• Title/Summary/Keyword: phospholipase C

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Effects of Protein Kinases on Phospholipase C Activation and Intracellular $Ca^{2+}$ Mobilization Induced by Endothelin-1 (Endothelin-1에 의한 phospholipase C 활성화와 세포내 $Ca^{2+}$ 이동에 미치는 protein kinase들의 효과)

  • 조중형;김현준;이윤혜;박진형;장용운;이승준;이준한;윤정이;김창종
    • YAKHAK HOEJI
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    • v.44 no.2
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    • pp.162-168
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    • 2000
  • To investigate the effects of protein kinases on endothelin-1-induced phospholipase C activation and $Ca^{2+}$ mobilization in Rat-2 fibroblast, we measured the formation of inositol phosphates and intracellular $Ca^{2+}$ concentration with [$^3$H]inositol and Fura-2/AM, respectively. Endothelin-1 dose-dependently activated phospholipase C and increased intracellular $Ca^{2+}$ concentration. Protein kinase C activator PMA, significantly inhibited both phospholipase C activity and $Ca^{2+}$ mobilization induced by endothelin-1. Tyrosine kinase inhibitor, genistein, inhibited both. On the other hand, cyclic nucleotide (cAMP and cGMP) did not have any influence on the signaling pathway of phospholipase C-Ca$^{2+}$ mobilization induced by endothelin-1. These results suggest that protein kinase C and tyrosine kinase counteract on the signaling pathway of phospholipase C-Ca$^{2+}$ mobilization induced by endothelin-1 in Rat-2 fibroblast. fibroblast.

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Liposome-Based Assay for Phospholipase C

  • 임수정;고유찬;이은옥;김종국
    • Bulletin of the Korean Chemical Society
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    • v.18 no.7
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    • pp.761-766
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    • 1997
  • Phospholipase C from Clostridium perfringens is known to catalyze the hydrolysis of phospholipids in biological membranes. In this study, a simple and sensitive method for assaying phospholipase C was developed by using liposomes entrapping calcein as a fluorescent marker. Phospholipase C-induced lysis of liposomes was determined by measuring the fluorescence intensity of calcein released out from liposomes, Various liposomes with different compositions were prepared by reverse-phase evaporation method to investigate the effect of liposomal composition on the lytic activity of phospholipase C. The calcein-entrapping efficiency of liposomes was affected by the chain length of fatty acid in phosphatidylcholine constituting liposomes. The lytic activity of phospholipase C was the highest against liposomes prepared with eggPC. The lytic activity decreased with increasing chain length of fatty acid in phosphatidylcholine. Incorporation of cholesterol more than 20% into the liposomal bilayer inhibited the phospholipase C-induced lysis. The lysis of liposomes was more greatly increased by the addition of 10 mM of calcium. The lytic activity of phospholipase C was also affected by the surface charge of liposomes. Taken together, it was concluded that reverse-phase evaporation vesicles composed of dipalmitoylphosphatidylcholine and cholesterol in the molar ratio of 9 : 1 allowed to detect the lowest concentration of phospholipase C (0.10 μg/assay volume). This study suggested that the use of liposomes can provide a simple, sensitive and inexpensive method for assaying phospholipase C.

The Action of ATP on Phospholipase $A_2$Activation in C6 Cells (C6세포에서 phospholipase $A_2$활성에 대한 ATP의 작용)

  • 심상수;김명준;윤신희;김창종;조양혁
    • YAKHAK HOEJI
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    • v.45 no.4
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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.

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Isolation of Scopafungin, a Potent Inhibitor of Phospholipase C from Actinomycetes isolate No. 2511-5 (방선균 분리주 No. 2511-5로부터 포스포리파제 C 저해물질 스코파훈진의 분리)

  • 오원근;이현선;안순철;김보연;박문수;민태익;안종석
    • YAKHAK HOEJI
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    • v.39 no.5
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    • pp.554-559
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    • 1995
  • MT 2511-5 was purified as a inhibitor of phospholipase C(PLC) from culture broth of a Streptmyces sp. NO. 2511-5. It was identified as scopafungin, 36-membered marcrolide by physico-chemical and spectroscopic data. Its $IC_{50}$ was 30.mu.M against phospholipase C and it also showed inhibitory activity against some fungi.

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Bradykinin-Mediated Stimulation of Phospholipase D in Rabbit Kidney Proximal Tubule Cells

  • Park, Kyung-Hyup;Jung, Jee-Chang;Chung, Sung-Hyun
    • Biomolecules & Therapeutics
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    • v.2 no.1
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    • pp.39-46
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    • 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.

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Bioluminescent assay of Phospholipase C Using A Luminescent Marine Mutant Bacterium Vibrio harveyi M-17

  • Cho, Ki-Woong;Mo, Sang-Jun;Lee, Hyi-Seung;Park, Jung-Rae;Jongheon Shin
    • Journal of Microbiology
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    • v.38 no.3
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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.

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Action of Phospholipase $A_2$in Histamine Release from Mast Cells (비만세포에서 Histamine유리에 관여하는 Phospholipase $A_2$의 작용)

  • 이윤혜;이승준;서무현;장용운;윤정이
    • YAKHAK HOEJI
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    • v.45 no.3
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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.

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Inhibitory Effects of Verapamil and TMB-8 on Tonic Contraction Are Accompanied by Inhibition of Phospholipase C Activity in Intact Gastric Smooth Muscle Cells

  • Sim, Sang-Soo;Yoon, Shin-Hee;Hahn, Sang-June;Rhie, Duck-Joo;Jo, Yang-Hyeok;Kim, Myung-Suk
    • The Korean Journal of Physiology
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    • v.29 no.1
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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.

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Activation of formyl peptide receptor 2 by WKYMVm enhances emergency granulopoiesis through phospholipase C activity

  • Kim, Hyung Sik;Park, Min Young;Lee, Sung Kyun;Park, Joon Seong;Lee, Ha Young;Bae, Yoe-Sik
    • BMB Reports
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    • v.51 no.8
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    • pp.418-423
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    • 2018
  • Emergency granulopoiesis is a very important strategy to supply efficient neutrophil number in response to infection. However, molecular mechanism involved in this process remains unclear. Here, we found that administration of WKYMVm, an immune modulating peptide, to septic mice strongly increased neutrophil number through augmented emergency granulopoiesis. WKYMVm-induced emergency granulopoiesis was blocked not only by a formyl peptide receptor 2 (FPR2) antagonist (WRW4), but also by FPR2 deficiency. As progenitors of neutrophils, $Lin^-c-kit^+Sca-1^-$ cells expressed FPR2. WKYMVm-induced emergency granulopoiesis was also blocked by a phospholipase C inhibitor (U-73122). These results suggest that WKYMVm can stimulate emergency granulopoiesis via FPR2 and phospholipase C enzymatic activity.

Bacillus cereus에 의한 Phospholipase C (PLC) 생산

  • Seo, Guk-Hwa;Lee, Jong-Il;Bornscheuer, Uwe T.
    • 한국생물공학회:학술대회논문집
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    • 2002.04a
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    • pp.232-234
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    • 2002
  • Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. This study focuses on the production of PLC by B. cereus and recombinant E. coli with fusion protein gene (plc::gfp). Fermentation processes have been monitored by a 2-dimensional fluorescence sensor.

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