• Title, Summary, Keyword: Phospholipase $A_2$

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Characterization of Chinese Cabbage Phospholipase D by a Multistirring Batch System Bioreactor (다중 교반형 생물반응기에 의한 배추 Phospholipase D의 특성연구)

  • 박동훈;정의호이해익이상영
    • KSBB Journal
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    • v.5 no.2
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    • pp.119-124
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    • 1990
  • Phospholipase D catalyzes the phosphatidohydrolysis and transphosphatidylation of phospholipid in the biological systems. In this study we were partially purified phospholipase D from Chinese cabbage and the characterization of the enzyme was carried out in a multistirring batch system bioreactor. The enzyme showed optimum activity at pH ,5.6, highest activity at 37$^{\circ}C$ and Ca2+ is important for the enzyme activity. Optimum concentrations of Ca2+ for phosphatidohydrolysis was 20 mM and for transphosphatidylation was 40 mM, respectively. Some organic solvents such as diethylether, isopropylether and butylacetate were activated the enzyme activity. On the other hand, EDTA, Ba2+, Mn2+ and Zn2+ showed inhibitory effect on the enzyme activity. The base acceptors in transphosphatidylation by the Chinese cabbage phospholipase D were tested. Various poly-and monohydroxy alcohols were found to be active.

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Production and Characterization of Extracellular Phospholipase D from Streptomyces sp. YU100

  • Lim, Si-Kyu;Choi, Jae-Woong;Chung, Min-Ho;Lee, Eun-Tae;Khang, Yong-Ho;Kim, Sang-Dal;Nam, Doo-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.12 no.2
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    • pp.189-195
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    • 2002
  • Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and calcium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at $28^{\circ}C$ for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate precipitation and DEAE-Sepharose CL-6B column chromatography, which produced a major band of 57 kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reaction temperature was elevated to $60^{\circ}C$. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as emulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.

Functional Properties of Enzymatically Modified Egg Yolk Powder Produced by Phospholipase $A_2$ Treatment

  • Kim, Mi-Ra;Shim, Jae-Yong;Park, Ki-Hwan;Imm, Jee-Young
    • Food Science and Biotechnology
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    • v.17 no.6
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    • pp.1289-1293
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    • 2008
  • Fresh egg yolk (EY) was enzymatically modified using phospholipase $A_2$ ($PLA_2$) to produce an enzymatically modified-egg yolk powder (EM-EYP). The EM-EYP offered significantly higher emulsifying activity, emulsion stability, protein solubility, and mayonnaise stability than the control EYP. By employing $PLA_2$ in the enzymatic modification process, structural changes occurred in the phospholipids and lipoproteins of the yolk, and cleavage of apo-high density lipoprotein (HDL) components (Mw 105 kDa) was detected by sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE). Based on its functional properties, EM-EYP has great potential as a replacement for fresh EY in the production of processed food products such as mayonnaise.

Production of Intracellular Calcium Oscillation by Phospholipase C Zeta Activation in Mammalian Eggs

  • Yoon, Sook-Young;Kang, Da-Won
    • Development and Reproduction
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    • v.15 no.3
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    • pp.197-204
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    • 2011
  • Egg activation is a crucial step that initiates embryo development upon breaking the meiotic arrest. In mammalian, egg activation is accomplished by fusion with sperm, which induces the repeated intracellular $Ca^{2+}$- increases ($[Ca^{2+}]_i$ oscillation). Researches in mammals support the view of the $[Ca^{2+}]_i$ oscillation and egg activation is triggered by a protein factor from sperm that causes $[Ca^{2+}]_i$ release from endoplasmic reticulum, intracellular $[Ca^{2+}]_i$ store, by persistently activation of phosphoinositide pathway. It represents that the sperm factor generates production of inositol trisphosphate ($IP_3$). Recently a sperm specific form of phospholipase C zeta, referred to as PLCZ was identified. In this paper, we confer the evidence that PLCZ represent the sperm factor that induces $[Ca^{2+}]_i$ oscillation and egg activation and discuss the correlation of PLCZ and infertility.

Role of Diacyl Glycerol (DAG) in Caprine Sperm Acrosomal Exocytosis Induced by Progesterone

  • Somanath, P.R.;Gandhi, K.K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.8
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    • pp.1091-1097
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    • 2002
  • Capacitated goat spermatozoa generated diacyl glycerol (DAG) when suspended in Krebs-Ringer bicarbonate medium and induced by progesterone or $Ca^{2+}$ ionophore A23187. We have added Sn-1-oleoyl-2-acetyl glycerol externally, to study the effect of DAG in goat sperm acrosomal exocytosis. Addition of neomycin abolished the DAG generating capacity of progesterone in a dose dependent manner, suggesting the involvement of a phosphoinositidase C activated phospholipase C system in the process. The level of increase in phosphatidic acid was considerably low and was produced well after the DAG generation thereby suggesting the involvement of a DAG kinase which phosphorylates DAG to produce PA. The inhibition of progesterone mediated effect by inhibitors of $GABA_A/Cl^{-}$ channel and $Ca^{2+}$ channels further supports the evidence that the events of binding of agonist to the receptor(s), opening of $Ca^{2+}$ channels and the activation of phospholipase C are reconciled to perform the function of acrosome reaction in capacitated goat spermatozoa.

Homogeneity of Phospholipase C of Bovine Uterus and Seminal Vesicle Compared with Brain Isozymes (소의 자궁 및 고환에서 Phospholipase C의 분리 및 뇌 Isozyme과의 비교 연구)

  • Kim, Jung-Hye;Rhee, Sue-Goo;Lee, Ki-Yung
    • Yeungnam University Journal of Medicine
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    • v.5 no.2
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    • pp.37-45
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    • 1988
  • Phosphoinositide-specific phospholipase C(PI-PLC) is a second messenger of signal transducer on cell membrane. In the previous study, PLC of bovine brain has been purified three isozymes. In this paper, uterus and seminal vesicle have been purified. Two peaks of PI-PLC activity were resolved when bovine uterus and seminal vesicle proteins were chromatographed on a DEAE and phenyl TSK 5PW HPLC column. Each two peak was compared with PI-PLC I, IT and ill from bovine brain and we got the retension time on HPLC. The peak fractions with PLC activity were tested homogeneity with brain PLC monoclonal antibodies(Mab). Mab-labeled affigels were bounded in the range of 73.8%~97.5% with PLC I, IT and III. Homogeneity of fractions were revealed that DEAE F-1 and phenyl F-1-I were highest level of PLC III in uterus and seminal vesicle and DEAE F-2 and phenyl F-2-I were mixed PLC I and II.

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Loss of phospholipase D2 impairs VEGF-induced angiogenesis

  • Lee, Chang Sup;Ghim, Jaewang;Song, Parkyong;Suh, Pann-Ghill;Ryu, Sung Ho
    • BMB Reports
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    • v.49 no.3
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    • pp.191-196
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    • 2016
  • Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and critical for normal embryonic development and repair of pathophysiological conditions in adults. Although phospholipase D (PLD) activity has been implicated in angiogenic processes, its role in VEGF signaling during angiogenesis in mammals is unclear. Here, we found that silencing of PLD2 by siRNA blocked VEGF-mediated signaling in immortalized human umbilical vein endothelial cells (iHUVECs). Also, VEGF-induced endothelial cell survival, proliferation, migration, and tube formation were inhibited by PLD2 silencing. Furthermore, while Pld2-knockout mice exhibited normal development, loss of PLD2 inhibited VEGF-mediated ex vivo angiogenesis. These findings suggest that PLD2 functions as a key mediator in the VEGF-mediated angiogenic functions of endothelial cells.

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.

Phospholipase $A_2$-Catalyzed Transesterification of Phosphatidylcholine with Nervonic Acid in Organic Solvent

  • Park, Chang-Won;Park, Ki-Won;Han, Jeong-Jun;Chung, Guk-Hoon;Rhee, Joon-Shick
    • Journal of Microbiology and Biotechnology
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    • v.10 no.5
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    • pp.721-723
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    • 2000
  • The phospholipase $A_2$-catalyzed transesterification of phosphatidylcholine (PC, 95%) with nervonic acid (NA, 95%) was successfully carried out in an organic solvent. The maximum yield after 48 h was 10.3% (w/w) at $50^{\circ}C$ with an initial water activity ($a_w$) of 0.16, and a molar ratio of NA to PC of 20 in 5 ml ethyl acetate.

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Role and Action Mechanism of Secretory phospholipase $A_2$ in Macrophage Activation

  • Baek, Suk-Hwan
    • Proceedings of the PSK Conference
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    • pp.179-180
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    • 2002
  • The phospholipase $A_2$($PLA_2$) family represents a diverse group of enzymes that hydrolyze sn-2 fatty acid from the cell membrane. Several mammalian cytosolic $PLA_2$ and secretory $PLA_2$(s$PLA_2$) have been characterized and classified into different families. At present, 12 distinct sPLA$_2$s have been identified in mammals and classified into different groups, depending on their primary structures as characterized by the number and position of cysteine residues. (omitted)

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