Isolation of Isocitrate Lyase from Malonate-Grown Pseudomonas fluorescens ATCC 11250

장세헌,김유삼,Jang, Sei-Heon,Kim, Yu-Sam 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.2

Ps. fluorescens을 malonate가 함유된 배지에서 배양하였을 때 glyoxylate cycle의 주 효소인 isocitrate lyase가 유도되었다. malonyl-CoA decarboxylase가 같은 조건에서 유도된다는 사실을 고려할 때 malonate는 다음과 같은 경로를 따라 대사가 이루어 진다고 간주된다. malonate${\rightarrow}$malonyl-CoA${\rightarrow}$acetyl-CoA${\rightarrow}$glyoxylate cycle. malonate 대사 과정에서 유도된 isocitrate lyase을 황산암모니움에 의한 분별 침전, Sephadex G-200, DEAE-cellulose 컬럼 크로마토그라피에 의하여 약 30배 정제 하였고, 이때의 효소 회수율은 약 24%이었다. 이와 같이 얻은 isocitrate lyase를 polyacrylamide gel에서 전기 영동한 결과 2개의 띠를 나타냈으며, 그 중 하나의 띠에만 효소의 활성이 검출되었다. Sephadex G-200 컬럼 크로마토그라피에 의해 측정된 이 효소의 분자의 크기는 155,000 dalton이었다. 이 효소의 최적 pH는 7.5이었다. 기질인 isocitrate에 대하여 반응속도를 측정한 결과 Michaelis-Menten 반응속도론에 부합하였으며, 이때의 Km 및 Vmax는 각각 0.27 mM과 4.14 ${\mu}$moles/min/mg이었다. 효소 활성이 SH 저해물질인 p-chloromercuric phenylsulfonic acid에 의하여 억제되었다. 따라서 효소 활성 부위 또는 그 근처에 SH기 가 있음을 의미한다. Malonate-grown Pseudomonas fluorescens induced isocitrate lyase which is one of key enzymes of glyoxylate cycle. In consideration of the previous investigation that molonate-grown Pseudomonas also induced malonyl-CoA decarboxylase, the following metabolic pathway is suggested; malonate-malonyl-CoA-acetyl-CoA-glyoxylate cycle. The malonate induced isocitrate lyase was purified nearly 30 folds by the combination of ammonium sulfate precipitation, gel filtration and DEAE-cellulose ion exchange chromatography. Polyacrylamide gel electrophoresis showed two major bands, which one contained the enzymatic activity. The molecular weight of the enzyme was 155,0000 and its pH optimum was pH 7.5. The enzyme showed a typical Michaelis-Menten substrate saturation, with an apparent Km and Vmax of 0.27 mM and 4.14 ${\mu}$moles/min/mg, respectively. Thiol-directed reagent, p-chloromercuric phenyl sulfonic acid, inhibited this enzyme.

Malonate 배지에 배양한 Pseudomonas fluorescens 로 부터 Isocitrate Lyase 의 정제

장세헌,김유삼 ( Sei Heon Jang,Yu Sam Kim ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.2

Malonate-grown Pseudomonas fluorescens induced isocitrate lyase which is one of key enzymes of glyoxylate cycle. In consideration of the previous investigation that molonate-grown Pseudomonas also induced malonyl-CoA decarboxylase, the following metabolic pathway is suggested; malonate-malonyl-CoA-acetyl-CoA-glyoxylate cycle. The malonate induced isocitrate lyase was purified nearly 30 folds by the combination of ammonium sulfate precipitation, gel filtration and DEAE-cellulose ion exchange chromatography. Polyacrylamide gel electrophoresis showed two major bands, which one contained the enzymatic activity. The molecular weight of the enzyme was 155, 0000 and its pH optimum was pH 7. 5. The enayme showed a typical Michaelis-Menten substrate saturation, with an apparent Km and Vmax of 0.27 mM and 4. 14 μmole/min/㎎, respectively. Thiol-directed reagent, p-chloromercuric phenyl sulfonic acid, inhibited this enzyme.

Novel insight into the role of thiamine for the growth of a lichen-associated Arctic bacterium, Sphingomonas sp., in the light

팜눙,팜코이,이창우,장세헌,Pham, Nhung,Pham, Khoi,Lee, ChangWoo,Jang, Sei-Heon The Microbiological Society of Korea 2019 미생물학회지 Vol.55 No.1

Bacteria in the polar region are under strong light and ultraviolet radiation. In this study, we investigated the effects of light on the growth of a psychrophilic bacterium, Sphingomonas sp. PAMC 26621, isolated from an Arctic lichen Cetraria sp. The growth of the strain in the light was lower than that in the dark. Surprisingly, thiamine increased the growth of Sphingomonas sp. PAMC 26621 in M9 minimal medium under light conditions. Thiamine increased the growth of the strain in a concentration-dependent manner along with ascorbic acid. N-acetylcysteine had no effect on the growth of the strain in the light. Thiamine and ascorbic acid also increased the activities of glucose-6-phosphate dehydrogenase and superoxide dismutase. The results of this study indicate that thiamine provided by the lichen symbiosis system plays an important role in light-induced oxidative stress in this Arctic bacterium as an antioxidant. Our study provide insight into the biochemistry and physiology of Arctic bacteria under strong light and ultraviolet radiation. 극지에 서식하는 세균은 강한 빛과 자외선을 받는다. 이 연구에서 우리는 북극에 서식하는 지의류 Cetraria sp.에서 분리한 호냉성 세균 Sphingomonas sp. PAMC 26621의 생장에 빛이 미치는 영향을 조사하였다. 이 세균은 암조건에서 명조건에서 보다 생장이 느렸다. 놀랍게도, 이 세균은 M9 최소배지에 티아민 혹은 아스코브르산을 첨가하면 명조건에서 생장이 증가하였지만, N-acetylcysteine을 첨가한 배지에서는 생장의 변화가 없었다. 첨가한 티아민과 아스코브르산은 포도당-6 인산 탈수소효소와 항산화 효소의 활성을 증가시켰다. 이 연구의 결과는 지의류와의 공생에서 제공된 티아민이 Sphingomonas sp. PAMC26621의 빛에 의한 산화적 스트레스를 완화시키는 항산화제 역할을 함을 의미한다. 이 연구는 강한 빛과 자외선이 만연한 북극에 서식하는 세균에 대한 생리적, 생화학적 관점에서 고찰할 점을 제시한다.

Identification of Phosphoproteins Induced by AT1 Receptor Blocker Losartan

Chang Woo Lee(이창우),Mijin Kim(김미진),Sei-Heon Jang(장세헌) 한국생명과학회 2008 생명과학회지 Vol.18 No.7

안지오텐신Ⅱ 수용체(AT1 수용체)는 혈관수축과 체내 전해질이온 조절에 중요한 역할을 한다. AT1 수용체 길항제(ARB)는 고혈압 치료에 이용되며 최근에는 당뇨병을 포함한 대사질환에 효능이 있음이 알려져 있다. 이 연구에서는 ARB 처리 후 세포 내 인산화단백질에 인산화가 일어나는지를 antibody array를 이용하여 실험하였다. 아미노산세린 및 트레오닌에 인산화되는 단백질 6개, 티로신에 인산화되는 단백질 12개에 대한 항체를 선정하여 nitrocellulose membrane에 부착시켰다. AT1 수용체를 발현한 COS-1 세포에 로사탄(losartan)을 처리하였을 때 small GTPase인 RhoA의 세린 잔기에 인산화가 20% 증가함을 관찰하였다. RhoA는 세포골격의 재배열에 중요한 역할을 하며 세린 잔기에 인산화가 되면 활성이 억제된다. 본 연구결과로부터 ARB가 AT1 수용체에 의한 혈관수축을 억제할 뿐만 아니라 새로운 세포 신호를 생성함을 알 수 있다. The angiotensin Ⅱ receptor (AT₁R) antagonists are effective in treating patients with hypertension and showed beneficial effects in diabetes and other metabolic diseases. The beneficial effects of AT₁R antagonists are mainly considered to be from inhibition of Ang Ⅱ-AT₁R signaling pathway such as the activation of NADPH oxidase and the generation of reactive oxygen species. In this study, we examined whether antagonist of the AT1R could account for phosphorylation of proteins in cells using antibody array. We have selected 6 proteins with Ser/Thr-phosphorylation sites and 12 proteins with Tyr-phosphorylation sites based on literature search. Upon AT1R antagonist losartan treatment to serum-starved COS-1 cells, there was ~20% increase of Ser phosphorylation in small GTPase RhoA. RhoA is known to be responsible for cytoskeleton rearrangement and is down-regulated upon Ser phosphorylation in vivo. Our finding provides a new insight into the mechanism and signaling pathway of the AT1R antagonist in cells.

Field Bioassay for Longhorn Pine Sawyer Beetle Monochamus alternatus (Coleoptera: Cerambycidae) in Korea Based on Aggregation Pheromone 2-(Undecyloxy)ethanol

Sung-Min Lee(이성민),Do Kyung Hong(홍도경),Jongseong Park(박종성),Jinho Lee(이진호),Sei-Heon Jang(장세헌),ChangWoo Lee(이창우) 한국생명과학회 2015 생명과학회지 Vol.25 No.12

소나무재선충(Bursaphelenchus xylophilus)은 소나무재선충병을 유발하여 한국의 소나무 숲에 심각한 영향을 미치고 있다. 소나무재선충에 감염된 소나무는 일반적으로 훈증 또는 파쇄처리되지만 소나무재선충병 피해지역의 방제효과를 검증할 수 있는 효과적인 방법은 아직 없다. 본 연구에서는 소나무재선충병 방제효과 검증에 적합한 솔수염하늘소 집합페로몬의 농도를 알아보기 위해 2-(Undecyloxy)ethanol, 알파-피넨, 에탄올을 이용하여 경상북도 포항시의 소나무재선충병 피해지역에서 필드테스트를 시행하였다. 총 27마리의 솔수염하늘소가 유인되었으며 고농도(700 mg)의 집합페로몬을 사용한 트랩이 가장 효과적이었다. 집합페로몬 2-(Undecyloxy)ethanol은 솔수염하늘소 개체수 조사와 소나무재선충병 방제효과 검증에 사용할 수 있을 것으로 사료된다. The pinewood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) poses a serious threat to pine forests in Europe and East Asia, leading to a debilitating pine wilt disease. Infected pine trees in Korea are generally fumigated or crushed to small wood chips after felling. Although pine wilt disease often recurs in pest management sites, there are no adequate means to monitor the effectiveness of pest control measures in those sites. Recently, a male-produced aggregation pheromone, 2-(undecyloxy)ethanol, was shown to be useful for attracting several Monochamus species, which are vectors for the pinewood nematodes. In this study, we investigated the abilities of 2-(undecyloxy)ethanol at three different doses (175, 350, and 700 mg), as well as host plant volatiles (α-pinene and ethanol), to attract M. alternatus (Coleoptera: Cerambycidae) at a pine forest in Pohang, Korea where infected pine trees had been cut down and fumigated. Twenty-seven M. alternatus were captured in cross-vane panel traps made of polyethylene terephthalate bottles and acrylic sheets. The results indicate that a high dose of 2-(undecyloxy)ethanol (700 mg per trap) is the most effective for attracting M. alternatus. The aggregation pheromone could be used to monitor the effectiveness of pest control measures as well as M. alternatus populations.

계배 추출물에 의해 분화된 계배 근관의 RhoA 유전자 클로닝

양재섭,장세헌,강봉석,조영준,유병제,박종광 한국유전학회 1997 Genes & Genomics Vol.19 No.1

The differentiation of skeletal muscle cells includes the morphogenesis from mononucleated myoblasts to multinucleated myotubes by cell fusion and regulation of gene expression at the transcriptional, posttranscriptional, and translational levels. We have screened a cDNA library from chick embryonic myotubes by differential plaque hybridization to isolate genes involved in myoblast differentiation in vitro. In this study, we found a cDNA clone, pJS323, that hybridized with 1.9 and 1.5kb transcripts enhanced about 15-fold when myoblasts were grown in differentiation media (8102), suggesting that these transcripts were accumulated during the myogenic differentiation in vitro. The deduced amino acid sequence of pJS323 cDNA showed a perfect homology to the partial sequence of rhoA, which is known as ras-related GTP binding protein of human, bovine, and canine. Our results suggest that pJS323 gene may be a chicken rhoA and play a role in the myoblast differentiation.

Production of Monoclonal Antibodies to Yeast Mitichondrial RNA Polymerase Specificity Factor

Kim, Kil-Woong,Jang, Sei-Heon 大邱大學校附設 基礎科學硏究所 1998 基礎科學硏究 Vol.14 No.2

Transcription of mitochondrial DNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mitochondrial RNA polymerase specificity factor which is a 43 kDa polypeptide encoded by the nuclear MTF1 gene. Mtf1p has only limited amino acid sequence homologue to bacterial sigma factors, but functions in many ways like sigma in that it is required for promoter recognition and initiation of transcription. To analyze the core-binding region of Mtf1p, monoclonal antibodies to this protein was prepared. Recombinant Mtf1p overproduced in E. coif was purified to near homogeneity and used to raise monoclonal antibodies (mAbs). From fused cells screened for Mtf1p's mAbs by immunodot blot analysis, 10 positive clones were initially isolated. Furhter analysis of positive clones by Western blotting resulted in 4 mAbs of Mtf1p.

Cloning and Nucleotide Sequence of cDNA CHK23 for Translationally Controlled Tumor Protein p23 Homolog in Chick Embryonic Myoblasts

양재섭,장세헌,강봉석,조영준,유병제 한국유전학회 1996 Genes & Genomics Vol.18 No.2

The differentiation of skeletal muscle cells includes fusion of mononucleated myoblasts into multinucleated myotubes and regulation of differentiation-related gene expression at the transcriptional, translational, and posttranslational levels. In order to study the muscle gene regulation during the differentiation of cultured chicken embryonic myoblasts, we had constructed cDNA library from myotubes and identified a developmentally regulated gene, named CHK23, from the myotube cDNA library by differential plaque hybridization. Using this cDNA clone as a probe, we observed that the amount of CHK23 transcripts is more abundant in the cells cultured in differentiation media(8102) than those cultured in growth media(811) or inhibition media(910) as judged from RNA dot blot analysis. Northern analysis showed that a 900 nucleotide transcript of CHK23 was preferentially detected in myotubes. Thus, CHK23 transcripts apparently accumulated during differentiation in vitro. The CHK23 has an open reading frame that codes for a protein of 172 amino acids. The deduced amino acid sequence from CHK23 cDNA was 88% and 90% identical to those of a growth-related tumor protein p23 in murine and human tumor cells. This striking sequence similarity among the mouse, human and chicken p23 suggests that these proteins may have very similar, if not identical, functions.