• Title, Summary, Keyword: 인지질분해효소

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Fatty Acid Composition of Different tissues of Spodoptera exigua Larvae and a Role of Cellular Phospholipase A2 (파밤나방 유충의 조직별 지방산 구성과 세포성 인지질분해효소의 역할)

  • Kim, Yonggyun;Lee, Seunghee;Seo, Seunghwan;Kim, Kunwoo
    • Korean journal of applied entomology
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    • v.55 no.2
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    • pp.129-138
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    • 2016
  • Eicosanoids are a group of C20 oxygenated polyunsaturated fatty acids (PUFAs). To monitor biosynthetic precursors of these PUFAs, this study extracted fatty acids from different tissues of the beet armyworm, Spodoptera exigua, and assessed their compositions using GC/MS. Fifth instar larvae were dissected to isolate different tissues of gut, fat body, hemocytes, and integument. From each tissue, total lipids were extracted and fractionated into neutral lipid (NL), glycolipid (GL), and phospholipid (PL). Most tissues contained palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3). However, their compositions were different among tissues and lipid types. Fat body and hemocytes possessed other type of fatty acids such as myristic acid (14:0) and three unknown fatty acids. Among lipid types, PL contained relatively high levels of linolenic acid than NL and GL, while it had lower saturated fatty acids. Total unsaturated fatty acid composition was varied among tissues and lipid types. PL was rich in unsaturated fatty acids in fat body, gut, and hemocytes. There was a significant influence of calcium-independent phospholipase $A_2$ ($iPLA_2$) on maintaining fatty acid composition because RNA interference of $iPLA_2$ expression significantly modified fatty acid compositions in NL and PL. However, this study did not detect arachidonic acid, a main eicosanoid biosynthesis precursor, in all tissues. This suggests an alternative biosynthesis of eicosanoids in insects, which is distinct from the biosynthetic pathway of mammals.

Effect of Cellular Phospholipase A2 Inhibition on Enhancement of Bt Insecticidal Activity (세포성 인지질분해효소 활성 억제에 따른 비티 살충력 증가 효과)

  • Eom, Seonghyeon;Park, Jiyeong;Kim, Kunwoo;Kim, Yonggyun
    • Korean journal of applied entomology
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    • v.53 no.3
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    • pp.271-280
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    • 2014
  • Some bacterial metabolites of Xenorhabdus nematophila (Xn) inhibit phospholipase $A_2$ ($PLA_2$) activity to shutdown eicosanoid biosynthesis in target insects. However, little has been known about the target insect $PLA_2$ of these bacterial metabolites. Eight bacterial metabolites identified in Xn culture broth exhibited significant insecticidal activities against larvae of both lepidopteran species of Plutella xylostella and Spodoptera exigua. Moreover, these bacterial metabolites significantly enhanced insecticidal activities of Bacillus thuringiensis (Bt). To determine target $PLA_2$, we cloned and over-expressed cellular $PLA_2$ ($SecPLA_2$) of S. exigua. Purified $SecPLA_2$ catalyzed phospholipids derived from the fat body and released several polyunsaturated fatty acids. Most Xn metabolites significantly inhibited $SecPLA_2$ activity, but were different in their inhibitory activities. There was a positive correlation between the inhibition of $SecPLA_2$ and the enhancement of Bt insecticidal activity. These results indicate that $SecPLA_2$ is a molecular target inhibited by Xn metabolite.

Hydrolysis of Phosphatidylcholine in Aerosol-OT/Isooctane Reversed Micelles by Phospholipase $A_2$ (역미셀계내에서 인지질분해효소 $A_2$에 의한 레시친의 가수분해)

  • Chang, Pahn-Shick
    • Korean Journal of Food Science and Technology
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    • v.29 no.1
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    • pp.26-31
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    • 1997
  • Bee venom (Apis mellifera) phospholipase $A_2$ solubilized in reversed micelles containing small amount of water stabilized by surfactant could catalyze the hydrolysis of dipalmitoyl phosphatidylcholine (DPPC). A sensitive and simple high performance liquid chromatographic (HPLC) methodology of phospholipase $A_2$ assay for the hydrolysis of DPPC was developed. Kinetic analysis of the phospholipase $A_2$-catalyzed reaction was found to be possible in reversed micelles. Among the surfactants and organic solvents tested, aerosol-OT and isooctane were most effective for the hydrolysis of DPPC in reversed micelles. Optimal temperature, optimal pH, $K_{m,app.},\;V_{max.,app.}$ and activation energy were determined to be $35{\sim}40^{\circ}C$, 7.0, 8.73 mM, 2.83 units/㎎ protein and 12.31 kcal/mole, respectively. The hydrolysis activity was dependent on water content and maximum activity was obtained at R value (=[water]/[aerosol-OT]) of 10.0.

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Phase Asymmetry Effect on Vesicle Fusion Induced by Phospholipase D (인지질분해효소D에 의해 유도된 소낭 융합에 대한 상 비대칭의 영향)

  • Park, Jin-Won
    • Korean Chemical Engineering Research
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    • v.53 no.6
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    • pp.672-676
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    • 2015
  • Spherical phospholipid bilayers, vesicles, were formed with respect to phase of each layer via a double emulsion technique. The conversion of phosphatidylcholine (PC) to phosphatidic acid (PA) at the outer layer, caused by phospholipase D (PLD), induced a curvature change in the vesicles, which eventually led them to fuse each other. The effect of the lipid layer physical-properties on the PLD-induced vesicle fusion was investigated using the fluorescence intensity change. 8-Aminonaphthalene-1,3,6-trisulfonic acid disodium salt(ANTS) and p-Xylene-bis(N-pyridinium bromide)(DPX) were encapsulated in the vesicles, respectively, for the quantification of the fusion. The fluorescence scale was calibrated with the fluorescence of a 1/1 mixture of ANTS and DPX vesicles in NaCl buffer taken as 100% fluorescence (0% fusion) and the vesicles containing both ANTS and DPX as 0% fluorescence (100% fusion), considering the leakage into the medium studied directly in a separate experiment using vesicles containing both ANTS and DPX. It was observed that the fusion occurred to the liquid-phase of the inner layer only. The fusion behaviors were very similar for both solid and liquid of the outer layer. However, the leakage was faster for the solid-phase outer-layer than the liquid-phase outer-layer. The difference in the leakage seems to be caused by the lipid concentration and the lateral diffusivity in the layer.

Role of Phospholipase $A_2$ on lipid peroxidation (과산화지질 형성에 있어서 Phospholipase $A_2$의 역할)

  • 황화신;정규찬;장현옥
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.341-341
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    • 1994
  • 생체막의 주요 구성성분인 인지질의 2번 위치에 결합한 불포화지방산은 각종 전이금속이나 각종 활성산소들의 공격을 쉽게 받아 지질과산화반응이 일어나서 생체에 유독한 화합물을 생성하게 된다. 생체는 이러한 기구의 해독을 위하여 크게 2가지 방어기전을 갖고 있다. 즉 Vitamin- C, $\alpha$-tocopherol, flavonoid, SOD, catalase 등과 같이 생성된 활성산소를 제거시키는 기구와. 활성산소에 의해 생성된 과산화물을 제거시키는 기구로 glutathione peroxidase (GPX)가 알려졌으며 GPX에 의해 독성이 낮은 수산화물까지 환원시키는 기구가 보고되었다. 그러나 인지질의 과산화물 그대로는 GPX의 기질이 쥘수 없으므로, 산화된 지방산을 절단하는 효소에 대한 기구의 해석이 요구되고 있다. 최근 여러질병에 관련되어 있는 인지질 2번위치의 지방산을 분해하는 phospholipase $A_2$ (PLA$_2$)가 과산화지질의 분해에 관여한다는 주장이 제기되었다. 따라서 본 연구에서는 rat liver microsome에 $CCl_4$투여로 일어나는 과산화반응에 있어서 PLA$_2$의 역할을 규명하기 위하여 본 실험을 행하였다.

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Cellular Localization and Translocation of Duplication and Alternative Splicing Variants of Olive Flounder Phospholipase C-δ1 (넙치 3가지 타입 인지질가수분해효소(PLC-δ1)의 세포 내 위치 및 이동)

  • Kim, Na Young;Kim, Moo-Sang;Jung, Sung Hee;Kim, Myoung Sug;Cho, Mi Young;Chung, oon Ki;Ahn, Sang Jung
    • Journal of Life Science
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    • v.27 no.11
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    • pp.1369-1375
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    • 2017
  • The purpose of this study was to investigate the cellular characterization of phospholipase C-${\delta}1$ in olive flounders (Paralichthys olivaceus). In general, phospholipase C signaling pathways are distributed in nuclei at plasma membranes and in cytoplasms, although the pathways' nuclear localization mechanisms are unclear. P. olivaceus duplicates type-A PoPLC-${\delta}1$ (PoPLC-${\delta}1A$), which has a high similarity to the human isoform PLC-${\delta}$; type-B PoPLC-${\delta}1$ (PoPLC-${\delta}1B$ [Sf]), which has a low similarity to the human isoform PLC-${\delta}$ and the alternative splice variant PoPLC-${\delta}1B$ (Lf), which has a nuclear localization signal (NLS) and a nuclear export signal (NES) for nuclear imports and exports, respectively. This study confirmed the effects of the cellular localization and translocation of GFP-tagged PoPLC-${\delta}1A$, PoPLC-${\delta}1B$ (Sf) and PoPLC-${\delta}1B$ (Lf). It administered treatments of $Ca^{2+}$ ionophore ionomycin and endoplasmic reticulum (ER)-$Ca^{2+}$ pump inhibitor thapsigargin to hirame natural-embryo (HINAE) cells. A laser-scanning confocal microscope was used. GFP-tagged PoPLC-${\delta}1A$ was distributed to the cellular organelles, rather than to the cytoplasms and cytomembranes, when PoPLC-${\delta}1B$ (Lf) and PoPLC-${\delta}1B$ (Sf) were localized at the plasma membranes. The treatments of ionomycin and thapsigargin showed the accumulation of PoPLC-${\delta}1A$ in the nuclei when PoPLC-${\delta}1B$ (Lf) nucleocytoplasmic shuttling and PoPLC-${\delta}1B$ (Sf) nucleocytoplasmic shuttling were not observed. The results were the first evidence that PoPLC-${\delta}1A$, which contains functional, intact NES sequences, has a main role in nucleocytoplasmic shuttling and translocation in fish.

Effect of Sodium deoxycholate and Sodium dodecy sulfate on Phospholipid Composition and Phospholiases of Rhizopus oryzae (Rhizopus oryzae의 인지질과 그 분해효소에 미치는 계면활성제의 영향)

  • 윤희주;조기승;최영길
    • Korean Journal of Microbiology
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    • v.24 no.1
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    • pp.38-45
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    • 1986
  • Effect of sodium deoxycholate and sodium dodecyl sulfate on Rhizopus oryzae were investigated. Morphological change was obtained by supplement of these surfactants into culture media during the sumerged culture. In accordance with morphological changes, composition of phospholipid was changed. In case of surfactant-free culture, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were measured more than 95% of total phospholipid. But cardiolipin and phosphatidylinositol were conspicuously increased by treatment of both sufactants. Presence of phospolipase A, C, and D were detected from mycelium. Phospholipase A and D were activated by supplement of sodium deoxycholate and C was activated by sodium dodecyl sulfate. These results were interpreted in respect of polymorphism of phospholipid and membrane stability against solubilization effect of surfactants.

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The role of CD14 and Toll-like receptors on the release of MMP-B in the LPS recognition pathway (지질 다당질 인지경로에서 기질금속단백분해효소-8 분비에 대한 CD14와 Toll-like receptors의 역할 연구)

  • Yang, Seung-Min;Kim, Tae-li;Seol, Yang-Jo;Lee, Yang-Moo;Ku, Young;Chung, Chong-Pyoung;Han, Soo-Boo;Rhyu, In-Chul
    • Journal of Periodontal and Implant Science
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    • v.36 no.3
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    • pp.579-590
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    • 2006
  • 1. 연구배경 교원질 분해작용을 하는 호중구의 세포질 효소인 기질금속단백분해효소-8은 치주질환, 류마티스 관절염, 그리고 궤양결장염과 같은 염증성 질환에서 농도가 증가한다고 알려져 있다. 최근에는 A. actinomycetemcomitans의 leukotoxin이 사람호중구에서 기질금속단백분해효소-8의 분비를 유도하는 것이 보고되었다. 이 연구의 목적은 선천면역 체계에서 세포표면 항원무리14, Toll-like 수용기, 그리고 $NF-{\kappa}$ B경로를 통하여 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비 여부와 세포기전을 알아보고자 하였다. 2. 연구재료 및 방법 건강한 개인 제공자(남자 13명, 여자 3명)로부터 얻은 개개인의 20ml 말초혈액을 제조사의 지침에 따라 호중구를 추출한 후 항세포표면 항원무리14와 함께 $4^{\circ}C$에서 30분간 전배양 한 후, $37^{\circ}C$에서 9시간 동안 배양시켰다. 추출한 호중구에 Toll-like 수용기 억제제 또는 $NF-{\kappa}$ B억제제인 TPCK를 첨가한 후 $37^{\circ}C$에서 1시간 동안 전배양하고 $37^{\circ}C$에서 9시간 동안 배양시켰다. 호중구에 세포뼈대 억제제인 cholchicine, nocodazole, demecolcine, 그리고 cytochalasin B를 A. actinomycetemcomitans의 지질다당질과 함께 $37^{\circ}C$에서 9시간 동안 배양시켰다. 기질금속단백분해효소-8 분비량은 효소면역측정법을 통해 결정하였다. 통계처리는 일원배치 분산분석법을 이용하였다(p<0.05). 3. 결과 A. actinomycetemcomitans 지질다당질은 기질금속단백분해효소-8의 분비를 증가시켰다. 기질금속단백분해효소-8의 분비는 항세포표면 항원무리14에 의해서 억제되었지만, 항 Toll-like 수용기2, 항 Toll-like 수용기4 항체는 억제시키지 못했다. $NF-{\kappa}$ B 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 $NF-{\kappa}$ B 결합 활성도와 기질금속단백분해효소-8 분비를 억제하였다. 미세섬유 중합반응 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비를 억제시켰으나, 미세관 중합반응억제제는 억제시키지 못했다. 4. 결론 위의 연구결과를 종합하여 볼 때, 기질금속단백분해효소-8은 A. actinomycetemcomitans의 지질다당질로 유도되며, 세포표면 항원무리-$NF-{\kappa}$ B 경로를 통하여 분비되고, 이 분비 과정은 미세섬유 계통이 관여하는 것으로 보인다.

The Functional Role of Phospholipase D Isozymes in Apoptosis (세포사멸에서 Phospholipase D 동위효소의 기능적 역할)

  • Min, Do Sik
    • Journal of Life Science
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    • v.24 no.12
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    • pp.1378-1382
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    • 2014
  • Phospholipase D (PLD) catalyzes the hydrolysis of phospholipid to phosphatidic acid (PA), a lipid secondary messenger. Two forms of PLD isozymes, phosphatidylcholine-specific PLD1 and PLD2, have been identified. PLD has emerged as a critical regulator of cell proliferation and survival signaling, and dysregulation of PLD occurs in a various illnesses, including cancer. PLD activity is essential for cell survival and protection from apoptosis. Overexpression of PLD isozymes or PLD-generated PA attenuates the expression of apoptotic genes and confers resistance to apoptosis. The apoptosis-related molecular mechanisms of PLD remain largely unknown. Recently, the dynamics of PLD turnover during apoptosis have been reported. The cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site, and PLD2 is cleaved two sites at the front of the N-terminus. The cleavage of PLD1 reduces its enzymatic activity, probably via the dissociation of two catalytic motifs, whereas the cleavage of PLD2 does not affect the catalytic motifs and its activity. Thus, PLD2 maintains antiapoptotic capacity, despite its cleavage. Therefore, the differential cleavage pattern of PLD isozymes by caspase affects its enzymatic activity and antiapoptotic function. Thus, PLD is considered a potential target for cancer therapy. We summarize recent studies regarding the functional role of PLD in apoptosis.

Reactivity of Phospholipase D toward Phosphatidylcholines with Different Length of Acyl Chains (길이가 틀린 아실사슬을 갖는 콜린 인지질에 대한 포스포리파제 D의 반응성)

  • Koh, Eun-Hie;Park, Insook
    • Journal of the Korean Chemical Society
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    • v.40 no.9
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    • pp.630-634
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    • 1996
  • In order to explore a substrate specificity for cabbage phospholipase D, we examined the PLD reactivity toward the phosphatidylcholines with different chain length of acyl groups. The selected acyl chains were the saturated fatty acid of $C_8:0,\;C_{12}:0,\;C_{16}:0,\;C_{20}:0$. The reactivity of these phospholipids were dependent largely on the ratio of PC : SDS. The PC : SDS ratio showing the optimal PLD activity were found to be 1:1.4, 1:2.2, 1:2.5, and 1:3.6 respectively as the increase of the acyl chain length. Likewise the optimum temperature for the maximal PLD activity were altered markedly to 25$^{\circ}C$, 30$^{\circ}C$, 35$^{\circ}C$, 45$^{\circ}C$ when the length of acyl chains increased. On the contrary the pH and concentration of $Ca^{2+}$ necessary for the optimum PLD activity were not altered significantly. The kinetic parameter $V_{max}$ for short acyl chain substrate was greater than the values for the longer acyl chain, which indicates the fastest rate of hydrolysis. By the same token, the reactivity of longer chain substrate became slower for the hydrolysis activity.

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