• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.184-184
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• 1996

Nitric Oxide Synthase(NO synthase: EC.1.14.13.39)는 생체내에서 L-arginine을 기질로 하여 nitric oxide(NO)와 L-citrulline의 생성을 매개하는 효소로서 뇌, 간장, 신장, 체장등 대부분의 주요장기와 근육세포, 신경세포 등 거의 모든 조직에 분포하고 있다. NO synthase에 의해 생성되는 NO는 혈관이완작용, 신경전달 물질로서의 작용, 면역 담당세포에서의 세포 독작용 등 많은 생리현상에 중요한 역할을 하는 것으로 알려져 있다. 특히 체장에서는 췌외분비 기능의 항진에 있어 세포내 cGMP level의 변동이 NO와 연관된다는 사실에 주목하고 있으며 본연구실에서도 이에 관한 연구가 진행중이다. 따라서 본 연구에서는 소 췌조직의 100,000$\times$g cytosol을 효소원으로 하여 다음과 같이 NO synthase의 분리, 정제를 시행하였다. Ammonium sulfate로 30%(176g solid ammonium sulfate/$\ell$) 포화, 침전 후 2',5'-ADP agarose 및 calmodulin-agarose affinity chromatography를 연속적으로 시행하여 NO synthase를 분리하였으며 electrophoresis상에서 약 160kd의 분자량을 나타내었다.

• BMB Reports
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• 제30권6호
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• pp.443-447
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• 1997

We have developed a novel method for assays of spermidine and spermine synthase (aminopropyltransferase) activities using antibody against 5'-deoxy-5'-methylthioadenosine (MTA). A new assay is reported here which is based on the observation that MTA is formed as a stoichiometric by-product of the spermidine and spermine synthases reactions. In order to determine MTA, a radioimmunoassay method with sensitivity and rapidity was used. (Lee and Cho, 1997). In this assay, adenine must be added in the reaction mixture, since it effectively inhibits the action of MTA phosphorylase by which MTA is metabolized. This assay is a improvement in term of sensitivity and time saving, compared to the currently used methods. It has a level of sensitivity (100 fmol) sufficient to monitor aminopropyltransferase activities in incubations containing as little as $10{\mu}g$ protein prepared from rat tissue homogenate. The results obtained showed that this method is particularly useful for cultured cells with low enzyme concentration. Moreover, this assay has the advantage which allows studies using alternative substrates (other amines). Spermidine synthase activity was high in rat liver, but low in rat kidney. The activity of spermine synthase was in most rat tissues very low as compared to that of spermidine synthase, but was high in brain.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.668-673
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• 2013

A recombinant E. coli co-expressing ALA synthase (hemA), NADP-dependent malic enzyme (maeB), and dicarboxylic acid transporter (dctA) was reported to synthesize porphyrin derivatives including iron-containing heme. To enhance the synthesis of bacterial heme, five genes of the porphyrin biosynthetic pathway [pantothenate kinase (coaA), ALA dehydratase (hemB), 1-hydroxymethylbilane synthase (hemC), uroporphyrinogen III synthase (hemD), and uroporphyrinogen III decarboxylase (hemE)] were amplified in the recombinant E. coli co-expressing hemA-maeB-dctA. Pantothenate kinase expression enabled the recombinant E. coli to accumulate intracellular CoA. Intracellular ALA was the most enhanced by uroporphyrinogen III synthase expression, porphobilinogen was the most enhanced by ALA dehydratase expression, uroporphyrin and coproporphyrin were the most enhanced by 1-hydroxymethylbilane synthase expression. The strain co-expressing coaA, hemA, maeB, and dctA produced heme of $0.49{\mu}mol/g$-DCW, which was twice as much from the strain without coaA expression. Further pathway gene amplifications for the porphyrin derivatives are discussed based on the results.

• Journal of the Korean Wood Science and Technology
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• 제46권4호
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• pp.415-422
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• 2018

Terpenoids have a wide range of biological functions and have extensive applications in the pharmaceutical, cosmetic, and flavoring industry. The white-rot fungus, Polyporus brumalis, is able to synthesize terpenoids via terpene synthase, which catalyzes an important step that forms a large variety of sesquiterpene products from farnesyl pyrophosphate (FPP). To improve the production of sesquiterpenes, the terpene synthase gene was isolated from Polyporus brumalis and was heterologously transformed into a Pichia pastoris strain. The open reading frame of the isolated gene (approximately 1.2 kb) was inserted into Pichia pastoris to obtain a recombinant enzyme. Five transformants were obtained and the expression of terpene synthase was analyzed at the transcript level by reverse transcription PCR (polymerase chain reaction) and at the protein level by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Expression of the terpene synthase gene product was elevated in the transformants and as expected the molecular weight of the protein was approximately 45 kDa. These recombinant enzymes have potential practical applications and future studies should focus on their functional characterization.

• BMB Reports
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• 제41권1호
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• pp.72-78
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• 2008

Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.

• Toxicological Research
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• 제24권3호
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• pp.169-174
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• 2008

This study examined the mechanisms underlying the effects of the vasorelaxation active substance(VAS), dimethyladenosine-5'-L-arabinose, and its partial purification fraction on nitric oxide synthase in improving erectile dysfunction with particular focus on the nitric oxide (NO)-cGMP pathways. Two rat models, 9-month-old SD rats and 11-month-old SD rats, were given VAS(40 mg/kg per day) for 4 days, The aqueous fraction of silworm male pupae extract; semi-purified VAS(100 mg/kg per day) for 10 days, respectively. The NOS activities of the following three enzymes were examined: neuronal NO synthase(nNOS), inducible NOS(iNOS), endothelial NOS(eNOS), vascular endothelial growth factor on endothelial cells(VEGF) and anti-inflammation effect of Tumor necrosis factor-$\alpha$. The results showed increases in the nitric oxide synthase activities. Western blotting of the tissue homogenate showed an increase in the nNOS level in the brain and tongue, and an increase in the endothelial NO synthase(eNOS) level in penis. However, there was little association with VEGF production in HUVEC endothelial cells and no relationship with TNF-$\alpha$ which showed low levels.

• Journal of Ginseng Research
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• 제32권2호
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• pp.114-119
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• 2008

Terpene synthase plays a key role in biosynthesis of triterpene saponins (ginsenosides) and is intermediate in the biosynthesis of a number of secondary metabolites. A terpene synthase (PgTPS) cDNA was isolated and characterized from the root of Panax ginseng c.A. Meyer. The deduced amino acid sequence of PgTPS showed a similarity with A. deliciosa (AAX16121) 61%, V. vinifera (AAS66357) 61%, L. hirsutum (AAG41891) 55%, M. truncatula (AAV36464) 52%. And the segment of a terpene synthase gene was amplified by reverse transcriptase-polymerase chain reaction (RTPCR). We studied expression of terpene synthase under stressful conditions like chilling, salt, UV, and heavy metal stress treatment. Expression of PgTPS was increased gradually after exposure to stresses such as chilling, salt, and UV illumination. But its transcription seems to be reduced by cadmium and copper treatment.

• 분석과학
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• 제5권4호
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• pp.477-484
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• 1992

형질 전환된 사철느타리버섯으로부터 초원심분리 및 설탕밀도 기울기법으로 44% 층에서 미토콘드리아를 분리정제하였다. ATPase와 ATP synthase의 최적 활성조건은 각각 pH 7.4, $60^{\circ}C$ 및 pH 7.5, $57^{\circ}C$였고, km값은 11.6mM과 8.4mM였다. ATPase는 기질농도 5~6mM의 ATP에서, ATP synthase는 5~10mM ADP 농도에서 활성이 높으며, 그 이상의 농도에서는 기질 저해를 받았다. ATPase/ATP synthase 모두 $Mg^{2+}$ 의존성 효소로 $G_{418}$으로 비경쟁적인 저해를 받았다. 효소의 아미노산 분석결과 hydrophobic 아미노산 잔기는 50.5%, small 아미노산 잔기는 56.1%, hydrogen bonding 아미노산 잔기는 43.7%, helix breaking 아미노산 잔기는 55.2%였다. 인지질을 분석한 결과 phosphatidyl glycerol, phosphatidyl choline 및 phosphatidyl ethanolamine으로 구성되었고 phosphatidyl serine과 phosphatidyl inositol은 전혀 없었다. 포화 지방산은 palmitate(51.31%)와 stearate(18.32%)의 함량이 많았고, 불포화 지방산($C_{18:1}$, $C_{18:2}$ 및 $C_{16:1}$)의 함량도 많았다.

• 생명과학회지
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• 제22권8호
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• pp.1024-1033
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• 2012

Kocuria gwangalliensis로부터 카로티노이드 생합성 경로의 첫 번째 단계 기질인 geranylgeranyl pyrophosphate (GGPP)를 생합성하는 GGPP synthase (CrtE)를 암호화하고 있는 crtE를 클로닝 하여 이를 KgGGPP로 명명하였다. 기존 세균에서 밝혀진 GGPP synthase의 아미노산 서열을 NCBI에서 검색하여 KgGGPP synthase의 아미노산 서열과 비교한 결과 Kocuria rhizophila와 59.6%의 상동성을 가지는 것을 확인하였다. crtE 유전자를 대장균에서 발현 시키기 위하여 pCcrtE 재조합 DNA를 구축하였고, 이를 대장균에서 발현시킨 결과 약 41 kDa의 재조합 단백질이 과발현 됨을 확인 할 수 있었으며, 이 단백질은 기존 세균에서 밝혀진 GGPP synthase와 유사한 분자량을 가지고 있다는 것을 알 수 있었다. CrtE 재조합 단백질의 활성을 분석하기 위하여 대장균 내에서 라이코펜의 생합성을 유도 하였다. 대장균의 경우 메발론산 경로를 통하여 FPP와 IPP를 생합성 하지만 crtE, crtB, crtI 유전자가 없기 때문에 라이코펜을 생합성 하지는 못한다. 대장균 내에서 라이코펜의 생합성을 위해서는 crtE, crtB, crtI 유전자의 발현이 필수적으로 요구되기 때문에 crtB, crtI 유전자의 경우는 P. haeundaensis에서 유래한 유전자를 이용하여 pRScrtBI 재조합 DNA를 구축하여 그 발현을 유도하였다. 상기 두 재조합 DNA를 대장균에서 공발현 시켰으며, HPLC 분석법을 이용하여 대장균 내에서 라이코펜의 생산 유무에 따른 KgGGPP synthase의 활성을 분석하였다.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1970-1975
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• 2007

Squalene synthase plays an important role in the cholesterol biosynthetic pathway. Inhibiting this enzyme in hypercholesterolemia can lower not only plasma cholesterol but also plasma triglyceride levels. A squalene synthase inhibitor was screened from Prunus mume fruit, and then purified via sequential processes of ethanol extraction, HP-20 column chromatography, ethyl acetate extraction, silica gel column chromatography, and crystallization. The squalene synthase inhibitor was identified as chlorogenic acid with a molecular mass of 354 Da and a molecular formula of $C_{16}H_{18}O_9$ based on UV spectrophotometry, $^1H$ and $^{13}C$ NMRs, and mass spectrometry. Chlorogenic acid inhibited the squalene synthase of pig liver with an $IC_{50}$ level of 100 nM. Since chlorogenic acid was an effective inhibitor against the squalene synthase of an animal source, it may be a potential therapeutic agent for hypercholesterolemia.