• 미생물학회지
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• 제41권3호
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• pp.157-163
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• 2005

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.

• 한국식품영양학회지
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• 제6권3호
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• pp.169-177
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• 1993

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 5. 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 NHtCl, 50M acetyl phosphate, 5mM ADP, 40M MgCl2, 300mM potassium phosphate buffer (pH 7.5), 5mM MnCl2, Under this condition, the most effective concentrations of enzyme were 70unit/ml glutamine synthetase and 99unit/ml acetate kinase. Under the optinum conditions, 98% of 100mM glutamate was converted to glutamine within 6 hours.

• BMB Reports
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• 제40권5호
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• pp.708-714
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• 2007

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

• 한국식품영양학회지
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• 제6권3호
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• pp.158-168
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• 1993

For the conversion of WAD to NADP, Immobilized Brevibacterium ammoniagenes cells with NAD kinase was coupled with ATP-generating system by acetate kinase. The membrane permeability of B. ammoniagenes was improved by toluene treatment of cells. The toluene treated B. ammoniagenes cells were immobilized for stable enzyme activity. Partially purified acetate kinase was used in the reaction system. The optimum conditions for the efficient conversion of UAD to WADP by energy-coupled system were investigated. B. ammoniagenes cells treated with toluene for the Improvement of membrane permeability showed 4.5 fold improved permeability in the conversion of NAD to NADP compared with Intact cells. 3% k-carrageenan as the immobilization matrix of B. ammoniagenes showed the best efficiency for the conversion of NAD to NADP The optimum conditions for the WAR to WARP conversion reaction coupled nth ATP-generating system were 10mM acetylphosphate, 5mM ADP 200mM inorganic phosphate, 10mM MgCl2, 250mg/ml Immobilized cells, 49.3mUnit/ml acetate kinase, pH 7.5 and 37$^{\circ}C$. Under the optimum conditions, 72% of 5mM(340mg/ml ) NAD was converted to UADP In 12 hours.

• BMB Reports
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• 제28권2호
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• pp.107-111
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• 1995

In malonate grown Pseudomonas fluorescens, malonate decarboxylase and acetyl-CoA synthetase were induced, whereas in Acinetobacter calcoaceticus malonate decarboxylase, acetate kinase, and phosphate acetyltransferase were induced. In both bacteria malonate decarboxylase was the first, key enzyme catalyzing the decarboxylation of malonate to acetate, and it was localized in the periplasmic space. Acetate thus formed was metabolized to acetyl-CoA directly by acetyl-CoA synthetase in Pseudomonas, and to acetyl-CoA via acetyl phosphate by acetate kinase and phosphate acetyltransferase in Acinetobacter.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1544-1553
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• 2013

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.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1127-1134
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• 2009

Escherichia coli excretes acetate during aerobic growth on glycolytic carbon sources, which has been explained as an overflow metabolism when the carbon flux into the cell exceeds the capacity of central metabolic pathways. Nonacetogenic growth of E. coli on gluconeogenic carbon sources like succinate or in carbon-limited slow growth conditions is believed an evidence for the explanation. However, we found that a strain defected in the acs (acetyl Co-A synthetase) gene, the product of which is involved in scavenging acetate, accumulated acetate even in succinate medium and in carbon-limited low growth rate condition, where as its isogenic parental strain did not. The acs promoter was inducible in noncatabolite repression condition, whereas the expression of the ackA-pta operon encoding acetate kinase and phosphotransacetylase for acetate synthesis was constitutive. Results in this study suggest that E. coli excretes and scavenges acetate simultaneously in the carbon-limited low growth condition and in nonacetogenic carbon source, and the activity of the acetate consumption pathway directly affects the accumulation level of acetate in the culture broth.

• Archives of Pharmacal Research
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• 제18권2호
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• pp.90-99
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• 1995

The effect of protein kinase C inhibitors, sturosporine and 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine(H7) on in vitro differentiation of erythroid progenitor cells which were isolated from spleens of mice infected with the anemia-inducing strain of Friend virus were examined. Erythropoietin-mediated differentitation of erythroid progenitor cells, as determined by the incorporation of $^{59}Fe$ into protoporphyrin, was inhibited by staurosporine and H7 in a concentration -dependent manner. Scatchard analysis of the $^3H-phorbol-12$, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of $^3H-phorbol-12$, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of $^3H-phorbol-12$, 13-dibutyrate binding sites per cell was approximately $3.7\times10^5$. Cytosonic protein kinase C was isolated from erthroid progenitor cells and then purified by sequential column chromatogrphy. Two isoforms of protein kinase C were found. Photoaffinity labeling of the purified protein kinase C samples with $^3H-phorbol-12$12-myristate 13-acetate followed by analysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autofluorography showed radiolabeled 82-KDa pepticles. Rediolabeling of the 82-KDa peptides with $^3H-phorbol-12$myristate 13-acete was almost completely blocked by excess unlabeled phorbol 12-myristate 13-acetate was almost 12-muristate 13-acetate-promoted phosphorylation with the puyrified protein kinase C samples showed that the phosphorylation of 82-KDa peptides was increased as the concentration of phorbol 12-myristate 13-acetate was increased from $10^{-8}M{\;}to{\;}10^{-4}$M. In light of the findings that erythroid progenitor cells possessed an abundance of protein kinase C and that stauroporine and H7 inhibited erythroid differentiation, it seemed likely that protein kinase C would play a role in the erythroid progenitor cell development.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2034-2042
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• 2015

A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25℃ for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'-triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

• The Korean Journal of Physiology and Pharmacology
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• 제17권1호
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• pp.51-56
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• 2013

Many intracellular proteins and signaling cascades contribute to the sensitivity of N-methyl-D-aspartate receptors (NMDARs). One such putative contributor is the serine/threonine kinase, protein kinase C (PKC). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes activation of extracellular signal-regulated kinase (ERK) and promotes the formation of new spines in cultured hippocampal neurons. The purpose of this study was to examine which PKC isoforms are responsible for the PMA-induced augmentation of long-term potentiation (LTP) in the CA1 stratum radiatum of the hippocampus in vitro and verify that this facilitation requires NMDAR activation. We found that PMA enhanced the induction of LTP by a single episode of theta-burst stimulation in a concentration-dependent manner without affecting to magnitude of baseline field excitatory postsynaptic potentials. Facilitation of LTP by PMA (200 nM) was blocked by the nonspecific PKC inhibitor, Ro 31-8220 ($10{\mu}M$); the selective $PKC{\delta}$ inhibitor, rottlerin ($1{\mu}M$); and the $PKC{\varepsilon}$ inhibitor, TAT-${\varepsilon}V1$-2 peptide (500 nM). Moreover, the NMDAR blocker DL-APV ($50{\mu}M$) prevented enhancement of LTP by PMA. Our results suggest that PMA contributes to synaptic plasticity in the nervous system via activation of $PKC{\delta}$ and/or $PKC{\varepsilon}$, and confirm that NMDAR activity is required for this effect.