S p l e e n P h o s p h o l i p a s e s A 2 B y HIROMASA TOJO, TAKASHI ONO,
and MITSUHIRO OKAMOTO
Introduction Phospholipase A2 (PLA2 ; EC 220.127.116.11) stereospecifically hydrolyzes the fatty acyl ester bonds at the sn-2 position of glycerophospholipids. The enzymes exist in almost every type of cell studied so far and are distributed in diverse subcellular fractions. Detailed comparison of the molecular properties of PLA2 s purified to homogeneity from different subcellular locations may provide an insight into their functional differences. Rat or human spleen is a relatively rich source of PLA2 among tissues other than pancreas, and it contains PLA2s in both soluble and membrane-associated forms. Recently, the two forms of PLA2 were purified from rat spleen and characterized in detail. They were demonstrated to be distinct from each other: the soluble form was identical to pancreatic PLA 2, whereas the membrane-associated form was of viperid/crotalid type. L2 This chapter describes the assay method, purification procedures, and properties of the enzymes. Assay Method 1
Principle. PLA 2 specifically catalyzes the hydrolysis of the fatty acyl bond at position 2 of 3-sn-phosphoglycerides. The fatty acids released are extracted by the method of Dole and Meinertz 3 and then derivatized with 9-anthryldiazomethane4 (ADAM). The derivatized fatty acids are analyzed by reversed-phase high-performance liquid chromatography (HPLC). Reagents for Standard Assay 0.05% (w/v) 9-Anthryldiazomethane (Funakoshi Co., Tokyo, Japan), in ethyl acetate/methanol (I : 9, v/v) I H. Tojo, T. Ono, S. Kuramitsu, H. Kagamiyama, and M. Okamoto, J. Biol. Chem. 263, 5724 (1988). 2 T. Ono, H. Tojo, S. Kuramitsu, H. Kagamiyama, and M. Okamoto, J. Biol. Chem. 263, 5732 (1988). 3 V. P. Dole and H. Meinertz, J. Biol. Chem. 235, 2595 (1960). 4 N. Nimura and T. Kinoshita, Anal. Lett. 13, 191 (1980).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
SPLEEN PHOSPHOLIPASES A 2
5 mM 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in chloroform 100 mM Sodium deoxycholate in methanol 25 mM Calcium chloride 0.5 M Tris-HC1, 0.5 M NaC1, pH 8.5 0.5 mM Heptadecanoic acid in methanol Dole's reagent (n-heptane/2-propanol/2 N sulfuric acid, 10:40:1, v/v) n-Heptane Preparation of Substrate Solutions. Mixed micellar solutions of deoxycholate and phosphatidylethanolamine (PE) are prepared as reported. 5 Solutions of sodium deoxycholate in methanol and PE in chloroform are mixed to obtain the desired molar ratios. We have routinely employed a deoxycholate/PE ratio of 6; since an optimal molar ratio depends on the detergent-phospholipid combination, the ratio should be determined for each set of detergent and phospholipid. The solvents are evaporated under nitrogen, and then the samples are further dried in vacuo for about 30 min. An appropriate amount of the buffer is added, and the tubes are vortexed for 2 rain at about 37°. In the case of assay without detergent the substrate is sonicated 3 times for 20 sec in the buffer. Preparation of ADAM Solution. Since ADAM is relatively unstable in methanolic solution, the solution of ADAM used for the assay should be prepared immediately before use. For convenience, ADAM dissolved in a small volume of ethyl acetate (e.g., 25 mg/ml) is divided into small portions containing known amounts, then the solvent is evaporated. Store in darkness at - 3 5 °. Only the amount needed for the day's work is first dissolved in 1 volume of ethyl acetate, and then 9 volumes of methanol is added to make up the final concentration of ADAM to 0.05%. Procedures. The assay mixtures contain 5 mM CaClz, 1 mM 1-palmitoyl-2-oleoyl-PE, 6 mM deoxycholate, 0.1 M NaC1, 0. I M Tris-HC1, pH 8.5, and the enzyme sample, in a final volume of 50/zl. In a control tube CaCI 2 is replaced by EDTA (10 raM). The enzyme reaction is stopped by the addition of 200/zl of Dole's reagent. To the reaction mixture are added 120/zlof heptane and 70/xl of water, and then 5 nmol of heptadecanoic acid (10/zl) is added as an internal standard. The tubes are vortexed for about 20 sec. Fatty acids are extracted into the upper heptane phase. 3 Fifty microliters of the heptane layer is transferred to a 0.3-ml microvial, and the solvent is evaporated with an aspirator. Then 50/zl of the 0.05% ADAM solution is added to the vial. The vial is incubated for about 15 rain at room temperature, and then the derivatized fatty acids are analyzed on a Superspher RP-18 (4 × 50 mm, Merck, Darmstadt, FRG) or a Cos5 S. Yedgar, R. Hertz, and S. Gatt, Chem. Phys. Lipids 13, 404 (1974).
mosil 3C18 (2.1 x 50 mm, Nacalai Tesque, Kyoto, Japan) column with a UV detector at 254 nm. The solvent system employed is 95-98% acetonitrile at 1 ml/min. The samples were automatically injected with a Gilson (Villiers-le-Bel, France) autosampler 231-401.
Comments (I) This method is applicable to the assay with any phospholipid class containing long-chain fatty acids as a substrate. Many kinds of synthetic phospholipids with high purity are now commercially available (e.g., from Avanti Polar Lipids, Inc., Birmingham, AL). Hence, the substrate specificities of PLA2s are easily evaluated by this method. 1,2When 1-palmitoyl2-oleoylphosphatidylglycerol was used as a substrate, the oleic acid released by PLA2 reacted with ADAM significantly poorly at PG concentrations above 1 mM. Phosphatidylglycerol (PG) itself did not hinder the reaction. Although we did not further examine the reason for this, the problem could be circumvented by treatment of the heptane phase (100 /zl) with silicic acid (~5 mg). Some portions of phospholipids are extracted into the upper heptane layer together with fatty acids by the Dole procedure. When phosphatidylserine (PS) was used as a substrate, a carboxylate group of the extracted PS was derivatized with ADAM, and the PS ester was eluted faster than most of derivatized long-chain fatty acids on HPLC. The PS extracted in the heptane layer could, if necessary, be removed by the silicic acid treatment. (2) The positional specificity of phospholipase A action can be determined by this method with a mixed-acyl phospholipid as a substrate: the time courses of the hydrolysis at the sn-1 position and of that at the sn-2 position are followed separately and compared with the time courses of the hydrolysis by pancreatic PLA2, which exclusively hydrolyzes the ester bond at the sn-2 position. (3) When ADAM-derivatized fatty acids are separated on a HPLC column of small size, it is essential to keep the pre- and postcolumn dead volumes associated with a HPLC system as small as possible. Otherwise, the resolving power of the small column is severely hampered. Purification of Phospholipases A2
From Rat Spleen Supernatant Purification Strategy. The key step of the purification procedures is reversed-phase HPLC. The HPLC method is a powerful tool for enrichment and purification of trace amounts of cellular PLAEs, which are stable
SPLEEN PHOSPHOLIPASES A 2
in organic solvents. 6 Since we introduced the HPLC method to the purification of a soluble PLA 2from rat spleen, 6the method has been successfully applied to the purification of PLA2s of various origins including rat pancreas, 7 rat gastric mucosa, 8 human spleen, 9'1° rabbit 1~ and rat 12 ascites, human synovial fluid, 13-14ahuman platelets, 13and the macrophagelike cell line P388D~. 15The procedure described here includes some modifications to adapt the HPLC method reported previously for a relatively larger scale preparation. HPLC System. The HPLC system consists of two Gilson Model 302 liquid delivery modules and a sample injector with a 8-ml sample loop. For microbore HPLC, the system equipped with a Gilson 811 dynamic mixer with a 65-/zl mixing chamber is operated in the microflow mode. The effluent is monitored simultaneously at 280 and 210 nm with a Gilson 116 detector with a 1.6-~1 flow cell. A well-established solvent system, trifluoroacetic acid (TFA)/acetonitrile, is effective for the purification of PLA2s. The solvent system employs an acidic (pH - 2 . I) mobile phase, which usually maximizes the separation for most peptides and small proteins.16 No irreversible inactivation of the enzymes takes place during elution at the acidic pH in the presence of the organic solvent. 6 The eluants used are as follows: eluant A, 0.1% (v/v) TFA in water; eluant B, 95% (v/v) acetonitrile, 0.1% TFA.
Buffers Buffer A, 20 mM Tris-HCl, pH 7.4 Buffer B, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0 6 H. Tojo, T. Teramoto, T. Yamano, and M. Okamoto, Anal. Biochem. 137, 533 (1984). 7 T. Ono, H. Tojo, K. Inoue, H. Kagamiyama, T. Yamano, and M. Okamoto, J. Biochem. (Tokyo) 96, 785 (1984). 8 H. Tojo, T. Ono, and M. Okamoto, Biochem. Biophys. Res. Commun. 151, 1188 (1988). 9 K. Nakaguchi, J. Nishijima, M. Ogawa, T. Mori, H. Tojo, T. Yamano, and M. Okamoto, Enzyme (Basel) 35, 2 (1986). 10A. Kanda, T. Ono, N. Yoshida, H. Tojo, and M. Okamoto, Biochem. Biophys, Res. Commun. 163, 42 (1989). 11 S. Forst, J. Weiss, P. Elsbach, J. M. Maraganore, I. Reardon, and R. L. Heinrikson, Biochemistry 25, 8381 (1986). 12H. W. Chang, I. Kudo, M. Tomita, and K. Inoue, J. Biochem. (Tokyo) 102, 147 (1987). ~3R. M. Kramer, C. Hession, B. Johansen, G. Hayes, P. McGray, E. P. Chow, R. Tizard, and R. B. Pepinski, J. Biol. Chem. 264, 5768 (1989). t4 S. Hara, I. Kudo, H. W. Chang, K. Matsuta, T. Miyamoto, and K. Inoue, J. Biochem. (Tokyo) 105, 395 (1989). 14aj. j. Seilhamer, S. Plant, W. Pruzanski, J. Schilling, E. Stefanski, P. Vadas, and L. K. Johnson, J. Biochem. (Tokyo) 106, 38 (1989). ~5R. J. Ulevitch, Y. Watanabe, M. Sano, M. D. Lister, R. A. Deems, and E. A. Dennis, J. Biol. Chem. 263, 3079 (1988). z6 W. C. Mahoney and M. A. Hermodson, J. Biol. Chem. 255, 11199 (1980).
PHOSPHOLIPASE A 2
Buffer C, 20 mM 2-methyl-2-amino-l,3-propanediol, pH 9.5 Buffer D, 100 mM Tris-HCl, pH 7.4 Preparation of Tissue Extract. Rats are anesthetized by the intraperitoneal injection of pentobarbital. Spleens are removed, carefully prepared to avoid the involvement of pancreas tissues, and then stored at - 3 5 °. Frozen rat spleen tissue (500 g) is homogenized in 2 liters of buffer A containing 1 mM CaCI 2 with a blender. The homogenate is centrifuged at 108,000 g for 1 hr. The supernatant contains about 10% of the total PLA 2 activity, the pellet about 90%. The pellet is stored at - 35° for further use.
DEAE-Cellulose Treatment. The supernatant is applied to a DEAEcellulose column (8 x 30 cm) preequilibrated with buffer A. The resulting flow-through fractions which contain the PLA2 activity are pooled. The pH of the pooled fractions is adjusted to 6.0 by the addition of 1 N HCI, and then the PLA 2 is concentrated with a short S-Sepharose column (5 x 6 cm) preequilibrated with buffer B. The PLA2 activity is eluted with buffer D containing 0.5 M NaC1. Note: If the capacity of the DEAE-cellulose column is insufficient to remove contaminants, lowering pH of the flowthrough fractions may form significant amounts of precipitates. This resuits in very low recovery of the enzyme activity. TEAE-Cellulose Column Chromatography. The concentrated PLA2 fraction is dialyzed against buffer C. The resultant solution is applied to a TEAE-cellulose column (3 x 10 cm) preequilibrated with buffer C. The major part (86%) of the PLA z activity is eluted under these conditions, while a small part (14%) is bound to the column. The PLA 2 contained in the major flow-through fraction, which is named PLA2 S-I, is further purified. The enzyme activity bound to the column is eluted in a stepwise manner with buffer D containing 0.2 M NaC1. BioGel P-30 Column Chromatography. The pooled PLAz S-1 fraction is dialyzed against buffer B, and then the resultant solution is concentrated with a short S-Sepharose column (2 × 1.5 cm) as described above. The concentrated PLA2 fraction is then applied to a BioGel P-30 (minus 400 mesh) column (2 × 50 cm) preequilibrated with buffer A containing 0.5 M NaCI. A single peak of PLA2 S-1 activity appears at an elution volume of 80 ml. The fractions containing PLA2 activity are pooled. Reversed-Phase HPLC. The pooled fraction is injected onto a Cosmosil 5C8-300 column (4 x 250 mm) initially equilibrated with eluant A. After the column is washed for 20 min with eluant A, the enzyme is eluted from the column with a linear gradient of increasing acetonitrile concentration, from 0 to 15% in eluant B in 5 min and then from 15 to 40% in eluant B in 60 min. The PLA2 activity is eluted in a fraction corresponding to a single absorbance peak at about 27% acetonitrile in eluant B. A
SPLEEN PHOSPHOLIPASES A 2
TABLE 1 PURIFICATION OF PHOSPHOLIPASEA2 S-1 FROM RAT SPLEEN SUPERNATANT
Total protein (rag)
Total activity (t~rnol/rnin)
Specific activity (nmol/min/mg)
Homogenate Supernatant DEAE-cellulose TEAL-cellulose BioGel P-30 Reversed-phase HPLC
37,600 24,400 2340 752 9.66 0.042
246 17.6 9.33 5.60 3.96 3.2
6.54 0.72 3.99 7.45 410 76,200
-100 53,0 31.8 22.5 18.2
small amount (-20/zl/ml) of 1 M Tris is added to the pooled fractions to neutralize the enzyme solution, and then the fractions are stored at - 35 °. The presence of acetonitrile does not affect the PLA2 activity, but it somewhat prevents nonspecific adsorption of the enzyme to glass tubes or polypropylene tubes. Usually, this preparation is essentially homogeneous as judged by sodium dodecyl sulfate (SDS)-gel electrophoresis. However, when minor contaminants are present in the preparation, rechromatography of an aliquot (-5/.~g) on a small Cosmosil 5C8-300 column (1 × 150 or 2.1 x 30 mm) is effective for further purification. A summary of a typical enzyme purification is shown in Table I. From Rat Spleen Membrane Fraction 2 Buffers Buffer A, 10 mM Tris-HCl, pH 7.4 Buffer B, 10 mM Tris-HC1, 0.3% lithium dodecyl sulfate (LDS), pH 7.4 Buffer C, 10 mM Tris-HCl, 1 M NaCI, pH 7.4 Buffer D, 200 mM Tris-HC1, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS), pH 7.4 Solubilization. Various salts and detergents are tested for the ability to extract PLA2 from the splenic membrane fraction (108,000 g pellet). Extraction is carried out at 4° for 2 hr. A high concentration (up to 1 M) of KC1 hardly solubilizes the enzyme activity, whereas treatment with 1 M KBr extracts about 20% of the PLA2 activity in the membrane fraction. Interestingly, the PLAz activity in the membrane fraction of human spleen could be well solubilized (-70%) by the KBr treatment. 9 The reason for this species difference in the solubility of the enzymes is unknown at present. Since bromide is a chaotropic ion, the KBr treatment may perturb
PHOSPHOLIPASE A 2
hydrophobic interactions as well as ionic interactions between the enzyme and the membrane. After being solubilized by 1 M KBr, the human splenic PLA2 is purified without any detergent. 9,~° CHAPS and octylglucoside do not solubilize PLA2 from the membrane fraction of rat spleen in the concentration range of 0.5 to 1%. In contrast, treatment with a much stronger detergent, LDS (0.3%), extracts about 90% of the PLA2 activity. The PLA 2 activity shows no change in 0.3% LDS for at least several hours. Since LDS has a lower Kraft point than SDS, LDS does not precipitate at 4°. Hence, LDS is superior to SDS for the solubilization and further purification of PLA 2 at 4°. The 108,000 g pellet is homogenized with 10 volumes of buffer B with a blender, and then the mixture is stirred for 2 hr at 4°. The resultant solution is centrifuged at 108,000 g for 1 hr. DEAE-Cellulofine A M Treatment. The supernatant obtained is applied to a DEAE-Cellulofine AM column (6 x 10 cm, Seikagaku Kogyo, Tokyo, Japan) previously equilibrated with buffer A. Since the flow rate becomes gradually slower, during loading of the sample, because of the presence of LDS, the enzyme solution is thoroughly mixed with the gel by occasional stirring with a glass bar to gain a sufficient flow rate. The enzyme is adsorbed to the column under these conditions. The enzyme activity is eluted with buffer D containing 0.5 M Trizma sulfate, the pH of which is adjusted to 7.0 with 1 M Tris base. After being diluted 10-fold with buffer A to reduce the salt concentration, the enzyme fraction is applied to an SSepharose column (4 x 10 cm) preequilibrated with buffer A. The enzyme activity is eluted from the column with buffer C containing 5% CHAPS. Octyl-Sepharose Chromatography. The pooled PLA2 fraction is diluted 5-fold with buffer A to reduce the concentration of CHAPS. Then the appropriate amounts of lithium sulfate and Trizma sulfate are added to the enzyme solution to a final concentration of 0.5 M each, and the pH of the solution is adjusted to 7.0 with 1 M Tris base. The resultant solution is applied to an octyl-Sepharose column (2.5 × 20 cm) preequilibrated with buffer A containing 1 M lithium sulfate. Elution is performed with buffer A containing I% CHAPS. The enzyme solution is diluted 10-fold with buffer A and concentrated with a small S-Sepharose column (1 × 3 cm) previously equilibrated with buffer A. The column is washed with buffer A containing 0.3 M NaCI and 0.5% CHAPS and then eluted with buffer C containing 0.5% CHAPS. Gel Filtration on Cellulofine GCL 300-m Column. The concentrate is applied to a Cellulofine GCL 300-m (Seikagaku Kogyo) gel-filtration column (4.5 × 74 cm) preequilibrated with buffer D. A peak of PLA2 activity is eluted at an elution volume of 780 ml. S-Sepharose Chromatography. The pooled enzyme fraction is applied
SPLEEN PHOSPHOLIPASES A 2
TABLE II PURIFICATION OF PHOSPHOLIPASE A 2 M FROM RAT SPLEEN MEMBRANE FRACTIONa
Total protein (mg)
Crude membrane Extract DEAE-cellulose Octyl-Sepharose Cellulofine GCL 300-m S-Sepharose BioGel P-30
2700 2360 385 34.8 3.0 0.25 0.01
Total activity (~mol/min) 4.80 4.40 3.51 0.32 b 0.96 b 1.60 0.30
Specific activity (nmol/min/mg) 1.8 1.9 9. I 9.2 320 6400 30,000
Yield (%) 100 91.7 73.1 6.7 20.0 33.3 6.3
a Data from T. Ono e t al. 2 b The reason for the apparent decrease in the total activity was not further examined.
to an S-Sepharose column (0.8 × 8 cm) preequilibrated with buffer A. Elution is performed with a linear gradient from 0.3 to 0.8 M NaC1 in buffer A. The PLA2 activity is eluted at 0.5-0.6 M NaCI. The fractions containing the enzyme activity are pooled and diluted 5-fold with buffer A. Then the solution is concentrated with a small S-Sepharose column (0.7 × 1 cm) as described at the step of octyl-Sepharose chromatography. BioGel P-30 Gel Chromatography. The concentrate is applied to a BioGel P-30 column (2.2 × 45 cm) preequilibrated with buffer D. The PLA2 activity is eluted, at an elution volume near the total available column volume, much later than the position expected from the molecular weight estimated on SDS-gel electrophoresis. This may suggest the hydrophobic interaction of the enzyme with the gels. The purified enzyme, named PLA2 M, is concentrated with a small S-Sepharose column as described above and stored at - 35 °. Table II summarizes the results of a typical enzyme purification. Properties
Purity and Molecular Weight. The PLA 2 preparations (PLA 2 S-1 and PLA2 M) purified from the two sources were homogeneous as judged by SDS-gel electrophoresis and analytical HPLC. z,2 Both enzymes migrated on SDS gels as a single protein band to the same position as rat pancreatic PLA2: the apparent molecular weight of both enzymes was estimated to be 13,600, in agreement with the weights calculated from the amino acid sequences. PLA 2 S-1 was eluted as a sharp peak at the same retention time as pancreatic PLA2 on reversed-phase HPLC, whereas PLA 2 M was
PHOSPHOLIPASE A 2
eluted later, as a significantly broad peak, than PLA2 S-1. This indicated that PLA2 M has a molecular surface more hydrophobic than PLA2 S-1. Catalytic Properties. Purified PLA 2 S-1 and PLA 2 M absolutely required calcium ions for activity: the concentrations of calcium ion which give half-maximum velocity were 0.03 mM for PLA: S-1 and 0.5 mM for PLA 2 M.1'2 Optimal activities were found in the range of pH 8.0-10.5 for PLA 2 S-1 and pH 8.0-9.5 for PLA 2 M. The positional specificity of PLA 2 S-1 and PLA2 M regarding the hydrolysis of an acyl ester bond at the sn-2 position of phospholipids was confirmed by the HPLC method as described above. The substrate specificities of PLA2 S-1 and PLA 2 M were examined for sonicated phospholipid vesicles and for mixed micelles of phospholipids and bile salts (cholate or deoxycholate) at various molar ratios. In all cases, the substrate specificity of PLA 2 S-I was practically identical to that of pancreatic PLAz. The hydrolytic rate order for sonicated phospholipids in the case of PLA2 M was PG > PE = PS > phosphatidylcholine (PC), whereas that in the case of PLA/S-1 was PG > PE > PC > PS. The general trend in the specificity of PLA z M toward the mixed micelles was similar to that of PLA2 S-l: PG was the best substrate, PS the poorest. There was, however, a clear difference in the dependence of the specificity on the cholate/phospholipid (PE or PC) molar ratio. The highest specific activities were obtained at a cholate/PG ratio of 6 (0.5 mmol/min/mg) for PLA 2 S-1 and at cholate/PG ratios ranging from 2.5 to 7 (0.15 mmol/min/ mg) for PLA 2 M. Immunochemical Properties. PLA 2 S-1 is immunochemically identical to rat pancreatic PLA2, whereas there is no immunochemical similarity between PLA2 M and pancreatic PLA2 .~,2 An antipancreatic PLA2 antibody did not recognize PLA2 M, and conversely an anti-PLA2 M antibody did not recognize the pancreatic PLA 2 . Structural Properties. The amino acid compositions of PLA 2 S-1 and pancreatic PLA2 are very similar; in addition, the peptide maps and sequences of the amino-terminal 32 residues of the two enzymes are identical. The enzyme purified from rat spleen supernatant is, therefore, of the pancreatic type (group I). 1 When rat tissues were screened with the antipancreatic PLA 2 antibody 17or rat pancreatic PLA 2cDNA ~8as a probe, the pancreati~c type PLA 2 was found in gastric mucosa and lung as well as in spleen. The gastric enzyme and its zymogen were purified and character17 M. Okamoto, T. Ono, H. Tojo, and T. Yamano, Biochem. Biophys. Res. Commun. 128, 788 (1985). msO. Ohara, M. Tamaki, E. Nakamura, Y. Tsuruta, Y. Fujii, M. Shin, H. Teraoka, and M. Okamoto, J. Biochem. (Tokyo) 99, 733 (1986).
SPLEEN PHOSPHOLIPASES A 2
ized. 8 The cDNAs encoding pancreatic PLA2 were cloned from gastric mucosa and lung, and their nucleotide sequences were completely identical to that isolated from pancreas. 19The pancreatic type PLA 2 was found to exist also in human spleen. 10 The amino acid composition and peptide map of PLA2 M were quite different from those of pancreatic PLA2. The amino-terminal sequence of PLA 2M revealed the absence of Cys-11, characteristic of group II (viperid/ crotalid venom type) PLA2s. 2° Group I (pancreatic and elapid type) PLA2 s are known to have a disulfide pair between Cys-ll and Cys-77. 2° More recently, the complete amino acid sequence of PLA2 M was determined by nucleotide sequencing of PLA2 M cDNA 21 as well as by protein sequencing 22of PLA2 M. The results from both the methods were completely identical and confirmed that PLA2 M belongs to the group II PLA2 category, that is, PLA2 M has a Cys-50 and a carboxy-terminal extension of the seven residues terminating in a cysteine residue. The hydropathy profile of PLA 2 M did not show any plausible membrane binding site. The primary structure of PLA2 M was different only by four residues from that of rat platelet secretory PLA 2 .23 The PLA2 M cDNA contains a sequence similar to typical signal sequences for secretory proteins. The mechanism of anchoring of the enzyme on the membrane therefore remains to be clarified. The primary structure of PLA2 M purified from fiuman spleen was also determined by protein sequencing. 10The sequence was also homologous to that of group II enzymes and identical to that deduced from the nucleotide sequence of the corresponding gene cloned from a genomic library.13'24
19 T. Sakata, E. Nakamura, Y. Tsuruta, M. Tamaki, H. Teraoka, H. Tojo, T. Ono, and M, Okamoto, Biochim. Biophys. Acta 1007, 124 (1989). 20 R. L. Heinrikson, E. T. Krueger, and P. S. Keim, J. Biol. Chem. 252, 4913 (1977). 2i j. Ishizaki, O. Ohara, E. Nakamura, M. Tamaki, T. Ono, A. Kanda, N. Yoshida, H. Teraoka, H. Tojo, and M. Okamoto, Biochem. Biophys. Res. Commun. 162, 1030 (1989). 22 T. Ono, H. Tojo, N. Yoshida, and M. Okamoto submitted for publication. 23 M. Hayakawa, I. Kudo, M. Tomita, S. Nojima, and K. Inoue, J. Biochem. (Tokyo) 104, 767 (1988). 24 j. j. Seilhamer, W. Pruzanski, P. Vadas, S. Plant, J. A. Miller, J. Kloss, and L. K. Johnson, J. Biol. Chem. 264, 5335 (1989).