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requiring 10 mM Ca 2+ for optimal activity. The PLA 2 shows a great preference for the E. coli substrate and hydrolyzes phospholipid/deoxycholate dispersions and sonicated liposomes at rates that are 100- and 1000-fold slower, respectively. In deoxycholate dispersions phosphatidylethanolamine is the preferred substrate and is hydrolyzed at a 4 times faster rate than phosphatidylcholine. The corresponding gene of the PLA 2 has been isolated and sequenced. 7'~5The deduced amino acid sequence of the protein consists of 124 amino acids, contains structural features common to all known PLA 2 , and has a half-cystine pattern that is characteristic for Group II snake venom PLA2.16 The sequence is preceded by a signal peptide, confirming that the PLA2 is a secretory enzyme. It should be noted that there are intracellular (nonsecretory) PLA 2 enzymes of higher molecular weight (>>14,000) that are activated by submicromolar Ca 2÷ and prefer phosphatidylcholine substrates. These PLA2 are likely to represent distinct enzymes.~7,~8 The high molecular weight PLA 2 appear to be inactivated by acid treatment and thus are lost during the purification procedure described here. 15 j. j. Seilhamer, W. Pruzanski, P. Vadas, S. Plant, J. A. Miller, J. Kloss, and L. K. Johnson, J. Biol. Chem. 264, 5335 (1989). 16R. L. Heinrikson, E. T. Krueger, and P. S. Keim, J. Biol. Chem. 252, 4913 (1977). 17 R. M. Kramer, J. A. Jakubowski, and D. Deykin, Biochim. Biophys. Acta 959, 269 (1988). t8 L. Loeb and R. Gross, J. Biol. Chem. 261, 10467 (1986).

[36] P u r i f i c a t i o n o f M a m m a l i a n N o n p a n c r e a t i c E x t r a c e l l u l a r Phospholipases A 2 B y SHUNTARO H A R A , HYEUN WOOK CHANG, KAZUHIKO HORIGOME,

ICHIRO KUDO, and KEIZO INOUE Introduction

High levels of extracellular phospholipase A 2 have been found at inflamed sites in some experimental animals and humans with diseases. It has been reported that various kinds of inflammatory cells, such as platelets, macrophages, and polymorphonuclear leukocytes, secrete phospholipase A 2 extracellularly on stimulation. We have purified and characterized METHODSIN ENZYMOLOGY,VOL. 197

Copyright© 1991by AcademicPress.Inc. All rightsof reproductionin any formreserved.

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PHOSPHOLIPASE A 2

TABLE I CHARACTERISTICSOF PURIFIED MAMMALIANPANCREATICAND NONPANCREATICEXTRACELLULARPHOSPHOLIPASESA 2 Phospholipase A2 Characteristic

Nonpancreatic °

Pancreatic b

Molecular weight Group (pattern of S---S bonds) Amino acid composition Affinity for heparin Substrate specificity c Optimum pH Ca 2+ requirement Stability at pH 4.0 Effect of p-bromophenacyl bromide Effect of deoxycholate

14K II Basic High PE > PC Alkaline Yes Yes Inhibition Inhibition

14K I Acidic Low PE = PC Alkaline Yes Yes Inhibition Stimulation

a From rat platelets. b From porcine pancreas. c PE, Phosphatidylethanolamine; PC, phosphatidylcholine.

phospholipases A2 found at inflamed sites in humans 1,2and rats, 3 and those secreted from rat 4,5 and rabbit 6 platelets. Our observations together with the results of Forst et al., who purified an extracellular phospholipase A2 from rabbit ascites fluid,7 indicate that these nonpancreatic extracellular phospholipases A 2 share common properties, such as molecular weight, optimum pH, Ca 2÷ requirement, substrate specificity, detergent sensitivity, stability at acidic pH, and heparin affinity (Table I). These enzymes, however, show different substrate speciI S. Hara, I. Kudo, H. W. Chang, K. Matsuta, T. Miyamoto, and K. Inoue, J. Biochem. (Tokyo) 105, 395 (1989). 2 S. Hara, I. Kudo, K. Matsuta, T. Miyamoto, and K. Inoue, J. Biochem. (Tokyo) 104, 326 (1988). 3 H. W. Chang, I. Kudo, M. Tomita, and K.'Inoue, J. Biochem. (Tokyo) 102, 147 (1987). 4 K. Horigome, M. Hayakawa, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 101, 625 (1987). 5 M. Hayakawa, K. Horigome, I. Kudo, M. Tomita, S. Nojima, and K. Inoue, J. Biochem. (Tokyo) 101, 1311 (1987). 6 H. Mizushima, I. Kudo, K. Horigome, M. Murakami, M. Hayakawa, D. K. Kim, E. Kondo, M. Tomita, and K. Inoue, J. Biochem. (Tokyo) 105, 520 (1989). 7 S. Forst, J. Weiss, P. Elsbach, J. M. Maraganore, I. Reardon, and R. L. Heinrikson, Biochemistry 25, 8381 (1986).

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ficities and detergent sensitivities from those of mammalian pancreatic phospholipase A2. Analysis of the entire amino acid sequence of phospholipase A 2 purified from rat platelets 8 indicates that the nonpancreatic enzyme shows approximately 30% homology with the pancreatic enzyme and contains some well conserved residues also present in the pancreatic enzyme. Remarkable structural features commonly observed in the nonpancreatic enzymes are a high content of basic amino acids and the absence of Cys-11, which is strictly conserved in the group I family of phospholipases A2 (those from mammalian pancreatic juice and from the venom of elapid and hydrophid snakes). The enzymes may therefore belong to the group II family, to which Crotalidae and Viperidae snake venom enzymes belong. In this chapter, we describe methods for purification of phospholipases A 2 from supernatants of rat activated platelets, 4 human synovial fluid, 1 and rat peritoneal fluid. 3

Purification of Extracellular Phospholipase A2 Released from Rat Platelets The most distinct property commonly observed in mammalian nonpancreatic extracellular phospholipases A2 is a high affinity for heparin. Heparin-Sepharose affinity chromatography is therefore a powerful tool for the purification of these enzymes. Extracellular phospholipase Az released from rat platelets is purified about 770-fold to near homogeneity by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW [gel-filtration high-performance liquid chromatography (HPLC)]. Lysophosphatidylserine-specific lysophospholipase, which is also released from rat platelets, is separated from phospholipase A2 by chromatography on heparin-Sepharose.

Reagents Heparin-Sepharose CL-6B (Pharmacia Fine Chemicals, Uppsala, Sweden) TSK gel G2000SW (Tosoh, Tokyo, Japan) Heparin-Sepharose buffer: 10 mM Tris-HC1 (pH 7.4) containing 0.15 or 1.5 M KCI HPLC buffer: 0.2 M Na2SO4, 10 mM acetic acid, pH 3.0 s M. Hayakawa, I. Kudo, M. Tomita, S. Nojima, and K. Inoue, J. Biochem. (Tokyo) 104, 767 (1988).

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[36] PURIFICATION OF NONPANCREATIC PHOSPHOLIPASES A 2 381 requiring 10 mM Ca 2+ for optimal activity. The PLA 2 shows a great preference for the E...
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