• 약학회지
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• 제45권4호
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• pp.413-418
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• 2001

To investigate action of ATP on ischemia-induced brain injury, we measured phospholipase $A_2$activity and nitric oxide (NO) production in C6 cells. ATP alone did not have any influence on phospholipase $A_2$activity but increased NO production. Glutamate (1 mM) significantly increased phospholipase $A_2$activity whereas did not increased NO production. ATP significantly inhibited phospholipase $A_2$activation induced by 0.1 $\mu$M A23187, 1 mM glutamate and 1 mM $H_2O$$_2$, but did not inhibited 1 $\mu$M PMA-induced phospholipase $A_2$activation. From the above results, it is suggested that the action of ATP in C6 cells has dual actions, such as the inhibition of agonist-induced phospholipase $A_2$activation and the increase of NO production.

• 대한약침학회지
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• 제4권2호
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• pp.5-15
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• 2001

This study was carried out for production of neutral antibody to bee venom $(anti-phospholipase\;A_2IgY)$. Hen layings were injected repeatedly with bee venom and phospholipase $A_2$ with Freund's adjuvant. Specific antibody in egg yolk from immunized hen laying was separated, and purified, also immunological characteristics of anti phospholipase $A_2\;IgY$ was invested. The results were summarized as follows: 1. Phospholipase $A_2$ was showed single band at molecular weight 17,000 in SDS-PAGE and bee venom was showed two band at molecular weight 17,000 and under molecular weight 6,500 in SDS-PAGE. 2. During 70 days after hen immunized with bee venom and phospholipase $A_2$, antibodies(anti-bee venom IgY) to bee venom were showed poor ELISA value in egg yolk, but antibodies$(anti-Phospholipase\;A_2IgY)$ to phospholipase $A_2$ in egg yolk were increased ELISA value from 8 days or 15 days and found maximum ELISA value at 42 days. Also after booster at 49 days, ELISA value of anti Phospholipase $A_2\;IgY$ in egg yolk was supported at optical density(O.D) 1.0 level, continuously. 3. Titer of phospholipase $A_2\;IgY$ was showed 1: 32,000. 4. In double immunodiffusion test to phospholipase $A_2$ after double dilution of anti-phospholipase $A_2\;IgY$, only precipitation line was made in 1:1 dilution well of anti-Phospholipase $A_2\;IgY$. But In immunodiffusion test to anti-phospholipase $A_2\;IgY$ after double dilution of phospholipase $A_2$, Precipitation line to 250ul/ml well of phospholipase $A_2$ was showed. In double immunodiffusion test to bee venom(1mg/ml) after double dilution anti-phospholipase $A_2\;IgY$, all well without 1:32 dilution well were showed strong precipitation line. 5. In dot bloting test to anti-phospholipase $A_2\;IgY$ after diluting bee venom(0.5mg/ml), dot bloting color was showed clearly to $1/100(5{\mu}g/ml)$ in bee venom.

• 약학회지
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• 제41권2호
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• pp.212-219
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• 1997

Multiple forms of extracellular phospholipase $A_2$ have been detected in human amniotic fluid (HAF). When HAF was subjected to heparin-Sepharose column chromatography, phospholipase $A_2$ activity was detected in both heparin-non binding and binding fraction. The activity of heparin-non binding fraction was further purified by sequential uses of column chromatographies on butyl-Toy-opearl 650M and DEAE-Sephacel. DEAE-Sephacel fraction contained three different phospholipase $A_2$ activities (Peak I, II, III). The molecular weight of DEAE-Sephacel fraction phospholipase $A_2$ determined by SDS-PAGE were about 52KDa (Peak I). Peak II, III required micromolar $Ca^{2+}$ ion for its maximum activity, but Peak I enzyme showed calcium independent phospholipase $A_2$ activity and showed broad range of pH (6.0~10.0) optimum. All these enzymes were not recognized by a monoclonal antibody raised against phospholipase $A_2$ from human synovial fluid. These results suggest that HAF might contain multiple forms of extracellular phospholipase $A_2$, which may neither belong to the 14KDa group II phospholipase $A_2$ family nor cytosolic phospholipase $A_2$.

• 한국식품과학회지
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• 제29권1호
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• pp.26-31
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• 1997

물을 극미량 함유하는 미수계 효소반응계인 aerosol-OT/이소옥탄으로 구성된 역미셀계내에서 레시친의 일종인 DPPC (dipalmitoyl phosphatidylcholine)의 가수분해반응을 phospholipase $A_2$가 촉매할 수 있었다. 또한 phospholipase $A_2$에 의한 가수분해반응을 정량분석하기 위한 고성능 액체 크로마토그래피에 의한 예민하고 간편한 방법을 고안하였는 바, 이에 의하여 aerosol-OT/이소옥탄 역미셀계내에서 phospholipase $A_2$에 의한 가수분해반응의 동력학적인 분석이 가능하였다. 한편, 여러가지 유화제와 유기용매 중 aerosol-OT와 이소옥탄일 때 효소반응이 가장 효과적이었으며, 반응 적정온도는 $35{\sim}40^{\circ}C$, 적정 pH는 7.0이었다. 또한 역미셀계내에서의 가수분해반응은 수분 함량변화에 민감하였으며, R-값(=[water]/[aerosol-OT])이 10.0일 때 가장 높은 효소활성을 나타내었다. 효소반응을 위한 최적조건하에서의 $K_{m,app.}$ 및 $V_{max.,app.}$는 각각 8.73 mM, 2.83 units/㎎ protein이었으며, 역미셀계내에서 phospholipase $A_2$에 의한 DPPC가수분해반응의 활성화에너지는 12.31 kcal/mole로 산출되었다.

• 약학회지
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• 제45권3호
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• pp.287-292
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• 2001

To investigate whether phospholipase $A_2$pathway is involved in histamine release of rat peritoneal mast cells, we measured histamine release in the presence of various enzyme inhibitors involved in eicosanoid pathway, such as phospholipase $A_2$, cyclooxygenase and lipoxygenase. Phospholipase $A_2$inhibitors, manoalide and OPC, significantly inhibited histamine release induced by 100 $\mu$M ATP and 1$\mu$g/ml compound 48/80. Cyclooxygenase inhibitors, ibuprofen and indomethacin, significantly inhibited ATP-induced histamine release and lipoxygenase inhibitors, baicalein and caffeic acid, also significantly inhibited. To investigate the involvement of protein kinase in ATP- and compound 48/80-induced histamine release, we observed effects of protein kinase inhibitors on histamine release. Bisindolmaleimide (protein kinase C antagonist) dose-dependently inhibited both ATP and compound 48/80-induced histamine release. Tyrosine kinase inhibitors (methyl 2,5-dihydroxy cinnamate and genistein) dose-dependently inhibited ATP and compound 48/80-induced histamine release. Protein kinase C and tyrosine kinase seem to be involved in histamine release induced by ATP and compound 48/80. These results suggest that phospholipase $A_2$pathway as well as protein kinase C and tyrosine kinase are involved in histamine release of rat peritoneal mast cells by ATP and compound 48/80.

• 대한약리학회지
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• 제27권2호
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• pp.191-196
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• 1991

본 연구에서는 흰쥐의 수정란 착상시기에 estradiol이 prostaglandins(PGs) 합성의 전구체인 arachidonic acid를 생성하는데 관여하는 phospholipase $A_2(PLA_2)$의 활성도를 조절하므로써 PGs의 합성을 촉진하는가를 조사하여 다음과 같은 결과를 얻었다. 자궁의 $PLA_2$ 활성도는 수정란이 착상하는 시기인 임신 제 5일에 증가되었으며, 비착상부위에서보다는 착상부위에서 더 높은 것으로 나타났다. Delayed implantation model에서, $PLA_2$ 활성도는 estradiol을 투여한 지 11시간후에 증가되었으며, dbcAMP를 투여한지 8시간후에 증가되었다. 또한 estradiol을 투여하기 2시간전에 phosphodiesterase inhibitor인 theophylline을 투여하면 estradiol만 투여한것에 비하여 $PLA_2$ 활성도가 증가되었다. Estradiol 또는 dbcAMP와 함께 indomethacin을 투여하면 자궁의 PGs합성은 억제되었으나 $PLA_2$ 활성도에는 영향을 주지않았다. 이상의 결과로 보아 흰쥐의 착상시기에 estradiol은 cAMP를 매개로하여 자궁의 $PLA_2$ 활성도를 촉진하므로써 PGs의 합성을 증가시키는 것으로 생각된다.

• BMB Reports
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• 제30권2호
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• pp.101-105
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• 1997

Extracellular phospholipase $A_2$ activity has been detected in urine of patients with acute pyelonephritis (APN). This enzyme required micromolar $Ca^{2+}$ ion for its maximum activity and showed a broad range of pH (4.5~10) optimum. Urine enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). $PLA_2$ activity in the urine of patients with APN was about 5-fold higher than that of healthy individuals. When urine was subjected to heparinSepharose column chromatography, phospholipase $A_2$ activity was detected in both heparin-non-binding and binding fractions. Both phospholipase $A_2$ activities were sensitive less than a micromolar calcium concentration and did not react with anti-human 14-kDa group II phospholipase $A_2$ monoclonal antibody, HP-l. These findings suggest that two kinds of novel extracellular phospholipase $A_2$. which may not belong to the 14-kDa group II phospholipase $A_2$ family, exist in the urine of patients with APN.

• 한국식품영양과학회지
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• 제26권5호
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• pp.908-913
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• 1997

The purpose of this study was to investigate phospholipase $A_{2}$ activity and lipid peroxidation I streptozotocin induced diabetic rats. Sprague-Dawley male rats weighting-Dawley male rats weighting 300$\pm$10gm were randomly assigned to normal and STZ-induced diabetic group. Diabetes was induced by intravenous injection of 55mg/kg of STZ in sodium citrate buffer(pH 4.3). Animals were sacrificed at the 6th day of diabetic states. Body weight gains were lower in DM group. Phosphatidylcholine hydrolysis in liver was not significantly different between two groups, whereas phosphatidylethanolamine hydrolysis in liver was increased by 69% in DM group comparing with that of normal group. Liver microsomal phospholipase $A_{2}$ activity and level of TBARS was increased by 91%, 109% in DM group compared with that of normal group, respectively. The present results indicate that phospholipase $A_{2}$ activity is specific to PE hydrolysis, leading to lipid peroxidation process in STZ induced diabetic rats.

• 약학회지
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• 제49권5호
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• pp.405-409
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• 2005

To investigate anti-inflammatory activity of Noni extracts, we measured the phospholipase $A_2$ activity using both in vitro and in vivo system. Water soluble fraction of Noni extracts did not affect melittin-induced arachidonic acid release, whereas lipid soluble fraction inhibited it in a dose dependent manner in Raw 264.7 cells. The purified phos­pholipase $A_2$ activity was dose-dependently inhibited by lipid soluble fraction of Noni extracts but not by its water soluble fraction. Lipid peroxidation, myeloperoxidase and phospholipase $A_2$ activity in incised skin of mice were significantly increased as compared with those in non-incised skin, and these increase was attenuated by the treatment with Noni pow­der. Our data suggest that Noni extracts has anti-inflammatory activity, and this is, in part, caused by inhibitory activity of phospholipase $A_2$.

• Parasites, Hosts and Diseases
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• 제49권1호
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• pp.1-8
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• 2011

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase $A_2$ (PLA$_2$). and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA$_2$. Acanthamoeba exhibited optimal phospholipase activities at $37^{\circ}C$ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA$_2$-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.