• Journal of Pharmaceutical Investigation
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• 제36권5호
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• pp.315-320
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• 2006

Silymarin showed P-glycoprptein(P-gp) inhibitory activity as much as verapamil, a well-known P-gp inhibitor, by decreasing $IC_{50}$ value of daunomycin(DNM)($16.0{\pm}0.7{\mu}M$), increasing the DNM accumulation($224.9{\pm}3.2%$), and decreasing DNM efflux($58.5{\pm}6.7%$), concurrently. In this study, we clarified the mechanism of action of silymarin for P-gp inhibitory function. First, silymarin may bind to the ATP-binding site and thus, prevent ATP hydrolysis. Second, the P-gp inhibitory activity of silymarin is not related to changing the cellular P-gp level. Third, the cytotoxicity of silymarin was increased in the presence of verapamil, reflecting that silymarin is a competent P-gp substrate against verapamil in the P-gp-overexpressed adriamycin-resistant MCF-7 breast cancer(MCF-7/ADR) cells. Conclusively, silymarin had the P-gp inhibitory activity through the action of competent binding to the P-gp substrate-binding site. Therefore, silymarin can be a good candidate for safe and effective MDR reversing agent in clinical chemotherapy by administering concomitantly with anticancer drugs.

• KSBB Journal
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• 제9권2호
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• pp.127-132
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• 1994

Amyloglucosidase를 이용하여 설탕으로부터 fructooligosaccharides의 생산조건을 최적화하였다. Amyloglucosidase의 두 가지 활성 중 가수분해 활성에 대한 최적반응 pH 및 온도는 각각 4.0, $75^{\circ}C$ 이었고, 올리고당 합성에 대한 활성은 pH 5.5, 온도 $55^{\circ}C$에서 최고치를 나타내었다. 저농도 설탕에서는 가수분해 활성이 대단히 강하여 fructooligosaccharides의 합성능이 매우 낮았고, 550g/l 이상의 설탕을 기질로 이용하였을 때 최적반응조건에서 최대 41%의 fructooligosaccharides를 생산할 수 있었다. 또한 Amyloglucosidase는 설탕뿐만 아니라 fructooligosaccharides도 기질로 이용할 수 있었다.

• 생명과학회지
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• 제10권2호
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• pp.218-227
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• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.

• 한국전자통신학회논문지
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• 제11권10호
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• pp.969-982
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• 2016

최근 천연물로부터 신약 후보물질을 개발하려는 연구가 활발히 이루어지고 있다. 인체 내에서 천연물은 주로 효소에 의해 대사된다. 본 연구는 화합물의 인체내 대사반응과 주로 관련된 효소에 의한 대사반응의 특징을 연관규칙마이닝을 활용하여 분석한다. 화합물이 인체 내에서 효소 대사반응과 관련된 데이터를 BRENDA(: BRaunschweig ENzyme DAtabase)로부터 수집하였다. 수집된 데이터를 효소대사반응의 기본 틀에 근거하여, 대사물들을 기질대사물, 생성대사물, 억제대사물, 그리고 활성대사물들로 구분한다. 이러한 대사물들로 이루어진 기질대사물 트랜잭션, 생성대사물 트랜잭션, 그리고 모든 대사물들을 포함한 효소반응트랜잭션들을 구성하였다. 또한 종 정보를 반영한 6개의 트랜잭션들로 구성하였다. 연관규칙 마이닝을 활용하여 6개의 트랜잭션에서 빈발대사물 및 패턴을 분석하였다. 또한 대사물들 사이의 관련성을 분석하였다. 그 결과 효소대사반응에 참여하는 대사물들의 분포와 패턴을 식별할 수 있었다. 더욱이 기질에만 속하는 순수 기질대사물들을 식별하였고 이들 대부분이 아주 낮은 지지도임을 확인할 수 있었다. 연구결과는 순수 기질대사물은 효과적인 대사변환 예측 모델 개발에 활용될 수 있다.

• Toxicological Research
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• 제15권1호
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• pp.95-101
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• 1999

Cytochrome P450 (CYP) 3A4 metabolizes aflatoxin B1 (AFB1) to AFB1-exo-8,9-epoxide (8,9-epoxidation) and aflatoxin Q1 (AFQ1; 3$\alpha$-hydroxylation) simultaneously. We investigated whether each metabolite was formed via its own binding site of CAP3A4 active site. Kinetics of the formation of the two metabolites were sigmoidal and consistent with the kinetics of substrate activation. The HIll model predicted that two substrate binding wites are involved in the oxidationof AFB1 by CYP3A4. Dehydronifedipine, a metabolite of nifedipine generated by CYP3A4, inhibited the formation of AFQ1 without any inhibition in the formation of AFB1-exo-8,9-epoxidation. Dehydronifedipine was found to act as a reversible competitive inhibitor against 3$\alpha$-hydroxylation of AFB1. Vmax and S0.5 of the 8,9-epoxidation were not changed in the presence of 0, 50, or 100 $\mu\textrm{M}$ dehydronifedipine. S0.5 of 3$\alpha$-hydroxylation was increased from 58$\pm$4 $\mu\textrm{M}$ to 111$\pm$8 $\mu\textrm{M}$ in the presence of 100 $\mu\textrm{M}$ nifedipine whereas Vmax was not changed. These results suggest that there exist two independent binding sites in the active site of CAP3A4 . One binding site is responsible for AFB1-exo-8,9-epoxidation and the other is involved in 3$\alpha$-hydroxylation of AFB1. Dehydronifedipine might selectively bind to the site which is responsible for the formation of AFQ1 in the active site of CYP3A4.

• BMB Reports
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• 제29권6호
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• pp.519-524
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• 1996

Peptidylprolyl cis-trans isomerase (PPlase) catalyzes the cis-trans isomerization of prolyl peptide and facilitates the folding of cellular proteins and peptides. PPlase consists of two distinct immunophilins, each specifically binding to the immunosupressive drug cyclosporin A (CsA) or FK506, respectively. A PPlase was isolated and partially purified from porcine spleen. The molecular weight of porcine spleen PPlase was determined to be ~14,000 on the basis of SDS-PAGE. The purified enzyme was strongly inhibited by FK506, but not by CsA. The inhibition constant and the true concentration of enzyme preparations were determined by active site titration using the tight binding inhibitor FK506: $K_{i}=18.7$ nM and $E_{t}=172$ nM. The equilibrium ratio of conformer. [cis]/[trans], of prolyl peptide substrates (N-Suc-Ala-Xaa-Pro-Phe-p-NA) in anhydrous trifluoroethanol/LiCl solvent system varied from 0.24 to 0.85 depending on the nature of Xaa. Overall. in this solvent-salt system, the populations of the cis conformer of substrates in equilibrium are higher than in an aqueous solution so that the substantial error caused by high background absorption can be reduced. The reactivities of porcine spleen PPlase are shown to be highly sensitive to changes in the structure of substrates. Thus, $k_{cat}/K_m$ value for the most reactive substrate (Xaa Leu) is $4.007+10^{6}M^{1}s^{1}$ and, is 2,636 fold higher than that for the least reactive peptide substrate tested, Xaa=Glu.

• Food Science and Biotechnology
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• 제17권1호
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• pp.161-165
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• 2008

The trypsin inhibitor activities (TIA) of various potato cultivars were evaluated by measuring the inhibition of trypsin inhibitor activity using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. The TIA values of 5 potato cultivars (1.99 to 2.88 mg/g) were significantly different among cultivars (p<0.05). When the TIA values of commercially processed potatoes were determined, no TIA was detected. During cooking, the $IT_{50}$ (time required to reach 50% inhibition of TIA) values were decreased as heating temperature and time increased. The ITso of moist heating was estimated to be 0.34 min at $100^{\circ}C$, whereas for deep-fat frying the $IT_{50}$ was 0.13 min at $180^{\circ}C$ and 5.28 min for oven baking at $100^{\circ}C$. The $IT_{50}$ value of microwave cooking was 0.194 min at medium heat, and which was similar to that of pressure cooking at $120^{\circ}C$ (0.185 min). Moreover, there was a negative relationship between temperature (${\geq}80^{\circ}C$) and $IT_{50}$ values ($R^2=0.99$, p<0.01). The TIA of potato was completely inactivated by moist heating at $100^{\circ}C$ within 5 min, whereas the pressure cooking at $120^{\circ}C$ and deep-fat frying at $180^{\circ}C$ within 60 and 30 sec, respectively. Based on our results, deep-fat frying is the most effective cooking method to reduce TIA in potatoes.

• 한국수산과학회지
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• 제46권2호
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• pp.119-128
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• 2013

A protease inhibitor was fractionated from fish eggs using methods based on protein solubility. Fractionation efficiency was evaluated with regard to percent recovery and total inhibitory activity (U). The fractionation of protease inhibitor (PI) from egg extracts of skipjack tuna (ST, Katsuwonus pelamis), yellowfin tuna (YT, Thunnus albacores), and Alaska pollock (AP, Theragra chalcogramma) was performed by precipitation with cold acetone or ammonium sulfate (AS). Fractions exhibiting the strongest inhibitory activity contained 20-40% (v/v) cold acetone or 40-60% saturated AS fractions. AS fractionation was more effective in isolating PI than was precipitation with acetone. The total inhibitory activity and percent recovery of fraction obtained with AS 40-60% toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 4,976 U and 24.2% for ST, 3,331 U and 38.1% for YT, and 4,750 U and 43.8% for AP, respectively. In comparisons against six commercial proteases, 40-80% AS fractions, made by combining the 40-60% and 60-80% AS fractions from fish egg extract, exhibited the strongest inhibition of trypsin when using a casein substrate. These results suggest that fish eggs act as serine protease inhibitors and may be useful for protease inhibition in foodstuffs.

• Applied Biological Chemistry
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• 제33권2호
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• pp.109-115
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• 1990

대두 STI의 열처리에 의한 불활성화에 L-cysteine 및 sodium sulfite의 첨가 효과를 조사하고, 대두 품종별 STI활성을 측정하였으며 활성도와 단백질 및 cysteine함량 그러고 소화율과의 관계를 비교하였다. 열처리에 의한 STI의 불활성화에 L-cysteine과 sodium sulfite의 첨가는 불활성화 효과를 크게 증가시켰으며 대두 품종간 불활성화 정도는 차이를 보이지 않았으며 L-cysteine과 sodium sulfite의 첨가는 불활성화된 STI의 재활생화를 크게 억제하였다. 품종별 STI의 활성도는 대두분말 g당 $64.7{\sim}86.47\;TIU$의 범위에 있었으며, 장백>힐>장엽, 광교>단엽>백운>단경>팔달, 새알, 덕유>황금 이었으며, 단백질 함량과 STI의 활성도와는 상관관계(r=-0.192)가 없는 것으로 나타났다. 품종별 cysteine 함량은 힐, 장백, 단경, 단엽, 황금, 백운, 장엽, 새알, 덕유, 광교, 활달의 순서이었으며, cysteine의 함량은 $73.5{\sim}40.0\;{\mu}mole/g$ 대두분 이었다. 또한 cysteine함량과 STI 활성도 사이에 정의 상관(r=0.6568)을 나타냈다. 품종별 소화율은 광교, 백운, 팔달, 단경, 새알, 힐, 장엽, 덕유, 황금, 장백, 단엽의 순서이었으며 $81.9{\sim}76.7%$ 정도이었다. 또한 소화율과 STI활성도 사이에는 부의 상관(r=-0.7695)을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1385-1389
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• 2007

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly ($CTD^{GG}$) and gly-ser-ser-gly ($CTD^{GSSG}$) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), $^{15}N-HSQC$,\;and\;^{31}P-NMR$ revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.