• BMB Reports
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• 제33권3호
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• pp.228-233
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• 2000

The steady-state investigation of the mechanism of Dhydroxyisovalerate dehydrogenase was performed in order to understand this type of kinetic patterns. The initial velocity was measured with various amounts of both substrates, NADPH and 2-ketoisovalerate. Double reciprocal plots gave patterns that conversed on or near the abscissa. Binding studies indicated that NADPH bound first to the enzyme. The product $NADP^+$ was found to be a competitive inhibitor with respect to NADPH at a constant concentration of 2-ketoisovalerate. However, it showed noncompetitive inhibition against 2-ketoisovalerate at a fixed amount of NADPH. Another product, D-hydroxyisovalerate, was a non-competitive inhibitor versus NADPH and 2-ketoisovalerate at constant levels of 2-ketoisovalerate and NADPH, respectively. These results were comparable with an ordered bi-bi mechanism, in which NADPH bound first to the enzyme, followed by the binding of 2- ketoisovalerate. $NADP^+$ is the last product to be released. The ordered reaction manner of D-hydroxyisovalerate dehydrogenase from 2-ketoisovalerate to D-hydroxyisovalerate allows the accurate regulation of valine metabolism and it may lead to the regulation of total biosynthesis of enniatins in the Fusarium species.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.82-82
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• 1995

Regulation of enzyme synthesis by transcriptional and translational control systems provides rather stable adaptation to change of amino acid level in the growth medium, while manipulation of enzyme activity through endproduct feedback inhibition represents rather short-term and reversible ways of adjusting metabolic fluctuation of amino acid level. Various control mechanisms interplay to regulate genes encoding enzymes for amino acid biosynthesis in the yeast, Sacchromyces cerevisiae. When amino acids are in short supply, genes under a cross-pathway regulatory mechanism Or general amino acid control (general control) increase their action, in which Gcn4p is the major positive regulator of gene expression. When cells are cultured in minimal medium, basal level expression is also regulated by supplementary control elements, where inorganic phosphate level is additionally involved. Most of amino acid biosynthetic genes are also regulated by the level of endproduct of the pathway. This pathway-specific regulatory mechanism is called specific amino acid control (specific controD, under which gene expression is reduced when endproduct is present in the medium. Derepression of a gene through general control can be usually overridden by repression through specific control, where the endproduct level of that particular pathway is high and not limiting. In this presentation, regulatory factors for basal level expression and general control of yeast amino acid biosynthesis will be discussed, m addition to pathway-specific repression patterns and interaction between CrOSS- and specific-control mechanisms. Preliminary results are also presented from the investigation of the cloned genes in the threonine biosynthetic pathway of the yeast. yeast.

• 생명과학회지
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• 제23권10호
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• pp.1252-1259
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• 2013

인간 암세포의 생육에 미치는 혈전용해효소(BK-1)의 영향을 조사하기 위해, 세포증식, 생존력, 형태변화 및 apoptosis 유도 등을 포함한 여러 가지 생화학적 실험을 하였다. 그 결과, AGS 인간 위장 암세포 및 T24 인간 방광 암세포상에의 BK-17 처리는 그 암세포들의 생존력 및 생율을 농도의존적 방법으로 감소시켰다. 현미경 관찰은, BK-17 처리에 의한 항 생육 효과는 막 수축, 세포의 rounding up, apoptotic bodies와 같은 형태학적 변화를 나타내었다. 특히, RT-PCR과 Western blotting data는, BK-17 처리가 항 apoptosis Bcl-2 군들 특히 Bcl-2, and $Bcl-X_L$의 down-regulation 그리고 AGS 세포에서, apoptosis 촉진 매개체 Bax와 Bad의 up-regulation를 유도했다는 것을 보여주었다. BK-17에 의해 유도된 AGS 세포의 apoptosis는 caspase-3, caspase-8 그리고 caspase-9의 단백질가수분해 활성과 관련이 있었다. 이상의 결과를 볼 때, BK-17은 apoptotic cell death의 유도와 밀접한 관련이 있다는 것을 보여주고 있다.

• Molecules and Cells
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• 제37권3호
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• pp.234-240
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• 2014

Cisplatin is one of the most potent chemotherapy agents. However, its use is limited due to its toxicity in normal tissues, including the kidney and ear. In particular, nephrotoxicity induced by cisplatin is closely associated with oxidative stress and inflammation. Heme oxygenase-1(HO-1), the rate-limiting enzyme in the heme metabolism, has been implicated in a various cellular processes, such as inflammatory injury and anti-oxidant/oxidant homeostasis. Capsaicin is reported to have therapeutic potential in cisplatin-induced renal failures. However, the mechanisms underlying its protective effects on cisplatin-induced nephrotoxicity remain largely unknown. Herein, we demonstrated that administration of capsaicin ameliorates cisplatin-induced renal dysfunction by assessing the levels of serum creatinine and blood urea nitrogen (BUN) as well as tissue histology. In addition, capsaicin treatment attenuates the expression of inflammatory mediators and oxidative stress markers for renal damage. We also found that capsaicin induces HO-1 expression in kidney tissues and HK-2 cells. Notably, the protective effects of capsaicin were completely abrogated by treatment with either the HO inhibitor ZnPP IX or HO-1 knockdown in HK-2 cells. These results suggest that capsaicin has protective effects against cisplatin-induced renal dysfunction through induction of HO-1 as well as inhibition oxidative stress and inflammation.

• BMB Reports
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• 제38권6호
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• pp.633-638
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• 2005

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase $A_2$ enzymes, cyclooxygenase (COX) enzymes, and various lineage-specific terminal prostanoid synthases. Prostaglandin E synthase (PGES), which isomerizes COX-derived $PGH_2$ specifically to $PGE_2$, occurs in multiple forms with distinct enzymatic properties, expressions, localizations and functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli, is down-regulated by anti inflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate $PGE_2$ production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes.

• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.719-723
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• 2014

Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.

• 인지과학
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• 제1권2호
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• pp.361-376
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• 1989

카테콜아민 생합성에 관여하는 마지막 효소인 phenylethanolamine Nmethyltransferase는 norepinephrine 을 epinephrine으로 전환시키는 중요한 효소이다. PNMT효소의 발현은 epinephrine 신경세표의 발현에 필수적이다.따라서 PNMT 유전자를 크로닝하여 그 구조를 결정하고,유전자 발현연구를 하는 것은 상당히 중요한 일이다.그러나 최근에 저자가 bovine 및 human cDA 를 처음으로 분리하여 그 구조를 보고한 것 외에는 아직까지 인간과 백서 전체 genomic DNA 의 분리 보고는 없다.이에 저자들은 인간과 백서 PNMT유전자의 전체구조와 여러종(species)사이의 진화적인 관계를 규명하기 위해서 human 과 Rat genomic library 를 만들고,이 library 를 이용하여 bovine cDNA 를 probe로 13.1kb와 13.2kb길이의 인간과 백서의 genomic clone 을 분리 크리닝하는데 성공하여 유전자의 구조적 규명하였다.

• Biomolecules & Therapeutics
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• 제17권3호
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• pp.293-298
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• 2009

Korean mistletoe lectin (KML) is the major component found in Viscum album var. (coloratum), displaying anti-cancer and immunostimulating activities. Even though it has been shown to boost host immune defense mechanisms, the regulatory roles of KML on the functional activation of macrophages have not been fully elucidated. In this study, regulatory mechanism of KML on macrophage-mediated immune responses was examined in terms of KML-mediated signaling event. KML clearly induced mRNA expression of tumor necrosis factor (TNF)-$\alpha$, the generation of reactive oxygen species (ROS) and phagocytic uptake in RAW264.7 cells. All of these events were strongly suppressed by U0126, whereas TNF-$\alpha$ mRNA was not diminished by SB203580 and SP600125, indicating ERK as a central enzyme managing KML-induced up-regulation of macrophage functions. Indeed, KML strongly induced the phosphorylation of ERK in a time-dependent manner without altering its total level. Therefore, these data suggest that ERK may be a major signaling enzyme with regulatory property toward various KML-mediated macrophage responses.

• 유기물자원화
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• 제1권1호
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• pp.115-125
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• 1993

Euglena gracilis 에서 arginine deiminas는 mitochondrial matrix 내에 존재한다. 고도로 정제된 효소가 0.23 nM의 $K_m$ 값을 갖고 효소반응을 하기 위해서는 $Co^{2+}$가 필요하며, 이때 최적 pH는 9.3~10.3이었다. Gel filtration에 의해서 얻어진 조효소 단백질의 분자량은 87,000이었으며, SDS-acrylamide gel electrophoresis에 의해 효소는 48,000의 분자량을 갖는 2개의 동일한 subunit로 구성되어 있음이 밝혀졌다. Euglena의 arginine deiminas는 sulfhydryl inhibitors에 의해서 활성이 저지되었는데, 이는 sulfhydryl group이 효소의 활성부위에 관여함을 나타낸다. 이 sulfhydryl group은 arginine이 효소와 결합하는데 있어서 negative cooperativity를 나타내었다. ${\beta}-guanidinopropionate$, ${\gamma}-guanidinobutyrate$와 guanidinosuccinate는 효소의 활성을 저지시키지 않는데 반하여, $L-^{\alpha}-amino-{\beta}-guanidino-propionate$, D-arginine, 그리고 L-homoarginine은 효소의 활성을 강력하게 저지시켰다. Citrulline과 ornithine에 의해서도 상당한 정도의 효소활성저지가 관찰되었다. 우리는 Euglena의 arginine deiminase의 독특한 성질이 Euglena 라는 원생동물 내에서 arginine 대사의 조절에 어떻게 영향을 미치는지를 토의하고자 한다.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.483-493
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• 2011

An acid phosphatase (HppA) activated by $NH_4Cl$ was purified 192- and 34-fold from the periplasmic and membrane fractions of Helicobacter pylori, respectively. SDS-polyacrylamide gel electrophoresis revealed that HppA from the latter appears to be several kilodaltons larger in molecular mass than from the former by about 24 kDa. Under acidic conditions (pH${\leq}$4.5), the enzyme activity was entirely dependent on the presence of certain mono- and/or divalent metal cations (e.g., $K^+$,$ NH_4{^+}$, and/or $Ni^{2+}$). In particular, $Ni^{2+}$ appeared to lower the enzyme's $K_m$ for the substrates, without changing $V_{max}$. The purified enzyme showed differential specificity against nucleotide substrates with pH; for example, the enzyme hydrolyzed adenosine nucleotides more rapidly at pH 5.5 than at pH 6.0, and vice versa for CTP or TTP. Analyses of the enzyme's N-terminal sequence and of an $HppA^-$ H. pylori mutant revealed that the purified enzyme is identical to rHppA, a cloned H. pylori class C acid phosphatase, and shown to be the sole bacterial 5'-nucleotidase uniquely activated by $NH_4Cl$. In contrast to wild type, $HppA^-$ H. pylori cells grew more slowly. Strikingly, they imported $Mg^{2+}$ at a markedly lowered rate, but assimilated urea rapidly, with a subsequent increase in extracellular pH. Moreover, mutant cells were much more sensitive to extracellular potassium ions, as well as to metronidazole, omeprazole, or thiophenol, with considerably lowered MIC values, than wild-type cells. From these data, we suggest that the role of the acid phosphatase HppA in H. pylori may extend beyond 5'-nucleotidase function to include cation-flux as well as pH regulation on the cell envelope.