• BMB Reports
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• v.28 no.5
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• pp.387-391
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• 1995

In order to investigate whether phospholipase C (PLC) activity in oat celIs is regulated by Gprotein, we have characterized PLC in plasma membranes of oat tissues. To identify the purified plasma membrane, $K^+$-stimulated, $Mg^{2+}$-dependent ATPase activity was measured. The activity of ATPase was shown to be proportional to the concentration of membrane protein. To examine the PLC activity regulated by G-protein, we used the inside-out and outside-out plasma membrane mixture isolated from the oat cells. The plasma membrane mixture showed higher PLC activity than the one of the outside-out plasma membrane. This suggests that PLC activity is located at the cytoplasmic surface of plasma membrane. PLC activity in plasma membrane mixture was dependent on $Ca^{2+}$ with maximum activity at 100 ${\mu}m$ $Ca^{2+}$ and it was inhibited by 1 mM EGTA. Using Sep-pak $Accell^{TM}$ Plus QMA chromatography, we found that inositol 1,4,5-trisphosphate ($IP_3$) was produced in the presence of 10 ${\mu}m$ $Ca^{2+}$. The PLC activity in the membrane was enhanced by an activator of G-protein ($GTP{\gamma}S$) and not by an inhibitor ($GDP{\beta}S$). This indicates that a G-protein is involved in the activation of PLC in the plasma membrane of oat cells.

• Journal of Yeungnam Medical Science
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• v.9 no.2
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• pp.288-301
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• 1992

The objective of the present study was to identify the characteristics of phospholipase C (PLC) isozymes purified from bovine brain and to investigate their interrelationship with G protein. The purified PLC isozymes ${\beta}$, ${\gamma}$ and ${\delta}$ were obtained and the characteristics of PLC activity on various concentrations of free $Ca^{2+}$ were observed. The activity of PLC was increased with increasing $Ca^{2+}$ concentration and the activity PLC ${\delta}$ was increased higher in the presence of phosphatidyl choline(PC) than in the abscence of PC. For vesicle formation as the structure of cell membrane, cholic acid and deoxycholic acid as detergent on phosphatidylinositol bisphosphate($PIP_2$) substrate containing PC were used, and then the activity of PLC isozymes were increased with increasing concentration of cholate, from 0.2% to 1% and were increased slightly in deoxycholate. In the $PIP_2$ containing phospholipid and glycolipid as brain extract, the activity of PLC isozymes were checked in 0.2%-1% cholic acid. The activities of PLC isoyzmes were continuously increased up to 1% cholic acid. The quantitation of PLC isozymes from several bovine organs by radioimmunoassay was made. Brain was the most sufficient organ in terms of amount of PLC ${\beta}$and ${\delta}$. A large amount of PLC ${\delta}$ was existed in adrenal gland. The binding capacity of GTPrS and G protein was observed and other observations of the binding effect of GTPrS-G protein and PLC monoclonal Ab-Protein A from tissue homogenate with PLC were made. From the observation the binding capacity was revealed the range of 0.11%-1.49%. The effects of each type of G protein on the percent activity of purified PLC isozymes were observed. From the observation, activities of isozymes were increased in $Go{\alpha}$ & Gmix, and the activities of PLC ${\beta}$ and ${\delta}$ were increased in $G{\beta}{\gamma}$ and $Gi{\alpha}$. Activities of PLC ${\beta}$ and ${\gamma}$ were decreased in $Gt{\alpha}$ but PLC ${\delta}$ increased.

• Biomedical Science Letters
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• v.12 no.4
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• pp.419-425
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• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• Journal of Yeungnam Medical Science
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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.

• BMB Reports
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• v.40 no.6
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• pp.888-894
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• 2007

Src homology (SH) domains of phospholipase C-$\gamma1$ (PLC-$\gamma1$) impair NGF-mediated PC12 cells differentiation. However, whether the enzymatic activity is also implicated in this process remains elusive. Here, we report that the enzymatic activity of phospholipase C-$\gamma1$ (PLC-$\gamma1$) is at least partially involved to the blockage of neuronal differentiation via an abrogation of MAPK activation, as well as sustained Akt activation. By contrast, Overexpression of WT-PLC-$\gamma1$ exhibited sustained NGF-induced MAPK activation, and triggered transient Akt activation resulting in profound inhibition of neurite outgrowth. However, lipase-inactive mutant (LIM) PLC-$\gamma1$ cells fail to suppress neurite outgrowth, although it contains intact SH domains, specifically enhancing the expression of cyclin D1 and p21 proteins, which regulate the function of retinoblastoma Rb protein. These observations show that the lipase inactive mutant of PLC-$\gamma1$ does not alter NGF-induced neuronal differentiation via enzymatic inability and the modulation of cell cycle regulatory proteins independent on SH3 domain.

• Biomolecules & Therapeutics
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• v.1 no.1
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• pp.121-124
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• 1993

To elucidate the effect of antipsychotics and antidepressants on phosphoinositide(Pl) second massenger system, we studied the dose-dependent inhibition of the phosphoinositide-specific phospholipase C(PLC) isozymes, ${\beta}_1,\;{\gamma}_1$ and${\delta}_1,$ by fluphenazine and haloperidol as antipsychotics, and amitriptyline, maprotiline and mianserin as antidepressants. All the antipsychotics and antidepressants tested showed inhibition on at least one of the PLC isozymes with $IC_{50}$ at the concentration between 25 and $250 {\mu}M.$ Maprotiline, mianserin and amitriptyline inhibited 80 to 90% of the activities of all three PLC isozymes at the concentration of $250{\mu}M,$ while haloperidol and fluphenazine inhibited PLC ${\beta}_1$ and${\gamma}_1$ But baclofen didn't inhibit any PLC isozyme. These results suggested that PLC isozymes are inhibited by antipsychotics and antidepessants even though the concentration is high, and these drugs may affect PI signal transduction system by direct inhibition of PLC isozymes.

• BMB Reports
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• v.35 no.5
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• pp.508-512
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• 2002

Phospholipase C-gamma-2 ($PLC{\gamma}2$) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to $PLC{\gamma}2$ activation, we devised a quick method for obtaining sufficient $PLC{\gamma}2$. We obtained the full-length cDNA for human $PLC{\gamma}2$ and expressed it in E. coli using the expression vector pT5T. To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons. The protein expression was detected in a Western blot analysis by both anti-$PLC{\gamma}2$ antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal. Crude lysates that were prepared from bacteria that express $PLC{\gamma}2$ were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on $PLC{\gamma}2$ that is isolated from mammalian tissue, the recombinant enzyme was $Ca^{2+}$ dependent with optimal activity at 1-10 uM $Ca^{2+}$.

• Animal cells and systems
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• v.2 no.1
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• pp.87-91
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• 1998

The present study investigated the involvement of the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways during progesteroneinduced meiotic maturation in amphibian (Rana dybowskii) oocytes. Prosesterone-induced germinal vesicle breakdown (GVBD) of oocytes was significantly inhibited by a PKC inhibitor, staurosporine and a PLC inhibitor, U73122, in a dose-dependent manner. In contrast, U73343, an inactive analogue of U73122, was ineffective in suppressing GVBD. PKC activity in oocytes reached a maximum level at 30 min after progesterone stimulation and this elevated PKC activity was effectively suppressed by U73122 or staurosporine, suggesting that the activation of PKC enzyme is closely linked to PLC signaling during oocyte maturation. In addition, these inhib itors blocked the maturation promoting factor (MPF) activity which appeared in oocytes in response to progesterone, suggesting that PKC activation is an important signal for MPF activity. Therefore, this study demonstrates that the activation of PKC via PLC signaling is directly linked to an intracellular protein kinase cascade related to the appearance of MPF activity during meiotic maturation in amphibian (Rana dybowskii) oocytes.

• Human Ecology Research
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• v.54 no.4
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• pp.365-384
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• 2016

The purpose of this research is to apprehend what the power of professional learning community (PLC) to make home economics teachers participate is and how PLC activity contributes to reflective practice and change of participating home economics teachers by experiencing reflective practices. For this, self-reflective action research of Kemmis and McTaggart was conducted. Six home economics teachers participated voluntarily and totally 18 PLC sessions from May 31, 2013 to May 19, 2014 were held. Two themes of 'looking in classes' as a main practice theme and 'designing classes together' as a supporting theme were carried. Findings and conclusions of this study are as following. First, participants of PLC to get data and information on teaching and to solve problems with fellow teachers for better classes at first. However, they have become to comfort and sympathize each other about difficulties in school as home economics teachers. Second, through the PLC activity, they found they had uncomfortable belief about teaching and tried to practice solutions by critical and reflective thinking. Third, they put efforts in finding alternative framework of looking inside their classes for the fundamental improvement in teaching. For this, they formulated questionnaire to describe their own reflective practice through the alternative framework from a critical perspective in teaching, a view of student's learning, and a teacher's inner view for improvement of practice. Fourth, PLC activity for a year allowed them to combine theory and reality though reflective process by designing classes together and reflectively practicing them in classes.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.191-194
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• 2003

The phospholipase C (PLC) hydrolyzes the polar head groups such as phosphocholine or phosphoethanolamine residues esterified at the sn-3 position of phospholipids. Pichia pastoris can utilize methanol as a carbon source and produce recombinant proteins under the control of the strong, tightly-regulated alcohol oxidase (AOX) promoter. In this study, we developed recombinant P. pastoris system for PLC expression and analyzed PLC activity.