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Selective Release of Phospholipase A2 and Lysophosphatidylserine

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Selective Release of Phospholipase A2 and Lysophosphatidylserine J. Biochem. 101, 53-61 (1987) Selective Release of Phospholipase A2 and Lysophosphatidylserine - Specific Lysophospholipase from Rat Platelets1 Kazuhiko HORIGOME, Makio HAYAKAWA, Keizo INOUE ,2 and Shoshichi NOJIMA3 Department of Health Chemistry, Faculty of Pharmaceutical Sciences , The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, July 11, 1986 Rat platelets released phospholipase A2 and lysophospholipase upon activation with thrombin or ADP. The release of phospholipases was energy-dependent and was not in parallel with that of a known lysosomal marker enzyme, N-acetyl-f-o glucosaminidase. The phospholipases are derived from other granules (dense granules or a-granules) rather than lysosomal granules of the cells. All of the activities of both phospholipases in the cell free fraction obtained from the activated platelet reaction mixture was recovered in the supernatant after centrifugation at 105,000x g. The degree of hydrolysis of phospholipids by the phospholipase A2 followed the order: phosphatidylethanolamine (PE) > phosphatidylserine (PS) > phosphatidylcholine (PC). Phospholipase A2 shows a broad pH optimum (> pH 7.0) and absolutely requires Ca 2+. Lysophospholipase was specific to lysophos phatidylserine (lysoPS), and neither lysophosphatidylethanolamine (lysoPE) nor lysophosphatidylcholine (IysoPC) was hydrolyzed appreciably. Both 1-acyl and 2-acyl-lysophosphatidylserine were equally hydrolyzed. Lysophospholipase activity shows similar pH optimum to phospholipase A2. The lysophospholipase activity was lost easily at 60°C. The activity was reduced by the presence of EDTA, though low but distinct activity was observed even in the presence of EDTA. Addition of Cat} to the mixtures restores the full activity. Normal serum and plasma from man and several acyl position of a number of phospholipid sub animals contain phospholipid deacylating activity strates, was attributed to post heparin lipoprotein (1-8). While phospholipase A1, acting on the 1- lipase (1-3), serum and plasma also contain phos- 1 This work was supported in part by Grant-in-Aid for Scientific Research (No . 59122004) from the Ministry of Education, Science and Culture of Japan. 2 To whom all correspondence should be addressed . 3 Present address: Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko-machi, Tsukui-gun, Kana gawa 199-01, Japan. Abbreviations: lysoPS, lysophosphatidylserine; lysoPE, lysophosphatidylethanolamine; lysoPC, lysophosphatidyl choline; PS, phosphatidylserine; PE, phosphatidylethanolamine: PC, phosphatidylcholine; EDTA, ethylene diaminetetraacetic acid; PAF (platelet activating factor), 1-O-alkyl-2-acetyl-sn-3-glycerophosphocholine. Vol. 101. No. 1, 1987 53 54 K. HORIGOME, M. HAYAKAWA, K. INOUE, and S. NOJIMA pholipase A2 activity (4-8). Though the origin labeled phospholipids by purified snake venom and function of phospholipase A2 have not been phospholipase A2 (Naja naja) revealed that over established, it has been thought that phospholipase 98 % of the radioactivity in 1-acyl-2-[I-14C]linoleoyl A2 always circulates in the blood and participates glycerophospholipids was in position 2 and 95 in the turnover of plasma phospholipids, in immu of the radioactivity in 1-[1-14C]palmitoyl-2-acyl nologically mediated membrane damage, or in the glycerophospholipids was in position 1. The spe degradation of bacterial envelope phospholipids cific activity of labeled phospholipids were adjusted during killing of bacteria (6, 7). Smith and Silver to 2,000 dpm/nmol by dilution with non-labeled (9) previously reported the release of lysosomal egg yolk PC, PE prepared from egg yolk PC by acidic phospholipase A, from human platelet upon transphosphatidylation, or bovine brain PS. I stimulation. The activity could only be detected [14C]Palmitoyl-lysophospholipids were prepared by in the presence of detergent. Vadas and Hay (10) treatment of 1-[14C]palmitoyl-2-acylphospholipids reported the release of phospholipase A2 from with phospholipase A2 from snake venom (habu, spontaneously aggregated sheep platelets. Release Trimeresurus flavoviridis). 2-[14C]Linoleoyl lysoPS of phospholipase A2 from rabbit peritoneal neu was prepared from 1-acyl-2-[14C]linoleoyl PS by trophils (11) and mouse peritoneal macrophages treatment with Rhizopus delemer lipase, as already (12) stimulated by zymosan were also reported. described (16). In this paper, we found that rat platelets Platelet Activation-Bloods were taken by released phospholipase A2 upon stimulation. We cardiac puncture from ether-anaesthetized male also found that lysophospholipase specific to Wistar rats and mixed with 3.8 % (w/v) sodium lysoPS together with phospholipase A2, both of citrate in plastic syringes in a ratio of 1 part to 9 which were of a-granule origin, were released when parts of blood. The blood was centrifuged for 10 platelets were stimulated. min at 270x g to prepare platelet-rich plasma. 5-Hydroxy[side-chain-2-14C]tryptamine creatinin MATERIALS AND METHODS sulfate (serotonin, 0.1 yCi/ml) was added to label the content of the dense granules, and the incu Chemicals and Enzymes-[1-14C]Palmitic acid, bation was performed for 30 min at 22?C. After [1-14C]linoleic acid, and 5-hydroxy [side-chain-2 the addition of 1/5 (v/v) of 65 mM citric acid and 14C]tryptamine creatinin sulfate were purchased 85 mM sodium citrate in 2Y. (w/v) glucose (ACD from the Radiochemical Center, Amersham, U.K. solution), platelets were separated from plasma by Purified phospholipase A2 of Naja naja, thrombin, centrifugation at 1,500 x g for 10 min. Cells were ADP, and p-nitrophenyl-N-acetyl-fl-n-glucosami resuspended in 5 parts HEPES-buffered medium nide were purchased from Sigma Chemical Com (137 mM NaCl, 2.7 mM KCI, 10 mM HEPES, pany, St. Louis, Mo., U.S.A. A23187 was obtained 0.1 % (w/v) glucose) and 1 part ACD solution, and from Calbiochem-Behring, U.S.A., and NADH washed once more. Washed platelets were finally from Oriental Yeast Co., Tokyo. Rhyzopus de resuspended in HEPES-buffered medium. lemer lipase (pure grade, 6,000 units/mg) was from The procedure for measuring the release of Seikagaku Kogyo, Tokyo. All other reagents phospholipases was essentially the same as de used were of analytical grade. scribed previously for the release of platelet gran Preparation of Substrates--I -[1-14C]Palmitoyl ular proteins (17). Aliquots of the suspension of 2-acyl-glycerophosphocholine and1-acyl-2-[114C] platelets (1 •~ 109 cells/ml) in HEPES-buffered me linoleoyl-glycerophosphocholine were prepared by dium containing 2 mM CaCl2 were incubated with the method of Lands (13) with a slight modifica the indicated concentration of stimuli (thrombin, tion (14). Both PS and PE were prepared from ADP, A23187, or platelet activating factor (PAF)) the above described labeled PC by transphospha at 37?C. The incubation was terminated after tidylation catalyzed by phospholipase D according cooling in an ice bath, and cells were removed by to the methods of Confurius and Zwaal (15) with centrifugation at 1,200 •~ g for 2 min. The super a slight modification. All labeled substrate thus natant was removed, and aliquots were taken for prepared showed a single spot on silica gel thin measurement of the enzyme activity and [14C] layer plates (Merck, 5724). Hydrolysis of the - serotonin. J. Biochem. PHOSPHOLIPASE RELEASE FROM PLATELETS 55 Assay of Released Enzymes-Lactate dehydro genase and N-acetyl-ƒÀ-D-glucosaminidase were measured by the method of Lowry et al. (18) and that of Day et al. (19) respectively. The results of all assays were expressed as a percentage of the total activity measured in the presence of Triton X-100 at a final concentration of 0.17 % (w/v). Phospholipase activity was measured using phospholipids, of which either the sn-1 or sn-2 position was selectively labeled as substrate. The reaction mixture contained 100 mM Tris-HCI (pH 7.4), 4 mM CaCl2, and 401eM sonicated sub strates. After incubation with the enzyme, the Fig. 1. Release of phospholipase activity from platelets reaction was terminated by adding methanol, and upon stimulation with thrombin. Platelets (1 •~ 109 all lipids were extracted by the methods of Bligh cells/ml) were incubated in the presence of varying con and Dyer (20) and analyzed on thin-layer chro centrations of thrombin at 37?C for 5 min. Aliquots of matography plates as described previously (14). the supernatant obtained by centrifugation of the The radioactive spots corresponding to fatty acid, reaction mixture were examined for enzyme release. lysophospholipid, and phospholipid were scraped Phospholipase A2 (0), [14C]serotonin (0), N-acetyl-ƒÀ off from the plate into vials and counted in a glucosaminidase (U), and lactate dehydrogenase (0) Packard Liquid Scintillation Counter Spectrom release were measured as described in " MATERIALS eter. Phospholipase A2 assay were mainly per AND METHODS." Points are means of duplicate formed using 1-acyl-2-[14C]linoleoyl PS as substrate determinations. in a total volume of 0.25 ml. Other conditions were the same as described above. As phospho unit/ml. A maximum of about 80% of total lipase Al activity was poor in rat platelets, phos cellular phospholipase (activity detected after soni pholipase A2 activity was measured in some exper cation) was released. The release of phospholipase iments by the methods already described (14). The activity almost paralleled the release of serotonin. reaction was stopped by adding 1.25 ml of Dole's N-Acetyl-(l-D-glucosaminidase, a lysosomal en reagent (21) and the product, free
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