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RISS 인기검색어
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- 저자 : Trono, (검색결과 4 건)
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Soccio, Mario,Trono, Daniela,Laus, Maura N.,Pastore, Donato 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.4
An improved spectrophotometric assay of phospholipase $A_2$ ($PLA_2$) activity based on the coupled $PLA_2$/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine ($PC_{LIN}$) as substrate is reported. The $PLA_2$-mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 ($3{\mu}L/{\mu}mol$ phospholipid) as surfactant and of ethanol ($15{\mu}L/mL$ reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory $PLA_2$ from honey bee venom (HBV-$PLA_2$). The enzyme did not discriminate among $PC_{LIN}$, phosphatidylcholine, and phosphatidylethanolamine, but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine ($PC_{DILIN}$). Nevertheless, the use of $PC_{DILIN}$ is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine, are known to be oxidized by LOX. HBV-$PLA_2$ showed maximal activity at pH 9.0, hyperbolic kinetics ($K_m$, $74.2{\pm}2.9{\mu}M$; $V_{max}$, $827{\pm}7{\mu}mol/min/mg$ protein) and competitive inhibition ($K_i$ about $5{\mu}M$) by palmityl trifluoromethyl ketone, a classical $PLA_2$ inhibitor. Interestingly, the HBV-$PLA_2$/soybean LOX-1 coupled reactions also allow an accurate assay of $PC_{LIN}$ concentration. In the whole, these results demonstrate that this improved $PLA_2$/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.
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Mario Soccio,Donato Pastore,Daniela Trono,Maura N. Laus 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.4
An improved spectrophotometric assay of phospholipase A2 (PLA2) activity based on the coupled PLA2/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PCLIN) as substrate is reported. The PLA2-mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 (3 μL/μmol phospholipid) as surfactant and of ethanol (15 μL/mL reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory PLA2from honey bee venom (HBV-PLA2). The enzyme did not discriminate among PCLIN, phosphatidylcholine, and phosphatidylethanolamine,but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine (PCDILIN). Nevertheless,the use of PCDILIN is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine,are known to be oxidized by LOX. HBV-PLA2showed maximal activity at pH 9.0, hyperbolic kinetics (Km,74.2±2.9 μM; Vmax, 827±7 μmol/min/mg protein) and competitive inhibition (Ki about 5 μM) by palmityl trifluoromethyl ketone, a classical PLA2 inhibitor. Interestingly, the HBV-PLA2/soybean LOX-1 coupled reactions also allow an accurate assay of PCLIN concentration. In the whole, these results demonstrate that this improved PLA2/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.
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A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation
Singh, Kulwant,Cassano, Marco,Planet, Evarist,Sebastian, Soji,Jang, Suk Min,Sohi, Gurjeev,Faralli, Hervé,Choi, Jinmi,Youn, Hong-Duk,Dilworth, F. Jeffrey,Trono, Didier Cold Spring Harbor Laboratory Press 2015 Genes & development Vol.29 No.5
<P>The transcriptional activator MyoD serves as a master controller of myogenesis. Singh et al. identify KAP1/TRIM28 as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only coactivators such as p300 and LSD1 but also corepressors such as G9a and HDAC1, with promoter silencing as the net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the corepressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors.</P><P>The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2 (myocyte enhancer factor 2), MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although it is known to reflect the action of chromatin modifiers. Here, we identify KAP1 (KRAB [Kréééüppel-like associated box]-associated protein 1)/TRIM28 (tripartite motif protein 28) as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only coactivators such as p300 and LSD1 but also corepressors such as G9a and HDAC1 (histone deacetylase 1), with promoter silencing as the net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the corepressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.</P>
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