Immunochemical relatedness between secretory phospholipase A2 and intracellular·phospholipase A2

Vol. 128, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

April 30, 1985 Pages 788-794

Immunochemical Relatedness between Secretory Phospholipase A2

and Intracellular*Phospholipase A2

Mitsuhiro Okamoto, Takashi Ono, Hiromasa Tojo and Toshio Yamano

Department of Biochemistry, Osaka University Medical School, 4-3-57, Nakanoshima, Kita-ku, Osaka 530, Japan

Received March 19, 1985

SUMMARY The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immune-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-

pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2. D 1985 Academic Press, Inc.

Phospholipase A2 [EC 3.1.1.41 hydrolyzes a fatty acyl ester bond at the sn-2 -

position of glycerophospholipids. The enzyme has been shown to exist in

almost every cell studied so far (for reviews see Refs. l-4). With the excep-

tion of the enzyme localized in lysosomes, phospholipase A2 is active under

alkaline conditions and requires Ca+2 for its action. The Ca+2-dependent

phospholipases A2 occurring in mammalian tissues are classified into two

groups in terms of their localization. Pancreatic phospholipase A2 is

biosynthesized in pancreatic acinar cells as an inactive proenzyme. The

prophospholipase A2 is secreted into the duodenlrm via the pancreatic duct

where it is converted into the active enzyme by tryptic digestion. On the

other hand, all cells are known to possess a phospholipase A2 of their own.

This type, called hereafter intracellular phospholipase A2, is associated with

all biological membranes where it is engaged in phospholipid turnover of the

membranes. Release of arachidonic acid from phospholipids catalyzed by this

enzyme is thought to be one of the regulatory steps in the biosynthesis of

prostanoids. In spite of these important roles ascribed to the intracellular

0006-291X/85 $1.50 Cowright 0 1985 by Academic Press, Inc. AN rights of reproduction in any form reserved. 788


phospholipase A2, not much information on this enzyme has been accumulated so

far, because its cellular content and its specific activity are very low.

and Although a comparative study of the intracellular

ses A2 from protein chemical aspects is desired,

this direction.

litt

We have recently purified rat pancreatic phosphol

pancreatic phospholipa-

e work has been done in

pase A2 and its proen-

zyme (51, and a Ca2+-dependent phospholipase A2 of intracellular origin from

rat spleen (6,7). Because we were able to obtain antiserum directed against

the purified rat pancreatic phospholipase A2, we have now initiated a com-

parative study on the pancreatic phospholipase A2 and the intracellular

phospholipase A2 from immunochemical aspects. In this communication, we

demonstrate the immunochemical relatedness between the two enzymes by using

the anti-rat pancreatic phospholipase A2 antibody as a probe.

MATERIALS AND METHODS

Enzyme preparations : Rat pancreatic phospholipase A2 and its proenzyme were purified to homogeneity as described previously (5). When rat spleens were stored under frozen conditions, Ca2+-dependent phospholipase A2 activity was

usually found in the cytosolic fraction as well as in the membranous fraction. The phospholipase A2 in the cytosolic fraction was purified to homogeneity as

described previously (7). ‘Ihe membrane-associated phospholipase A2 activity

was easily solubilized when the microsomes were incubated with a buffer of high ionic strength (0.01 M Na acetate-1M KBr, pH 5.5, for instance). ‘Ihe solubilized enzyme was purified to homogeneity as described for the purifica- tion of the supernatant enzyme (7) with some modification. The two types of the purified splenic hospholipase A2 had similar enzymic properties as to the

specific activity, Ca II+- requirement and optimum pH for the enzyme action. Human pancreatic phospholipase A2 was purified from pancreatic juice as

described previously (8) with some modification.

Anti-rat pancreatic phospholipase A2 antibody: A rabbit was immunized by sub- cutaneous injection with the purified rat pancreatic phospholipase A2 (500 ng) in Freund’s complete adjuvant. After 4 weeks, a booster injection was given with the antigen in incomplete adjuvant, and antiserum was obtained on the seventh day after the injection. IgG was purified from the antiserum by ammo- nium sulfate fractionation followed by gel exclusion chromatography on a column of Sephacryl S-300.

Immunoelectrophoresis: This was performed on 1% agarose gel plates. The buffer used was 25 mM Tris-HCl buffer, pH 8.0, containing 0.1% sodium azide. Counter immunoelectrophoresis was carried out according to the method described by Gocke and Howe (9).

Inhibition of the enzyme activity by the antibody: Phospholipase A2 was incubated with various amounts of IgG in 170 ~1 of 0.1 M Tris-HCl buffer, pH 7.4, for 3 h at 5 ‘C. After the incubation, aliquots were taken for assaying of the remaining activity of phospholipase A2. Adjustment was made so that each assay tube contained 88% of the incubated IgG.

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Enzyme activity: Phospholipase A2 activity was determined as reported previously (6)) and expressed as the amount of radioactive fatty acid released from Z-[lk]linoleoyl-phosphatidylethanolamine per min.

RESULTS AND DISCUSSION

Immunoelectrophoretic evidence for immunochemical relatedness between

pancreatic phospholipase A2 and splenic phospholipase A2

Immunoelectrophoresis was conducted to examine the immunochemical rela-

tionship between the pancreatic phospholipase A2 and the splenic phospholipase

A2 - As shown in Fig. lA, the pancreatic phospholipase A2 formed a sharp immu-

noprecipitin band with the antibody at a point slightly away from the origin

to the cathodic side under these conditions. The proenzyme of pancreatic

phospholipase A2 formed a broader immunoprecipitin band with the antibody at a

+

Figure 1. A. Immunoelectrophoretic pattern of phospholipase A2. me proenzyme of pancreatic phospholipase A2 ( PROP, 3 pg), pancreatic phospholipase A2

(P 0.9 ng) and splenic phospholipase A2 purified from the cytosolic fraction i SC, 3 ~8) were placed in the wells, and electrophoresis was conducted horizontally at 10 mA for 2 h. After the electrophoresis, anti-rat pancreatic phospholipase A2 antibody ( F , 24.3 m&ml) was placed in the troughs. B. Counter immunoelectrophoretic pattern of splenic phospholipase A2. The splenic enzyme purified from the cytosolic fraction ( SC , 3 up) and that from the microsomal fraction ( Qq, 3 pg) were placed in the upper wells. Anti-rat pancreatic phospholipase A2 antibody ( F , about 100 pg) was placed in the lower wells. Electrophoresis was cond,ucted vertically at 10 mA for 2 h. After the electrophoresis, the agarose gel was incubated in physiological saline for 4 days, and then it was stained with Coomassie brilliant blue.

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Vol. 128, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNKATIONS

point further away to the cathodic side, indicating that the proenzyme is

immunochemically similar to the active pancreatic phospholipase A2, but that

it is more positively charged under these conditions. Surprisingly, an immu-

noprecipitin band appeared on the track of the splenic phospholipase A2. The

band emerged on the cathodic side. The band was fuzzy compared to that formed

between the pancreatic phospholipase A2 and the antibody, indicating that the

splenic phospholipase A2 and the pancreatic phospholipase A2 are immunochemi-

tally similar, but not identical.

The immunochemical cross-reactivity of the anti-pancreatic phospholipase

A2 antibody with the splenic phospholipase A2 was confirmed by counter immu-

noelectrophoresis as shown in Fig. 1B. The splenic enzyme was purified to

homogeneity both from the soluble fraction and from the microsomal fraction

(see MATERIALS AND METHODS). The two splenic phospholipases A2 were placed in

wells on an agarose gel plate. The antibody was placed in a different well on

each track, and then electrophoresis was conducted. ‘Ihe antibody and the

antigen would migrate in the opposite directions under these conditions, and

would form a precipitin band where they met in the gel. As shown, the two

splenic enzymes, whether they were purified from the soluble fraction or from

the membrane fraction, clearly formed immunoprecipitin bands with the anti-

pancreatic phospholipase A2 antibody.

Inhibition of the phospholipase A2 activity by the anti-pancreatic phospholi-

pase A2 antibody

Figure 2A shows the effect of the anti-pancreatic phospholipase A2 anti-

body on the activity of the pancreatic phospholipase A2. The antibody up to

30 ug under these particular experimental conditions had little effect on the

enzyme activity. However, from more than 50 ~g of the antibody, the enzyme

began to be inhibited, and 130 ug of the antibody almost completely inhibited

the enzyme activity. IgG prepared from a preimmune rabbit did not inhibit the

activity, on the contrary, it apparently enhanced the enzyme activity when

added in a large amount. When a similar amount of bovine serum albumin

instead of normal IgG was added to the incubation mixture, similar apparent

791


OO xx) 203 ml 0 03 ml xx) ANTIBODY tug)

Figure 2. Effect of anti-pancreatic phospholipase A2 antibody on phospholipase - . A2 activity. A. Rat pancreatic phospholipase A2 (0.9 ug) was incubated with the indicated amounts of anti-rat pancreatic phospholipase A2 antibody (open circles), or normal IgG prepared from preimmune rabbits (closed circles), in 170 ul of 0.1 M Tris-HCl buffer, pH 7.4, for 3 h at 5 ‘C. After the incubation, aliquots were taken for the phospholipase A2 activity assay. The results are expressed as percentages of the activity of the enzyme incubated without IgG. ‘Ihe enzyme used had a specific activity of 50 urn01 fatty acid released/min/mg. B. Rat splenic phospholipase A2 (9 ug) purified from the microsomal fraction was incubated with anti-rat pancreatic phospholipase A2 antibody (open circles), or normal IgG (closed circles), as described above. Tbe enzyme used had a specific activity of 0.5 umol fatty acid released/min/mg .

activation was observed. Moreover, when an experiment in which the total

amount of protein in the incubation mixture was kept at 350 ug by the addition

of bovine serum albumin in addition to the enzyme and IgG was performed, no

apparent activation by normal IgG was observed (data not shown). Tnerefore,

the activation observed in the presence of a large amount of normal IgG may be

nonspecific, and may be due to its protective effect as to denaturation of the

enzyme on incubation at a low protein concentration.

Figure 2B shows the effect of the antibody on the activity of the splenic

phospholipase A2 purified from the microsomal fraction. The antibody at more

than 50 ug reduced the enzyme activity to 30%. No further inhibition was

observed in the presence of up to 350 ug of the antibody.

Taken together, the results shown in Fig. 2 indicate that both pancreatic

phospholipase A2 and splenic phospholipase A2 are recognized by the antibody

directed against the pancreatic phospholipase A2, and their activities are

inhibited in the presence of a large amount of the antibody. It should be

noted that the mode of inhibition of the splenic enzyme was different from

that of the pancreatic enzyme. Concerning the immuno-specificity of the anti-

792


body > we should point out that the activity of human pancreatic phospholipase

A2 was little affected by the anti-rat pancreatic phospholipase A2 antibody

(data not shown).

General discussion

The results presented herein clearly indicate the immunochemical related-

ness between the rat pancreatic phospholipase Ap and the rat splenic phospho-

lipase AZ. To our knowledge this is the first report of the similarity

between the pancreatic phospholipase A2 and the intracellular phospholipase

A2 from an immunological point of view. In order to further confirm the

findings, we have to raise an antibody against the splenic phospholipase

Ap and use the antibody to demonstrate its immuno-crossreactivity with the

pancreatic enzyme. Unfortunately, the small quantity of the splenic phospho-

lipase A2 available at present is hampering this line of research.

Basic enzymic properties of the rat pancreatic phospholipase A2 and the

rat splenic phospholipase A2 are quite similar to each other (5-7). For

example, their molecular weights are about 15,000, the apparent optimum pH of

their enzyme actions is pH 8-9, the apparent optimum Ca2+ concentration

required for their enzyme actions is approximately 5 mM, the concentration of

deoxycholate required for their enzyme actions is O.l%, and both enzymes are

stable under acidic conditions. Furthermore, as reported in this com-

munication, the two enzymes are immunochemically similar to each other. A

major difference between the two enzymes is in their specific activity. The

purified rat pancreatic phospholipase A2 had a specific activity of greater

than 50 nmol/min/mg in our assay system, whereas the purified splenic phospho-

lipase A2 had a specific activity of about 0.5 umol/min/mg. How this dif-

ference can be explained on the basis of the protein chemistry of the two

enzymes remains to be clarified.

ACKNOWLEDGMENTS: This work was in part supported by research grants from the Science and Technology Agency of Japan and from the Ministry of Education, Science and Culture of Japan.

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Verheij, H.M., Slotboom, A.J., and De Haas, G.H. (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203.

Van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246. Van den Bosch, H. (1982) Phospholipids, pp. 313-357, Elsevier, Amsterdam. Slotboom, A.J., Verheij, H.M., and De Haas, G.H. (1982) Phospholipids,

pp. 359-434, Elsevier, Amsterdam. Ono, T., Tojo, H., Inoue, K., Kagamiyama, H., Yamano, T., and Okamoto, M.

(1984) J. Biochem. 96, 785-792. Teramoto, T., Tojo, H., Yamano, T., and Okamoto, M. (1983) J. Biochem. 93,

1353-1360. Tojo, H., Teramoto, T., Yamano, T., and Okamoto, M. (1984) Anal. Biochem.

137, 533-537. Nishijima, J., Okamoto, M., Ogawa, M., Kosaki, G., and Yamano, T. (1983)

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Immunochemical relatedness between secretory phospholipase A2 and intracellular·phospholipase A2