• Journal of Microbiology
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• 제38권3호
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• pp.150-155
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• 2000

A bioluminescent assay method for detecting the activity of phospholipase C(PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2- dimyristoyl glycerol was further hydrolyzed with lipase from Candida ecylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hdrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the were estalished to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.354-361
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• 2005

Vibrio fluvialis, an enteropathogenic bacterium, produces a phospholipase which is thought to be an important factor in the pathogenesis of disease. In this study, the phospholipase gene (vfp) was identified from V fluvialis (KCTC 2473) and its sequence was determined. The entire open reading frame was composed of 1,689 nuc1eotides and 563 amino acids. The phospholipase gene (vfp) was overexpressed in Escherichia coli as a his-tag fused protein. This recombinant protein (rVFP58) was solubilized with 6 M urea and purified by Ni-NTA affinity chromatography. The action mode of rVFP58 was determined by TLC and GC-MS, and it showed phospholipase B activity, which had both phospholipase A and lysophospholipase activities. The rVFP58 showed a maximum activity at pH around 9- 10 and temperature of about 40OC, and it was stable under alkaline condition over pH 9. The cytotoxicity of rVFP58 was evaluated, using a fish cell line, CHSE-2l4, and was found to cause significant cell death after 14 h of exposure to 250 $\mu$g of the protein.

• Archives of Pharmacal Research
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• 제26권6호
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• pp.478-481
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• 2003

Phospholipase D is a ubiquitous enzyme that plays an important role in various lipid mediated cellular signaling pathways and produces rare phospholipids, phosphatidylethanol or phosphatidylbutanol, instead of phosphatidic acid with unique catalytic activity transphosphatidylation in the presence of primary alcohols. The reaction products, phosphatidylethanol or phosphatidylbutanol are used as markers of in vitro phospholipase D activity in many studies. For the sensitive detection of the phospholipase D products, we developed an advanced lipid extraction method that facilitates recovery of the compounds. With the new method, the activity change of phosaholipase D by agonists could be detected more easily and the recovery rate was also increased. The increase of detected enzyme activity change was about double fold compared to the conventional lipid extraction method. This method provides selective force for the phospholipase D products in the extraction procedure.

• The Korean Journal of Physiology and Pharmacology
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• 제14권6호
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• pp.427-433
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• 2010

This study was conducted to investigate the effects of extremely low frequency electromagnetic fields (EMF) on signal pathway in plasma membrane of cultured cells (RAW 264.7 cells and RBL 2H3 cells), by measuring the activity of phospholipase $A_2$ ($PLA_2$), phospholipase C (PLC) and phospholipase D (PLD). The cells were exposed to the EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h. The basal and $0.5\;{\mu}M$ melittin-induced arachidonic acid release was not affected by EMF in both cells. In cell-free $PLA_2$ assay, we failed to observe tbe change of $cPLA_2$ and $sPLA_2$ activity. Also both PLC and PLD activities did not show any change in the two cell lines exposed to EMF. This study suggests that the exposure condition of EMF (60 Hz, 0.1 or 1 mT) which is 2.4 fold higher than the limit of occupational exposure does not induce phospholipases-associated signal pathway in RAW 264.7 cells and RBL 2H3 cells.

• Journal of Acupuncture Research
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• 제18권1호
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• pp.129-145
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• 2001

Objectives : To characterize the antitumorigenic potential of three representative bee venom components, Melittin, Apamin, and Phospholipase A2, their effects on cell proliferation and apotosis of the human melanoma cell line SK-MEL-2 were analyzed using molecular biological approaches. Methodes & Results : To determine the doses of the drugs that do not induce cytotoxic damage to this cell line, cell viability was examined by MTT assay. While SK-MEL-2 cells treated with 0.5 - 2.0㎍/㎖ of each drug showed no recognizable cytotoxic effect, marked reductions of cell viability were detected at concentrations over 5.0㎍/㎖. [3H]thymidine incorporation assay for cell proliferation demonstrated that DNA replication of SK-MEL-2 cells is inhibited by Apamin and Phospholipase A2 in a dose-dependent manner. Consistent with this result, the cells were accumulated at the G1 phase of the cell cycle after treatment with Apamin and Phospholipase A2, whereas no detectable change in cell proliferation was identified by Melittin treatment. In addition, tryphan blue exclusion and flow cytometric analyses showed that all of these drugs can trigger apoptotic cell death of SK-MEL-2, suggesting that Melittin, Apamin, and Phospholipase A2 have antitumorigenic potential through the suppression of cell growth and/or induction of apoptosis. Qantitative RT-PCR analysis revealed that Apamin and Phospholipase A2 inhibit expression of growth-promoting genes such as c-Jun, c-Fos, and Cyciin D1. Furthermore, Phospholipase A2 induced tumor suppressors p53 and p21/Wafl. In addition, all three drugs were found to activate expression of a representative apoptosis-inducing gene Bax while expression of apoptosis-suppressing Bcl-2 and Bcl-XL genes was not changed. Taken together, this study strongly suggests that Metittin, Apamin, and Phosphalipase A2 may have antitumorigenic activities, which are associated with its growth-inhibiting and/or apoptosis-inducing potentials.

• The Korean Journal of Physiology
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• 제29권1호
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• pp.29-37
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• 1995

Gastric smooth muscle of guinea pigs was used to investigate whether the inhibitory effect of calcium antagonists on tonic contraction was accompanied by inhibition of phospholipase C activity. Tonic contraction and $[^{3}H]$ inositol phosphate (IP) formation in response to acetylcholine were measured after pretreatment with verapamil, nifedipine, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxy-benzoate (TMB-8) or EGTA. Verapamil $(10\;{\mu}M)$, TMB-8 $(10\;{\mu}M)$ or EGTA (2 mM) significantly inhibited acetylcholine $(1\;{\mu}M)$-stimulated tonic contraction but nifedipine (100 nM) did not. Acetylcholine dose-dependently increased the formation of $[^{3}H]IP$. This effect was not observed in the presence of 2 mM EGTA. Both verapamil and TMB-8 significantly inhibited $[^{3}H]IP$ formation induced by $10\;{\mu}M$ acetylcholine, whereas nifedipine did not. In a subsequent study, we measured phospholipase C activity in gastric muscle cell homogenate and in permeabilized cells to determine whether calcium antagonists could inhibit the activity directly. The calcium antagonists did not change the phospholipase C activity of the cell homogenate or the permeabilized cells. But EGTA decreased phospholipase C activity by 50%. These results suggest that the inhibitory effects of verapamil and TMB-8 on acetylcholine-stimulated tonic contraction may be accompanied by inhibition of phospholipase C activity.

• Clinical and Experimental Reproductive Medicine
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• 제36권2호
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• pp.81-88
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• 2009

• 약학회지
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• 제38권1호
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• pp.97-101
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• 1994

Platelets play a very important role in nomal hemostasis and their functions are more enhanced in various pathogenic states than in normal state. Especially it has been postulated that abnormal platelet and endothelium function might be major factors of microcirculatory disturbance in diabetes mellitus. Hydroxybrazilin, a phenolic constituent of Hematoxylon campechianum has been examined for its inhibitory effect on platelet aggregation. Its antiaggregatory effect might be mediated through the decrease of ATP release from dense granule and those of thromboxane $A_2$ production in normal and streptozotocin induced diabetic rats. The present study was undertaken to gain insight into the mechanism that hydroxybrazilin inhibited thromboxane $A_2$ production in platelets. Thus we measured the effect of hydroxybrazilin on phospholipase $A_2$, a rate limiting step of thromboxane $A_2$ production, in normal and streptozotocin induced diabetic rats. Hydroxybrazilin significantly inhibited the platelet phospholipase $A_2$ activity in normal and streptozotocin induced diabetic rats.

• 생약학회지
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• 제33권1호통권128호
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• pp.49-52
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• 2002

Amentoflavone, naturally occurring biflavonoid, isolated from the leaves of Ginko biloba, selectively inhibited human seceretory phospholipase $A_2$. This compound potently and irreversibly inhibited human group IIA in a dose dependent manner with an $IC_50$ about $3\;{\mu}M$. Amentoflavone inhibited phospholipase $A_2$ by a noncompetitive manner with the apparent Ki value of $1{\times}10^{-5}M$. In addition, the inhibitory activity of amentoflavone is rather specific against group IIA phospholipase $A_2$ than group IB phospholipase $A_2$. Furthermore, this compound strong inhibit histamine release from $A_{23187}$ treated rat peritoneal mast cells. These results indicate naturally occurring biflavonoid represents a novel anti-inflammatory agent.

• 미생물학회지
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• 제40권3호
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• pp.211-216
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• 2004

세포외 phospholipase D (PLD)를 과량 생산하는 균주 JE-11을 토양으로부터 분리하였다. 16S rDNA에 의한 분석과 형태적, 생리적 특성을 조사한 결과 이 균은 Streptomyces somaliensis로 동정되었다. 선발한 S. somaliensis로 부터 PLD를 암호화하는 유전자(sspld) 분리하고 염기서열을 조사하였다. Open reading frame을 분석한 결과 33개의 아미노산으로 이루어진 분비 signal peptide와 505개의 아미노산으로 구성된 PLD단백질을 암호화하는 것으로 예상되었다. 또한, sspld의 염기 서열로부터 유추된 단백질 서열은 기존에 보고된 다른 Streptomyces PLD들과 70-88％의 서열 유사성을 보였다. 이 PLD는 96-98％(㏖/㏖)의 수율로서, Phosphatidylcholine을 glycerol과 serine을 기질로 하여 각각 phosphatidylglycerol 과phosphatidylserine으로 전환을 하였으나, 알코올 공여체인 inositol과 ethanolamine과는 반응하지 않았다.