.6 72.6 91.2 54.8 35.4 0 0 0

1.55

95.2 79.9

0.086

not all of bone marrow cells other than megakaryocytes were also positively stained.

Fig. 2. Detection of phospholipase A, by immunoblotting. 50 pg of rat platelet lysate (lanes 1 and 3). or 1 pg of purified rat platelet phosphohpase A, (lanes 2 and 4) were electrophoresed in SDS-polyacrylamide gel in the presence (lanes 1 and 2), or absence (lanes 3 and 4) of 2-ME, transferred to the membrane and blotted with antiserum R377 (A) or R385 (B), respectively, as described in Materials and Methods. Lanes 5 and 6 in (B) represent the protein pattern in the presence of 2-ME visualized with Coomassie brilliant blue. Lane 5 represents purified rat platelet phospholipase A, (1 pg); lane 6, rat platelet

lysate (50 pg as protein).

Determination of phospholipase A_, content by sandwich ELISA There are some demerits for the detection or quantitation of immunocrossreactive phospholipases A 2 by inhibition assay of enzymatic activity, since several investigators have indicated the presence of modulator(s) of enzymatic activity in crude preparations [26-321. Polyclonal antibodies were evaluated for their ability to detect and quantitate the ‘platelet type’ phospholipase A, even in complex protein mixtures. A sandwich ELISA was established using the solid-phase attached polyclonal antibody R377 and the soluble biotinylated monoclonal antibody MD7.1 which we have previously prepared and characterized [20]. A typical standard

TABLE

II

Determination sources

of content of ‘platelet type’ phospholipase

in various

The contents were calculated as described in the text. Purified platelet phospholipase A, was used as a standard. Sample

spleen, lung, bone marrow cells and inflamed site were inhibited partially (Table I). To localize the immunocrossreactive phospholipase A 2 in bone-marrow cells, immunohistochemical analysis was performed (Fig. 6). Megakaryocytes (Fig. 6, arrows), the precursor cells of platelets, were strongly stained by treatment with R377. In addition, most but

A,

Concentration of phospholipase (ng/ml), determined by:

Supernatant of platelets stimulated with thrombin Serum Peritoneal fluid experiment experiment

1 2

ELISA (n=3)

phospholipase activity

8500+900 1070* 50

6 600 1400

302* 68*

25 12

131 36

rat

A2

A2

38

B

A 2.0.

2. O-

2.

1..O-

1.

1.5 I 1.0

0.5

O

L 40

160

640

f

.oI-

Dilution

of Serum

Fig. 3. Crossreactivity of the antisera against rat platelet phospholipase A, with phospholipases A, from various sources. 2 pg/ml of phospholipases A, of porcine pancreas (A, E); Naja nu&~ (B, F); Croralus adamanfeus (C, G) or 0.5 pg/ml of phospholipase A, of rabbit platelet (D, H) were coated on poly(vinyl chloride) microtiter plates, respectively. After blocking with 1% BSA in PBS, they were incubated with R377 (A-D) or R385 (E-H), respectively, and developed with HRP-conjugated anti-rabbit IgG and orrho-phenylenediamine. Closed symbols represent antisera

and open symbols

represent

pre-immune

serum, respectively.

1oc

^o a> .z > .+

2

50

m

4 ._ ul

t?

50 Antibody

().tg)

100

c

100

50 Antibody

(pg)

Fig. 4. Effect of the polyclonal antibodies on phospholipase A, activity. Purified R377 (A) or R385 (B) were mixed with purified rat platelet phospholipase A, (0). rat peritoneal exudated cell lysate (A) or rat pancreas homogenate (m), respectively. Then, they were immediately added to phospholipase A, assay mixtures. 4 units of phospholipases A, were added to each of the samples. The results are expressed as percentages of the activity of the enzyme incubated without antibody.

39

5 r

50

=

2000

/y;;,.

.

j:

results were compared with the content evaluated on the basis of the enzyme activity (Table II). Both extracellular medium of the stimulated platelets and the serum gave almost the same values in both assays. In the case of peritoneal exudated fluid, however, values obtained from the immunological method were 2- to 3-fold higher than that from the enzymatic assay. These results may indicate the presence of endogenous inhibitor(s) of phospholipase A, in peritoneal fluid. Discussion

G-

N

OO

50

100 Antibody

,,,g,

Fig. 5. Effect of polyclonal antibodies on the interaction between phosphohpase A, and heparin. lodinated phospholipase A, (0.2 pg) was incubated with 100 ~1 of heparin-Sepharose CL-6B in 250 ~1 of 0.2%BSA-TBS in the presence of an indicated amount of R377 (H) or R385 (0) for 2 h at room temperature. The incubation mixture was then centrifuged at 200x g and the radioactivity in the supernatants was measured (left scale). The results are expressed as percentages of the radioactivity of the enzyme incubated with 100 ~1 of Sepharose CL-6B (Pharmacia) in the absence of antibodies (right scale).

curve is shown in Fig. 7. The minimal amount of rat platelet phospholipase A, detectable in this method was approx. 10 ng/ml (35 fmol per well). This method was next applied to quantitate the content of the phospholipase A, in several sources and the

Several reports have so far been made of antibodies elicited against phospholipases A,. Meijer et al. [33] prepared the polyclonal antibody against pancreatic phospholipase A,, which showed no crossreactivity with snake venom phospholipase A,. Likewise, Wurl and Kunze [34] found no immunochemical relationship between seminal and pancreatic phospholipases A *. Okamoto et al. [35] found that the anti-rat pancreatic phospholipase A, could react with ‘pancreas type’ phospholipase A, present in rat spleen. Senegas-Balas et al. [36] raised anti-swine intestinal phospholipase A, and anti-pancreatic enzyme antibodies and found no crossreactivity between them. Monoclonal antibodies against rat liver mitochondrial phospholipase A, [37] and rat platelet phospholipase A, [20] failed to react

Fig. 6. Immunohistochemical staining of rat bone-marrow cells (X 500) with the polyclonal antibodies against rat platelet phospholipase A,. Cells were fixed and processed for immunoperoxidase staining as described in Materials and Methods. (A) shows control staining using pre-immune serum, whereas (B) shows staining using antiserum R377. Arrows indicate megakaryocytes.

40

Phospholipase A2 (nglml)

Fig. 7. Standard curve for determination of phospholipase A, content by sandwich ELISA. Microtiter plates were blocked with 1 pg/ml of purified R377 for 2 h. After washing, wells were coated with 1% BSA for 2 h. After washing, various concentrations of purified rat platelet phospholipase A, was added and plates were further incubated for 1 h. After washing, 1 pg/ml of biotinylated monoclonal antibody MD7.1 was added and plates were incubated for 1 h, followed by washing and incubating with HRP-avidin for 1 h. Orrho-phenylenediamine was added after washing and absorbance was measured at 492 nm.

with enzymes of pancreas and snake venom origin. The lack of immunochemical relationships observed in these experiments may indicate that the epitopes on the enzymes differed and that immunologically common regions might not be recognized by these antibodies. The characteristics of R377 reported in this paper are in line with their observations. It was quite specific for rat platelet phospholipase A, and no crossreactivity was observed with the enzymes of pancreas or snake venom. Treatment of rat platelet phospholipase A, with 2-ME resulted in loss of its reactivity with R377, indicating that some conformational structures supported by intramolecular disulfide bonds may be involved in the epitopes of the enzyme, which could be recognized by R377. Another antibody, R385, which was elicited in a rabbit by injection of phospholipase A, denatured by treatment with 2-ME, showed quite different reactivity from that of R377. It reacted with both non-reduced and reduced forms of the phospholipase A, present in rat platelet lysates. In both preparations, a major band stained with R385 bore a molecular mass of about 14 kDa and might correspond to the phospholipase A,. Beside the main band, two minor bands of about 32 and 44 kDa were detected in the platelet lysates. These bands were only weakly stained with the protein staining and did not appear with either normal rabbit serum (data not shown) or the R377 antiserum. Masliah et al.

[38] reported similar observations with polyclonal antibodies against Naja naja venom phospholipase A,. They had immunized rabbits with 2-ME-denatured venom enzyme and prepared antiserum. In immunoblotting of mammalian cell lysates with the antiserum, they had detected three major protein bands of apparent molecular masses 14, 30 and 45 kDa, respectively. With regard to this, Apitz-Castro et al. [39] detected a 44 kDa phospholipase A, in human platelets and Loeb et al. [40] found a 30 kDa phospholipase A, in sheep platelets. There is a possibility that either the 32 or the 44 kDa band corresponds to isozymes of phospholipase A z present in platelets. The wide crossreactivity was another characteristics of R385. It crossreacted not only with ‘Group II’ enzymes (Crotalus adamanreus, rabbit platelet) but also with ‘Group I’ enzymes (porcine pancreas, Naja naja). The treatment of the enzyme with 2-ME may induce expression of unique epitopes different from those of intact enzyme, to which antibody R385 was elicited. The epitopes are expressed ubiquitously on various types of phospholipase A z, irrespective of the denaturation. The specific reactivity of R377 with rat platelet phospholipase A, enable us to investigate the distribution of immunologically related enzymes, which we called ‘platelet type’ phospholipases A,, in various sources. The inhibition assay of enzymatic activity by R377 revealed that ‘platelet type’ phospholipases A, exist in spleen, lung, bone marrow and inflamed sites. In this phospholipase A, assay, over 40 mg of protein was used for liver and kidney, while only 40 pg for pancreas homogenates. When purified rat platelet phospholipase A, was added to the crude tissue homogenates, such as liver or kidney, the activity of purified enzyme added was completely inhibited by the treatment with 100 pg of R377. The failure of R377 to inhibit phospholipase A, activity of liver or kidney is therefore not due to aspecific absorption of the antibody to the excess proteins. Partial inhibition of enzyme activity in bone-marrow, spleen or lung by R377 may indicate the existence of phospholipase(s) A, distinct from ‘platelet type’ phospholipase A 2. Indeed, rat spleen and lung were shown to contain ‘pancreas type’ phospholipase Az [41,42]. These results are consistent with our previous data obtained using monoclonal antibodies except for the reaction with peritoneal exudated cells: R377 reacted with the cells, whereas the monoclonal antibody did not [20]. This discrepancy could be explained as follows: the domain recognizable by the monoclonal antibody was blocked by some endogenous substance(s) in the crude protein mixtures of peritoneal cell lysates, while epitopes other than that reactive with the monoclonal antibody are still available for the polyclonal antibody R377. Alternatively, there may be phospholipase A, which is not identical but has a structure closely related to rat platelet phospholipase A,. The

41 latter possibility might be supported by the findings that a higher amount of antibodies was required for inhibiting phospholipase A, activity of peritoneal cells than that for enzymes of platelets. Recently, Aarsman et al. purified a phospholipase Az from rat liver mitochondria and found almost 100% sequence homology between the N-terminal 24 amino acids of liver mitochondrial enzyme and platelet enzyme [ 131. Furthermore, monoclonal antibodies raised against liver mitochondrial enzyme were shown to crossreact with platelet enzyme [37,43]. In our experiments, however, both polyclonal and monoclonal antibodies [20] raised against platelet enzyme failed to react with liver enzyme. This discrepancy could not be explained by the difference in assay conditions for enzymatic activity. We used phosphatidylserine as a substrate at pH 7.4 (Table I), whereas Aarsman et al. used phosphatidylethanolamine at pH 8.5. However, R377 showed no inhibition of phospholipase A, activity of liver mitochondrial fraction, even when examined under their assay conditions (data not shown). Further studies must be carried out to explain why our antibodies do not react with liver mitochondrial phospholipase A,. The endogenous modulators of phospholipase A, activity such as ‘lipocortins’ have been detected [26-311. Pepinsky et al. [28] isolated from rat peritoneal exudates a 37 kDa protein which inhibited phospholipase A, of snake venom. The endogenous activator, whose molecular mass was about 28 kDa, was reported by Clark et al. [32] in several mouse cell-lines. The enzyme activity measured could not reflect the content of the enzyme in crude protein mixtures, since it might be modified by the presence of modulators of phospholipase A, activity. Establishing a method of quantitating ‘rat platelet type’ phospholipases A, has been required. The combination of polyclonal antibody R377 and monoclonal antibody MD7.1 gave a sensitive sandwich ELBA which could be applied to phospholipase A, even in complex protein mixtures. The sensitivity (35 fmol per well) of the ELISA was within the range of phospholipase A, contents detected in inflamed sites. Peritoneal exudated fluids contain phospholipase A,, whose N-terminal sequence is identical to rat platelet enzyme [15]. Endogenous inhibitor(s) to the peritoneal enzyme may also exist in peritoneal fluid, because the recovery of total activity of the enzyme in the gel-filtration purification step was over 200%. Immunochemical quantitation gave a 2- to 3-fold higher content of the phospholipase A, than the estimation on the basis of enzymatic activity of purified platelet phospholipase A,. It should be noted that the peritoneal enzyme showed almost the same specific activity as the platelet enzyme when phosphatidylserine was used as a substrate: the specific activity of purified peritoneal enzyme was 19800 units/mg and that of purified platelet enzyme was 23000 units/mg. In the supernatant of activated rat platelets as well as

rat serum, no appreciable difference was observed between the two methods, indicating that such modulators activity might not exist in these of phospholipase A, samples. Some information has been accumulated about ‘platelet type’ phospholipases A, in inflamed sites of various species, including human H-191. Little information, however, has been available for understanding roles of phospholipase A, in the process of inflammation. Antibodies we have established may provide useful information about localization, dynamics or functions of phospholipases A, in inflamed sites. Acknowledgement This work was supported in part by Grant-in-Aid for Scientific Resarch (Nos 63480490 and 62870093) from the Ministry of Education, Science and Culture of Japan. References Van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246. Vadas, P. and Pruzanski, W. (1986) Lab. Invest. 55, 391-404. Pruzanski, W., Vadas, P.. Stephanski, E. and Urowitz, M.B. (1985) J. Rheumatol. 12, 211-216. 4 Vadas, P., Wasi, S., Movat, H.Z. and Hay, J.B. (1981) Nature 293, 5833585. 5 Maraganore, J.M. and Heinrikson, R.L. (1986) J. Biol. Chem. 261, 4797-4804. 6 Heinrikson, R.L., Krueger, E.T. and Keim, P.S. (1977) J. Biol. Chem. 252, 4913-4921. I Fleer, E.A.M.. Verheij, H.M. and De Haas, G.H. (1978) Eur. J. Biochem. 82, 261-269. 8 Slotboom, A.J., Verheij, H.M. and De Haas, G.H. (1982) in Phospholipids (Hawthorne, J.N. and Ancell, G.B., eds.), pp. 3599434, Elsevier, Amsterdam. 9 Horigome, K., Hayakawa, M.. Inoue, K. and Nojima, S. (1987) J. B&hem. 101, 625-631. 10 Hayakawa, M., Kudo, I., Tomita, M., Nojima. S. and Inoue, K. (1988) J. Biochem. 104, 767-772. 11 Horigome, K., Hayakawa, M., Nojima, S. and Inoue, K. (1987) J. B&hem. 101, 53-61. 12 Ono, T., Tojo, H., Kuramitsu, S., Kagamiyama, H. and Okamoto, M. (1988) J. Biol. Chem. 263, 5732-5738. 13 Aarsman, A.J., De Jong, J.G.N., Arnoldussen, E., Neys, F.W., Van Wassennaar, P.D. and Van Den Bosch, H. (1989) J. Biol. Chem. 264, 10008-10014. 14 Verger. R., Ferrato, F., Mansbach, C. and Pieroni, G. (1982) Biochemistry 21, 6883-6889. 15 Chang, H.W., Kudo, I., Tomita, M. and Inoue, K. (1987) J. Biochem. 102, 147-154. 16 Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, L. and Heinrikson, R.L. (1986) Biochemistry 25, 8381-8385. 17 Hara, S., Kudo, I., Chang, H.W., Matsuta, K., Miyamoto, T. and Inoue, K. (1989) J. Biochem. 105, 395-399. 18 Seilhamer, J.J., Pruzanski, W., Vadas, P., Plant, S., Miller, J.A., Kloss, J. and Johnson, L.K. (1989) J. BioI. Chem. 264, 5335-5338. 19 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. 20 Murakami, M., Kobayashi, T., Umeda, M., Kudo, I. and Inoue, K. (1988) J. Biochem. 104, 884-888.

42 21 Mizushima, H., Kudo, I., Horigome. K., Murakami, M., Hayakawa, M., Kim, D.-K., Kondo, E., Tomita, M. and Inoue, K. (1989) J. Biochem. 105. 520-525. 22 Forsgen, A. and Sjoquist, J. (1966) J. Immunol. 97. 822-827. 23 Fraker, P.J. and Speck, J.C. (1978) Biochem. Biophys. Res. Commun. 80, 849-857. 24 Yanoshita, R., Kudo, I., Ikizawa. K., Chang, H.W., Kobayashi, S., Ohno, M., Nojima, S. and Inoue, K. (1988) J. Biochem. 103, 815-819. 25 Chang, H.W., Kudo, I., Hara, S., Karasawa, K. and Inoue, K. (1986) J. Biochem. 100, 1099-1101. 26 Blackwell, G.J., Carnuccio, R., Di Rosa, M., Flower, R.J., Perente, L. and Persico, P. (1980) Nature 287, 1477149. 27 Hirata, F., Schiffmann, E., Venkatatsubramanian, K., Salomon, P. and Axelrod. J. (1980) Proc. Natl. Acad. Sci. USA 77, 253332536. 28 Wallner, B.P., Mattaliano, R.J., Hession, C.. Cate, R.L., Tizard,

29

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Immunochemical detection of 'platelet type' phospholipase A2 in the rat.

Polyclonal antibodies were raised against rat platelet phospholipase A2. One of them, designated as R377 was prepared by immunizing a rabbit with the ...
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