요각류 Paracyclopina nana Acetate Kinase의 클로닝 및 대장균에서의 발현

정상운,서정수,이영미,박태진,김일찬,박흠기,이재성,Jung Sang-Oun,Seo Jung Soo,Lee Young-Mi,Park Tae-Jin,Kim Il-Chan,Park Heum Gi,Lee Jae-Seong 한국미생물학회 2005 미생물학회지 Vol.41 No.3

요각류 Paracyclopina nana Acetate Kinase를 클로닝하였다. 전체 open reading frame은 1,200 bp이었으며, poly(A) signal sequence가 ORF에 내재되어 있었다. 분자계통학적 분석결과 P. nana acetate kinase 유전자는 진핵생물계 곰팡이류인 Aspegillus와 같은 branch를 형성하였고, P. nana acetate kinase가 다른 원핵미생물들의 acetate kinase와는 구별되며 fungi와 같은 branch에 존재하는 것을 확인하였다. 또한, E. coli를 이용하여 원핵세포 발현벡터를 이용한 단백질 발현 유도를 통하여 P. nana acetate kinase 단백질 분자량이 약 50 kDa에 이르는 것을 확인하였다. 이 자료는 본 요각류와 다른 생물의 acetate kinase 단백질의 생화학적 특성비교에 유용하게 쓰이리라 사료된다. The acetate kinase gene from the copepod Paracyclopina nana was cloned. The open reading frame (ORF) was 1,200 bp, and poly(A) signal sequence was located in the end of the ORF. After the molecular phylogenetic analysis of P nana acetate kinase gene, it was revealed that it formed the same branch with that of Aspergillus. Also P. nana acetate kinase showed the difference with those of other prokaryotic microorganisms but showed the same clade with those of fungi. We also confirmed that the recombinant protein of P. nana acetate kinase made approximately 50 kDa after expression of recombinant gene construct in E. coli. This may be useful to compare this protein to those of other organisms in biochemical characteristics.

Escherichia coli의 Glutamine Synthetase와 Acetate Kinase에 의한 Glutamine 생산

조정일 한국식품영양학회 1993 韓國食品營養學會誌 Vol.6 No.3

Glutamate로부터 glutamine으로 전환하는 효소인 glutamine synthetase는 cofactor로서 ATP를 요구하는 endergonic reaction이므로, glutamine 전환반응에 요되는 ATP의 효율적인 공급을 위해서 acetate kinase에 의한 ATP 생성계를 도입하였다. Glutamine synthetase의 효소윈으로 사용된 미생물은 glutamine synthetase의 활성이 강화된 E. coli K-12로 부터 사용하였으며, acetate kinase는 E. coli K-12로 부터 부분 정제하여 사용하였다. Acetate kinase에 의한 ATP 생성계를 도입한 glutamine 전환반응의 최적조건은 100mM glutamate, 100mM NH_4Cl, 50mM acetyl phosphate, 5mM ADP, 40mM MgCl_2, 300mM potassium phosphate buffer(pH7.5) 5mM MnCl_2, 70units/㎖ glutamine synthetase, 99units/㎖ acetate inase이었으며, 상기의 최족조건하에서 6시간째 98%의 최대전환율을 나타내었고 이때 생산량은 14.3g/ℓ 이었다. The conversion of glutamate by glutamine synthetase is the endergonic reaction that demands ATP as its energy source. In order to supply efficiently ATP that is demanded in the conversion of glutamate to glutamine. the ATP-generating system by acetate kinase partially purified from Escherichia coli K-12 was coupled with glutamine synthetase partially purified E. coli K-12 Pgln6. The optinum conditions of the coupled reaction were investigated. As the result, the highest conversion of glutamate to glutamine was shown in the reaction mixture containing 100mM glutamate, 100mM NH_4Cl, 50mM acetyl phosphate, 5mM ADP, 40mM MgCl_2, 300mM potassium phosphate buffer(pH 7.5), 5mM MnCl_2. Under this condition, the most effective concentrations of enzyme were 70unit/m Q. glutamine synthetase and 99unit/㎖ acetate kinase. Under the optinum conditions, 98% of 100mM glutamate was converted to glutamine within 6 hours.

Two-component Signal Transduction in Synechocystis sp. PCC 6803 under Phosphate Limitation: Role of Acetyl Phosphate

Juntarajumnong, Waraporn,Eaton-Rye, Julian J.,Incharoensakdi, Aran Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.5

The two-component signal transduction, which typically consists of a histidine kinase and a response regulator, is used by bacterial cells to sense changes in their environment. Previously, the SphS-SphR histidine kinase and response regulator pair of phosphate sensing signal transduction has been identified in Synechocystis sp. PCC 6803. In addition, some response regulators in bacteria have been shown to be cross regulated by low molecular weight phosphorylated compounds in the absence of the cognate histidine kinase. The ability of an endogenous acetyl phosphate to phosphorylate the response regulator, SphR in the absence of the cognate histidine kinase, SphS was therefore tested in Synechocystis sp. PCC 6803. The mutant lacking functional SphS and acetate kinase showed no detectable alkaline phosphatase activity under phosphate-limiting growth conditions. The results suggested that the endogenous acetyl phosphate accumulated inside the mutants could not activate the SphR via phosphorylation. On the other hand, exogenous acetyl phosphate could allow the mutant lacking functional acetate kinase and phosphotransacetylase to grow under phosphate-limiting conditions suggesting the role of acetyl phosphate as an energy source. Reverse transcription PCR demonstrated that the transcripts of acetate kinase and phospho-transacetylase genes in Synechocystis sp. PCC 6803 is up-regulated in response to phosphate limitation suggesting the importance of these two enzymes for energy metabolism in Synechocystis cells

Mechanistic target of rapamycin and an extracellular signalingregulated kinases 1 and 2 signaling participate in the process of acetate regulating lipid metabolism and hormone-sensitive lipase expression

Li Yujuan,Fu Chunyan,Liu Lei,Liu Yongxu,Li Fuchang 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.9

Objective: Acetate plays an important role in host lipid metabolism. However, the network of acetate-regulated lipid metabolism remains unclear. Previous studies show that mitogen-activated protein kinases (MAPKs) and mechanistic target of rapamycin (mTOR) play a crucial role in lipid metabolism. We hypothesize that acetate could affect MAPKs and/or mTOR signaling and then regulate lipid metabolism. The present study investigated whether any cross talk occurs among MAPKs, mTOR and acetate in regulating lipid metabolism.Methods: The ceramide C6 (an extracellular signaling-regulated kinases 1 and 2 [ERK1/2] activator) and MHY1485 (a mTOR activator) were used to treat rabbit adipose-derived stem cells (ADSCs) with or without acetate, respectively.Results: It indicated that acetate (9 mM) treatment for 48 h decreased the lipid deposition in rabbit ADSCs. Acetate treatment decreased significantly phosphorylated protein levels of ERK1/2 and mTOR but significantly increased mRNA level of hormone-sensitive lipase (HSL). Acetate treatment did not significantly alter the phosphorylated protein level of p38 MAPK and c-Jun aminoterminal kinase (JNK). Activation of ERK1/2 and mTOR by respective addition in media with ceramide C6 and MHY1485 significantly attenuated decreased lipid deposition and increased HSL expression caused by acetate.Conclusion: Our results suggest that ERK1/2 and mTOR signaling pathways are associated with acetate regulated HSL gene expression and lipid deposition.

Targeting Acetate Kinase: Inhibitors as Potential Bacteriostatics

( Saeme Asgari ),( Parvin Shariati ),( Azadeh Ebrahim Habibi ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.11

Despite the importance of acetate kinase in the metabolism of bacteria, limited structural studies have been carried out on this enzyme. In this study, a three-dimensional structure of the Escherichia coli acetate kinase was constructed by use of molecular modeling methods. In the next stage, by considering the structure of the catalytic intermediate, trifluoroethanol (TFE) and trifluoroethyl butyrate were proposed as potential inhibitors of the enzyme. The putative binding mode of these compounds was studied with the use of a docking program, which revealed that they can fit well into the enzyme. To study the role of these potential enzyme inhibitors in the metabolic pathway of E. coli, their effects on the growth of this bacterium were studied. The results showed that growth was considerably reduced in the presence of these inhibitors. Changes in the profile of the metabolic products were studied by proton nuclear magnetic resonance spectroscopy. Remarkable changes were observed in the quantity of acetate, but other products were less altered. In this study, inhibition of growth by the two inhibitors as reflected by a change in the metabolism of E. coli suggests the potential use of these compounds (particularly TFE) as bacteriostatic agents.

Anti-inflammatory effects of lindenenyl acetate via heme oxygenase-1 and AMPK in human periodontal ligament cells

Jeong, G.S.,Lee, D.S.,Li, B.,Kim, J.J.,Kim, E.C.,Kim, Y.C. North-Holland ; Elsevier Science Ltd 2011 european journal of pharmacology Vol.670 No.1

The molecular basis for the anti-inflammatory effects of lindenenyl acetate (LA) was investigated in the lipopolysaccharide (LPS)-stimulated human periodontal ligament (HPDL) cell model. LA concentration-dependently inhibited LPS-induced inducible nitric oxide synthase (iNOS) derived nitric oxide (NO) and cyclooxygenase-2 (COX-2) derived prostaglandin E2 (PGE<SUB>2</SUB>) production in HPDL cells. LA also attenuated the production of LPS-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12. LA stimulated heme oxygenase-1 (HO-1) protein expression and enzyme activity of HPDL cells in a dose-dependent manner. Pretreatment with the HO-1 inhibitor, tin protoporphyrin (SnPP), attenuated the inhibitory activities of LA on LPS-induced inflammatory NO, PGE<SUB>2</SUB>, IL-1β, TNF-α, IL-6 and IL-12 production. LA induced translocation of Nrf-2. Furthermore, an inhibitor of JNK MAPK abolished LA-induced HO-1 expression. LA exposure up-regulated the levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its upstream kinase activators, including LKB1 and Ca2+/calmodulin-dependent protein kinase kinase-II. Furthermore, compound C, a specific AMPK inhibitor, partially blocked the LA-induced anti-inflammatory effect. Taken together, these results indicate that LA has anti-inflammatory activity in HPDL cells that might be mediated by the HO-1, AMPK, JNK MAPK, and Nrf-2 pathways. Thus, LA may serve as a potential therapeutic agent in periodontal disease.

Chromium acetate stimulates adipogenesis through regulation of gene expression and phosphorylation of adenosine monophosphate-activated protein kinase in bovine intramuscular or subcutaneous adipocytes

Kim, Jongkyoo,Chung, Kiyong,Johnson, Bradley J. Asian Australasian Association of Animal Productio 2020 Animal Bioscience Vol.33 No.4

Objective: We hypothesized that Cr source can alter adipogenic-related transcriptional regulations and cell signaling. Therefore, the objective of the study was to evaluate the biological effects of chromium acetate (CrAc) on bovine intramuscular (IM) and subcutaneous (SC) adipose cells. Methods: Bovine preadipocytes isolated from two different adipose tissue depots; IM and SC were used to evaluate the effect of CrAc treatment during differentiation on adipogenic gene expression. Adipocytes were incubated with various doses of CrAc: 0 (differentiation media only, control), 0.1, 1, and 10 μM. Cells were harvested and then analyzed by real-time quantitative polymerase chain reaction in order to measure the quantity of adenosine monophosphate-activated protein kinase-α (AMPK-α), CCAAT enhancer binding protein-β (C/EBPβ), G protein-coupled receptor 41 (GPR41), GPR43, peroxisome proliferator-activated receptor-γ (PPARγ), and stearoyl CoA desaturase (SCD) mRNA relative to ribosomal protein subunit 9 (RPS9). The ratio of phosphorylated-AMPK (pAMPK) to AMPK was determined using a western blot technique in order to determine changing concentration. Results: The high dose (10 μM) of CrAc increased C/EBPβ, in both IM (p = 0.02) and SC (p = 0.02). Expression of PPARγ was upregulated by 10 μM of CrAc in IM but not in SC. Expression of SCD was also increased in both IM and SC with 10 μM of CrAc treatment. Addition of CrAc did not alter gene expression of glucose transporter 4, GPR41, or GPR43 in both IM and SC adipocytes. Addition of CrAc, resulted in a decreased pAMPKα to AMPKα ration (p<0.01) in IM. Conclusion: These data may indicate that Cr source may influence lipid filling in IM adipocytes via inhibitory action of AMPK phosphorylation and upregulating expression of adipogenic genes.

Role of Tyrosine Kinases in Vascular Contraction in Deoxycorticosterone Acetate-Salt Hypertensive Rats

Yeum, Cheol-Ho,Jun, Jae-Yeoul,Choi, Hyo-Sub The Korean Society of Pharmacology 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.5

It has been known that activation of tyrosine kinases is involved in signal transduction. Role of the tyrosine kinase in vascular smooth muscle contraction was examined in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy, one week after which they were subcutaneously implanted with DOCA (200 mg/kg) and supplied with 1% NaCl and 0.2% KCl drinking water for $4{\sim}6$ weeks. Control rats were treated the same except for that no DOCA was implanted. Helical strips of carotid arteries were mounted in organ baths for measurement of isometric force development. Genistein was used as a tyrosine kinase inhibitor. Concentration-response curves to 5-hydroxytryptamine (5-HT) shifted to the right by genistein in both DOCA-salt hypertensive and control rats. Although the sensitivity to genistein was similar between the two groups, the maximum force generation by 5-HT was less inhibited by genistein in arteries from DOCA-salt hypertensive rats than in those from controls. Genistein-induced relaxations were attenuated in arteries from DOCA-salt rats. Genistein affected the contraction to phorbol 12, 13-dibutyrate (PDBu) neither in DOCA-salt nor in control arteries. These observations suggest that tyrosine kinase is involved in 5-HT-induced vascular contraction, of which role is reduced in DOCA-salt hypertension.

Role of Tyrosine Kinases in Vascular Contraction in Deoxycorticosterone Acetate-Salt Hypertensive Rats

Cheol Ho Yeum,Jae Yeoul Jun,Hyo Sub Choi 대한생리학회-대한약리학회 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.5

<P> It has been known that activation of tyrosine kinases is involved in signal transduction. Role of the tyrosine kinase in vascular smooth muscle contraction was examined in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy, one week after which they were subcutaneously implanted with DOCA (200 mg/kg) and supplied with 1% NaCl and 0.2% KCl drinking water for 4∼6 weeks. Control rats were treated the same except for that no DOCA was implanted. Helical strips of carotid arteries were mounted in organ baths for measurement of isometric force development. Genistein was used as a tyrosine kinase inhibitor. Concentration-response curves to 5-hydroxytryptamine (5-HT) shifted to the right by genistein in both DOCA-salt hypertensive and control rats. Although the sensitivity to genistein was similar between the two groups, the maximum force generation by 5-HT was less inhibited by genistein in arteries from DOCA-salt hypertensive rats than in those from controls. Genistein-induced relaxations were attenuated in arteries from DOCA-salt rats. Genistein affected the contraction to phorbol 12, 13-dibutyrate (PDBu) neither in DOCA-salt nor in control arteries. These observations suggest that tyrosine kinase is involved in 5-HT-induced vascular contraction, of which role is reduced in DOCA-salt hypertension.

TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway

Fen Xue,Lifen Liu,Xueqiang Tao,Weipei Zhu 대한부인종양학회 2024 Journal of Gynecologic Oncology Vol.35 No.5

Objective: Src homology phosphotyrosin phosphatase 2 (SHP2) has been implicated in the progression of several cancer types. However, its function in endometrial cancer (EC) remains unclear. Here, we report that the ten-eleven translocation 3 (TET3)-mediated DNA demethylation modification is responsible for the oncogenic role of SHP2 in EC and explore the detailed mechanism. Methods: The transcriptomic differences between EC tissues and control tissues were analyzed using bioinformatics tools, followed by protein-protein interaction network establishment. EC cells were treated with shRNA targeting SHP2 alone or in combination with isoprocurcumenol, an epidermal growth factor receptor (EGFR) signaling activator. The cell biological behavior was examined using cell counting kit-8, colony formation, flow cytometry, scratch assay, and transwell assays, and the median inhibition concentration values to medroxyprogesterone acetate/gefitinib were calculated. The binding of TET3 to the SHP2 promoter was verified. EC cells with TET3 knockdown and combined with SHP2 overexpression were selected to construct tumor xenografts in mice. Results: TET3 and SHP2 were overexpressed in EC cells. TET3 bound to the SHP2 promoter, thereby increasing the DNA hydroxymethylation modification and activating SHP2 to induce the EGFR/extracellular signal-regulated kinase (ERK) pathway. Knockdown of TET3 or SHP2 inhibited EC cell malignant aggressiveness and impaired the EGFR/ERK pathway. Silencing of TET3 inhibited the tumorigenic capacity of EC cells, and ectopic expression of SHP2 or isoprocurcumenol reversed the inhibitory effect of TET3 knockdown on the biological activity of EC cells. Conclusion: TET3 promoted the DNA demethylation modification in the SHP2 promoter and activated SHP2, thus activating the EGFR/ERK pathway and leading to EC progression.