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

• Biomolecules & Therapeutics
• /
• 제2권1호
• /
• pp.39-46
• /
• 1994

The present study was undertaken to demonstrate whether or not bradykinin activates a phospholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [$^3$H]phosphatidic acid and [$^3$H]phosphatidylethanol we could elucidate the direct stimulation of phospholipase D by bradykinin. Bradykinin leads to a rapid increase in [$^3$H]phosphatidic acid and [$^3$H]diacylglycerol, and [$^3$H]phosphatidic acid formation preceded the formation of [$^3$H]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine by the action of phospholipase D, not from diacylglycerol by the action of diacylglycerol kinase. In addition, the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activated and regulated by extracellular calcium ion and pertussis toxin-insensitive G protein, respectively. It has also been shown that bradykinin may activate phospholipase D through protein kinase C-dependent pathway. In conclusion, we are now, for the first time, strongly suggesting that bradykinin-induced activation of phospholipase D in the rabbit kidney proximal tubule cells is mediated by a pertussis toxin-insensitive G protein and is dependent of protein kinase C.

• Journal of Photoscience
• /
• 제6권2호
• /
• pp.71-75
• /
• 1999

Tempting to further understand the molecular mechanism of pharmacological action of ethanol, we evaluated effects of phosphatidylethanol (PET) on inositol 1,4,5-triphosphate (IP3) level and protein kinase C (PKC) activity in cultured NG108-15 cells. PET increased intracellular concentration of IP$_3$. PET incorporation into membranes of NG108-15 cells had no effect on the phosphorylation of the PKC-specific substrate MBP$\_$4-14/, thus indicates that PET does not affect PKC activity in this system.

• Journal of Nutrition and Health
• /
• 제19권3호
• /
• pp.135-145
• /
• 1986

식이 $\omega$3 지방산이 신장의 지질구성 및 기능에 미치는 영향을 조사하기 위해 젖뗀 55-60 g의 흰쥐에게 고등어유, 들깨유, 콩기름을식이의 10% (W/W)포함하는 먹이를 조제하여 24일간 섭취시켰다. 고등어유군의 신장 인지질 지방산에 C20~C22 $\omega$ 3 지방산의 함량이 월등히 많은 반면 들깨유군은 콩기름군에 비해 약간 높을 뿐이어서 C18 : 3 ($\omega$3) 지방산으로 단기간에 쉽게 전환되지 않음을 보였다. C18 : 2($\omega$6) 지방산은 고등어군만 낮았고 C20 : 4($\omega$6)는 들깨유군에서는 낮았지만 고등어유군은 콩기름군과 같은 수준이었다. 또한 고등어유군에서 C18 : 1($\omega$9)을 포함한 단일 불포화 지방산이 높고 포화지방산 함량이 낮은 것도 특징이었다 . 인지질 종류별 분석결과에서 고등어유군이 sphingomyelin 이 높고 phosphatidylethanol-amine이 낮았다. 반면 신장 Cholest대기 함량은 고등어유군이 높았으며, 이러한 지질구성 변화는 신장 Na-K-ATPase 활성감소시킨 것이다.

• Archives of Pharmacal Research
• /
• 제17권6호
• /
• pp.405-410
• /
• 1994

The present study was undetertaken to demonstrate whether or not angiotensin II activates a phopholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [$^3H$] phosphatidic acid and [$^3H$]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholin by the action of phopholipase D, not from the action of diacylglycerol kinase on the diacylglycerol. In addition the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activited by extracellular calium ion. It has also been shown that angiotensin II may activate phosphoilpase D through protein kinase C-independent pathway.

• Bulletin of the Korean Chemical Society
• /
• 제17권10호
• /
• pp.905-908
• /
• 1996

A substrate specificity of cabbage phospholipase D (PLD) was studied using the synthetic phospholipids having different head groups. The phospholipids were synthesized from phosphatidylcholine and appropriate bases by transphosphatidylation of PLD. The bases used were ethanolamine, serine, ethanol and γ-hydroxybutyric acid. The phosphatidic acid, the product of PLD, was separated in TLC and measured densitometrically. The kinetic parameters were estimated for each substrate and the effects of pH, SDS, Ca2+ and other metal ions were examined. Vmax values found were 3.75, 2.36, 5.59, 1.63, 2.30 nmol/min/μg protein for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylethanol, and phosphatidylburytic acid, respectively. These results indicate a broad specificity of cabbage PLD toward phospholipids with different head groups. Particularly phosphatidylserine was most easily hydrolyzed by PLD and its activity did not depend on Ca2+.

• 한국수산과학회지
• /
• 제27권5호
• /
• pp.476-481
• /
• 1994

액젓 제조시 발효를 촉진하기 위하여 정어리를 기질로한 코오지를 제조하고 이것으로 부터 추출한 조효소액인 lipase의 pH 및 온도의존성과 코오지의 구성지질 및 지방산 조성을 조사하였다. Lipase의 최적 활성을 나타내는 pH는 $7{\sim}8$이었으며 pH 5.0 이하와 9.0 이상이 되면 활성은 급격히 감소하였고, 최적온도는 $40^{\circ}C$ 부근이였다. pH 및 온도에 변화에 따른 활성잔존율을 조사한 바 pH $6{\sim}9$ 범위에서 $90{\sim}100\%$, $40^{\circ}C$에서 1시간 가열처리했을때 약 $80\%$의 잔존율을 보였고 $50^{\circ}C$ 이상이 되면 잔존율은 크게 감소하여 $50^{\circ}C$는 $10\%$, $60^{\circ}C$는 $3\%$의 잔존율을 보였다. 코오지 지질 조성은 중성지질은 triglyceride가 $53.4\%$ 유리지방산은 $28.1\%$로 전체 중성 지질의 $81.5\%$를 차지하였고 인지질은 phosphatidylethanol이 $32.7\%$로 가장 높았고 그 다음이 phosphatidylinositol이 $25.5\%$, phosphatidylcholine이 $25.1\%$였고 그 외 lyso-phosphatidyl-choline, sphingomyeline 등도 $7.3\%$와 $3.5\%$정도 함유되어 있었다. 한편 총지질과 중성지질의 주요 지방산은 16:0, 22:6, 18:1n-9, 18:2n-6 및 20:5이였고 인지질의 경우는 22:6이 중성지질에 비해 월등히 높게 나타났고 그 다음이 16:0, 18:2n-6 및 20:5로 나타났다.

• BMB Reports
• /
• 제30권5호
• /
• pp.337-341
• /
• 1997

In order to investigate the role of a small GTP-binding protein RhoA in lysophosphatidic acid (LPA)-induced stress fiber formation, C3 ADP-ribosyltransferase was prepared by expressing in E. coli and then applied to Rat-2 fibroblasts. C3 transferase isolated from E. coli was as effective as the toxin from Clostridium botulinum in ADP-ribosylation of RhoA. Incubation of the cells with C3 transferase for 2 days induced ADP-ribosylation of RhoA by a dose-dependent manner, with a sub-maximal induction at $25\;{\mu}g/ml$. As expected, LPA-induced stress fiber formation was completely blocked by pre-incubation with C3 transferase for 2 days. However, exogenously added C3 transferase had no significant effect on the formation of phosphatidylethanol by LPA. These results suggested that phospholipase D was not activated by RhoA in the LPA-induced stress fiber formation.

• BMB Reports
• /
• 제29권1호
• /
• pp.11-16
• /
• 1996

The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the $Ca^{2+}$ ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation. $GTP{\gamma}S$ also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.

• 한국독성학회:학술대회논문집
• /
• 한국독성학회 1997년도 국제 심포지움 및 춘계 학술대회
• /
• pp.65-74
• /
• 1997

By using guinea pig lung mast cells, this study aimed to examine the effects of Aloe component(NY945) on the mediator releases caused by mast cell activation, and also aimed to assess the effects of NY945 on the mechanism of mediator releases in the mast cell activation. We partially purified mast cells from guinea pig lung tissues by using the enzyme digestion, the rough and the discontinuous density percoll gradient method. Mast cells were sensitized with $IgG_1$ (anti-OA) and challenged with ovalbumin. Histamine was assayed by fluorometric analyzer, leukotrienes by radioimmunoassay The phospholipase D activity was assessed more directly by the production of labeled phosphatidylethanol or phosphatidylbutanol which was produced by phospholipase D-mediated transphosphatidylation in the presence of ethanol or butanol. The amount of mass 1,2-diacylglycerol was measured by the [$^3H$]1,2-diacylgycerol produced when prelabeled with [$^3H$]myristic acid. In the mast cells prelabeled with L-[$^3H$]methyl methionine the phospholipid methylation was assessed by measuring the incorporation of the [$^3H$]methyl moiety into phospholipids. Pretreatment of NY945(10$\mu$g) significantly decreased histamine and leukotrienes releases during mast cell activation. The decrease of histamine release was stronger than that of leukotrienes during mast cell activation. The phospholipase D activity increased by the mast cell activation was decreased by the dose-dependent manner in the pretreatment of NY945. The amount of mass 1,2-diacylglycerol produced by activation of mast cells were decreased in the pretreatment of NY945. NY945 pretreatment strongly inhibited the incorporation of the [$^3H$]methyl moiety into phospholipids. The data suggest that NY945 purified from Aloe inhibits in part an increase of 1,2-diacylglycerol which is produced by activating mast cells with antigen-antibody complexes which is mediated via phosphatidylcholine-phospholipise D and phosphatidylinositole-phospholipise C systems, and then followed by the inhibition of histamine release. Furthermore, NY945 reduces the phosphatidylcholine production by inhibiting the methyltransfsrase I and II, which decrease the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrines.