Abstract
Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.
Highlights
Lysophosphatidic acid (LPA)1 is a key intermediate for de novo synthesis of phospholipids and triacylglycerol
Rat testis contains a phospholipase A1 (PLA1) activity toward PA (9), which was co-purified with Phospholipase A2 (PLA2) at earlier steps of purification but was not detectable after a SuperQ Toyopearl chromatography presumably because of its nonspecific adsorption onto the column
Purified enzyme showed a single band on a silver-stained gel (Fig. 1), ratios of the specific activity of PLA2 to lipase were constant at the final purification steps (Table I), and the two activities co-migrated on native gels required the presence of DTT in the assay mixtures and were inhibited by Methylarachidonyl fluorophosphonate (MAFP) and APMSF (Table II), suggesting that a single enzyme catalyzes PLA2 and monoacylglycerol lipase activity
Summary
Materials—The following glycerolipids were obtained from Avanti Polar Lipids, Inc.: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoylsn-glycero-3-phosphoinositol (POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoglycerol (POPG), 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphocholine (PAPC), 1-O-hexadecyl-2-oleoyl-sn-glycero-3phosphocholine (1-O-hexadecyl-OPC), LPC, lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). The assay mixtures contained 10 mM EDTA, substrate micelles, or emulsion (10-l stock solution), 0.1 M NaCl, 0.1 M Tris-HCl (pH 8.5 for lipase activity), or MES (pH 5.5 for PLA2 and LBPA activity), 6 mM DTT, and the enzyme sample in a final volume of 50 l. The assay mixtures contained cholesterol ester emulsion (10 l), either 10 mM DOC or 30 mM Triton X-100, 6 mM DTT, and 0.1 M NaCl, either 0.1 M Tris-HCl (pH 8.5) or MES (pH 5.5), and enzyme sample. The active PLA2 pools were purified further by HPLC on a Super SW 3000 column (4.6 ϫ 300 mm; Tosoh Corporation) pre-equilibrated with 20 mM Tris-HCl (pH 7.0) containing 300 mM NaCl, 0.1% C12E8, and 1 mM DTT at 0.1 ml/min. The PLA2 and monoacylglycerol lipase activities were determined as described under “Experimental Procedures” using 1 mM POPA/6 mM taurocholate and 1 mM monooleoylglycerol/6 mM taurocholate mixed micelles as substrates
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