• 미생물학회지
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• 제24권3호
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• pp.251-262
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• 1986

High-molecular-weight ${\beta}-glucosidase$ (EC 3.2.1.21) was purified from the culture filtrate of Trichoderma koningii through a four-step procedure including chromatography on Bio-Gel P-150, DEAE-Sephadex A-50 and SP-Sephadex C-50; and chromatofocusing on Polybuffer exchanger PBE 94. The molecular weight of the enzyme was determined to be about 101,000 by SDS-polyacrylamide gel electrophoreses, and the isoelectric point was estimated to be 4.96 by analytical isoelectric focusing. The temperature optimum for activity was about $55^{\circ}C$, and the pH optimumwas 3.5. The enzyme was considerably thermostable, for no loss of activity was observed when the enzyme was preincubated at $60^{\circ}C$ for 5h. Km values for cellobiose, gentiobiose, sophorose, salicin and $p-nitrophenyl-{\betha}-D-glucoside$ were 99.2, 14.7, 7.09, 3.15 and 0.70 mM, respectively, which indicates that the enzyme has much higher affinity towards $p-nitrophenyl-{\betha}-D-glucoside$ than towards the other substrates, especially cellobiose. Substrate inhibition by $p-nitrophenyl-{\betha}-D-glucoside$ and salicin was observed at the conecntrations exceeding 5mM. Gluconolactone was a powerful inhibitor against the action of the enzyme on $p-nitrophenyl-{\betha}-D-glucoside\;(K_i\;37.9\;{\mu}M)$, wherease glucose was much less effective ($K_i$ 1.95 mM). Inhibition was of the competitive type in each case. Transglucosylation activity was detected shen the readtion products formed from $p-nitrophenyl-{\betha}-D-glucoside$ by the enzyme were analysed using high-performance liquid chromatography.

• BMB Reports
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• 제30권5호
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• pp.370-377
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• 1997

A novel assay method is described for rapid quantitation of reaction rate of sterol ${\Delta}^7$-reductase (${\Delta}^7$-SR) which catalyzes reduction of the ${\Delta}^7$-double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-. ${\Delta}^7$-SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics ($K_m=210\;{\mu}M$, $V_{max}=1.93\;nmol/min/mg$) and inhibition of the rat hepatic ${\Delta}^7$-SR against well-studied cholesterol lowering agents such as triparanol ($IC_{50}=16\;{\mu}M$). 3-$\beta$-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) ($IC_{50}=5.2\;{\mu}M$), and trans-1.4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) ($IC_{50}=0.25\;{\mu}M$). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., ${\Delta}^7$-SR, sterol ${\Delta}^8$-isomerase, and sterol ${\Delta}^14$-reductase). ${\Delta}^7$-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound ($K_i=0.109\;{\mu}M$). Substrate specificity studies of the microsomal ${\Delta}^7$-SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. ${\Delta}^7$-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin ($\simeq$5.0-fold). Finally, microsomal ${\Delta}^7$-SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0~10%) precipitation, which should be suitable for further purification of the enzyme.

• Applied Biological Chemistry
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• 제30권3호
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• pp.285-290
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• 1987

사과의 건조, 가공 중의 갈변을 방지하기 위한 기초 조사로서 후지 사과로부터 추출한 crude peroxidase의 열불활성화와 갈변 저해제의 저해 효과 등을 조사하였다. Peroxidase의 최적 pH와 온도는 p-phenylenediamine과 $H_2O_2$를 기질로 하였을 때 각각 5.5와 $35^{\circ}C$이었고, 열불활성화 반응은 biphasic으로 heat labile fraction의 $E_a$와 Z값은 각각 48.2kcal/mol 과 $11.2^{\circ}C$, heat resistant fraction의 $E_a$와 Z값은 각각 36.3kcal/mol과 $14.9^{\circ}C$이었다. Peroxidase에 의한 갈변은 sodium diethyldithiocarbamate와 potassium metabisulfite는 10mM에서, L-cysteine과 ascorbic acid는 1mM에서 현저하게 저해되었다.

• The Korean Journal of Physiology and Pharmacology
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• 제11권2호
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• pp.71-77
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• 2007

Cisplatin treatment increases the excretion of inorganic phosphate in vivo. However, the mechanism by which cisplatin reduces phosphate uptake through renal proximal tubular cells has not yet been elucidated. We examined the effect of cisplatin on $Na^+$-dependent phosphate uptake in opossum kidney (OK) cells, an established proximal tubular cell line. Cells were exposed to cisplatin for an appropriate time period and phosphate uptake was measured using $[^{32}P]$-phosphate. Changes in the number of phosphate transporter in membranes were evaluated by kinetic analysis, $[^{14}C]$phosphonoformic acid binding, and Western blot analysis. Cisplatin inhibited phosphate uptake in a time- and dose-dependent manner, and also the $Na^+$-dependent uptake without altering $Na^+$-independent uptake. The cisplatin inhibition was not affected by the hydrogen peroxide scavenger catalase, but completely prevented by the hydroxyl radical scavenger dimethylthiourea. Antioxidants were ineffective in preventing the cisplatin-induced inhibition of phosphate uptake. Kinetic analysis indicated that cisplatin decreased Vmax of $Na^+$-dependent phosphate uptake without any change in the Km value. $Na^+$-dependent phosphonoformic acid binding was decreased by cisplatin treatment. Western blot analysis showed that cisplatin caused degradation of $Na^+$-dependent phosphate transporter protein. Taken together, these data suggest that cisplatin inhibits phosphate transport in renal proximal tubular cells through the reduction in the number of functional phosphate transport units. Such effects of cisplatin are mediated by production of hydroxyl radicals.

• 유기물자원화
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• 제15권4호
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• pp.131-137
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• 2007

황산염 환원조건에서의 리그노셀룰로오스의 분해에 대하여 고찰하였다. 특히, 리그닌에 대한 셀룰로오스의 비(C/L 비)를 각각 42.15, 4.59, 2.51, 1.14, 0.7로 하여 리그닌과 셀룰로오스의 상호작용에 대하여 고찰하였다. 셀룰로오스의 분해율은 1차 반응식에 의해 계산되어져, C/L 비가 감소할수록 반응상수는 감소하여 셀룰로오스의 분해에 대한 리그닌의 저해작용을 보여 주였다. 1차 반응식에 의한 반응상수와 리그닌의 함량의 증가에 대한 셀룰로오스의 분해율은 0.97의 $R^2$ 값을 가지며 로그힘수에 의해 표현할 수 있었다. 리그노셀룰로오스의 분해율 또한 C/L 비와 로그함수의 관계를 가지며 리그닌의 함량이 많을수록 감소하였다. 리그닌의 분해율은 C/L 비가 2.51 및 1.14인 조건에서 4.59 및 0.7의 조건보다 높게 나타나, 공동기질로서의 과도한 탄소원은 리그닌분해에 장애가 됨을 보여 주였다.

• 대한토목학회논문집
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• 제8권4호
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• pp.59-67
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• 1988

혐기성소화(嫌氣性消化)에 미치는 온도(溫度)의 영향(影響)을 가장 효과적으로 파악할 수 있는 체류시간(滯留時間) 5일(日)에서 인공(人工)슬러지를 대상으로 $35{\sim}55^{\circ}C$의 소화실험(消化實驗)을 행하였다. 소화온도증가(消化溫度增加)에 따라 메탄발효(醱酵)의 저해(沮害)가 감소하여, 중온(中溫) 및 중간영역(中間領域)의 온도(溫度)에서는 잔발효(酸醱酵)가 우세하였으나 $55^{\circ}C$에서는 활발한 메탄발효(醱酵)가 이루어졌다. 온도(溫度)의 변화(變化)는 미생물활성(微生物活性)뿐 아니라 슬러지의 물리(物理), 화학적(化學的) 특성(特性)에도 영향(影響)을 미친다고 추정된다. 또한 유입(流入) 슬러지의 희석(稀釋)에 의하여 소화저해(消化沮害)가 크게 감소하여 모든 온도(溫度)에서 활발한 메탄발효(醱酵)가 가능하였다. 소화효율(消化效率)은 수리학적(水理學的) 부하량외(負荷量外)에 유기물부하량(有機物負荷量)에도 지배받음을 알 수 있었다. 소화효율(消化效率)의 급격한 저해(沮害)가 발생된다고 보고된 $40{\sim}45^{\circ}C$에서도 뚜렷한 저해(沮害)는 없었다. 한편 소화온도증가(消化溫度增加)에 따라 소화(消化)슬러지의 침강특성(沈降特性)도 향상되었다.

• 대한토목학회논문집
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• 제7권3호
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• pp.1-11
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• 1987

중온소화(中溫消化)와 고온소화(高溫消化)의 차이점을 보다 명확히 규명하기 위하여 소화(消化)의 정지(停止)를 포함하는 폭넓은 실험을 행하였다. 연구결과, 정상소화(正常消化)와 소화조해(消化阻害)를 구분짓는 한계체류시간(限界滯留時間)이 존재하여, 중온(中溫)에서는 10일(日), 고온(高溫)에서는 5일(日)부근이었고 그 이하(以下)의 체류시간(滯留時間)에서는 소화(消化)의 조해(阻害)가 급격하게 발생하였는데 고온(高溫)에 비하여 중온(中溫)에서의 조해(阻害)정도가 매우 컸다. 중온소화(中溫消化)의 한계체류시간이상(限界滯留時間以上)에서는 소화(消化)상태가 거의 유사하였으나, 그이하(以下)의 체류시간(滯留時間)에서는 고온소화(高溫消化)가 월등히 양호(良好)하였으며, 고온(高溫)에서는 중온(中溫)에 비하여 소화(消化)슬러지발생량(發生量)이 감소하는 동시에 소화(消化)슬러지의 침강속도(沈降速度)가 크게 증가하고 여과비저항(濾過比抵抗)은 감소하였다. 한편 반응속도(反應速度)는 소화(消化)에 마치는 온도(溫度)와 체류시간(滯留時間)의 영향을 나타내는 데 유효하였다. 또한 온도(溫度)에 관계없이 반응속도(反應速度)가 증가함에 따라 가스발생속도(發生速度)가 선형적(線形的)으로 증가하였고 그 상관관계(相關關係)는 중온(中溫)과 고온(高溫)에서 거의 일치하였다.

• Bulletin of the Korean Chemical Society
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• 제32권12호
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• pp.4397-4402
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• 2011

The NF-${\kappa}B$ system of transcription factors plays a crucial role in inflammatory diseases, making it an important drug target. We combined quantitative structure activity relationships for predicting the activity of new compounds and quantitative dynamic models for the NF-${\kappa}B$ network with intracellular concentration models. GFA-MLR QSAR analysis was employed to determine the optimal QSAR equation. To validate the predictability of the $IKK{\beta}$ QSAR model for an external set of inhibitors, a set of ordinary differential equations and mass action kinetics were used for modeling the NF-${\kappa}B$ dynamic system. The reaction parameters were obtained from previously reported research. In the IKKb QSAR model, good cross-validated $q^2$ (0.782) and conventional $r^2$ (0.808) values demonstrated the correlation between the descriptors and each of their activities and reliably predicted the $IKK{\beta}$ activities. Using a developed simulation model of the NF-${\kappa}B$ signaling pathway, we demonstrated differences in $I{\kappa}B$ mRNA expression between normal and different inhibitory states. When the inhibition efficiency increased, inhibitor 1 (PS-1145) led to long-term oscillations. The combined computational modeling and NF-${\kappa}B$ dynamic simulations can be used to understand the inhibition mechanisms and thereby result in the design of mechanism-based inhibitors.

• Advances in Traditional Medicine
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• 제7권5호
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• pp.477-484
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• 2008

The present study was aimed at investigating the hypouricemic and xanthine oxidase inhibitory activities of the various fractions of the hydromethanolic extract of the leaves of Coccinia grandis L. Voigt (Cucurbitaceae). The leaves of this species was used in traditional medicinal system for the treatment of gout, rheumatism, jaundice, bronchitis, fever, skin eruptions, wounds, etc. The degree of xanthine oxidase inhibition was determined in vitro by measuring the increase in absorbance at 295 nm associated with uric acid formation. Among the fractions tested, the chloroform fraction exhibited highest potency ($IC_{50}$ $17.8\;{\mu}g/ml$). This was followed by the pet-ether ($IC_{50}$ $29.7\;{\mu}g/ml$), ethyl acetate ($IC_{50}$ $41.2\;{\mu}g/ml$) and residual ($IC_{50}$ $47\;{\mu}g/ml$) fractions. The $IC_{50}$ value of allopurinol was $6.1\;{\mu}g/ml$. In addition, the hypouricemic and hepatic xanthine oxidase (XO)/xanthine dehydrogenase (XDH) inhibitory activities of the fractions were examined in vivo using oxonate (280 mg/kg, i.p.) induced hyperuricemic mice. At a dose of 200 mg/kg orally for 7 days, the pet-ether, chloroform and ethyl acetate fractions produced a significant (P < 0.01) reduction in serum urate level and also inhibited hepatic XO/XDH activities when compared to hyperuricemic mice. These inhibitory effects were weaker than that observed for the standard drug, allopurinol (10 mg/kg, p.o.). Lineweaver-Burk analysis of the enzyme kinetics indicated that the mode of inhibition was of a mixed type. These results suggest that the use of Coccinia grandis leaves for the treatment of gout could be attributed to its XO inhibitory activity.

• The Korean Journal of Physiology and Pharmacology
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• 제10권5호
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• pp.243-249
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• 2006

The effect of genistein, widely used as a specific tyrosine kinase inhibitor, on rat brain Kv1.5 channels which were stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Genistein inhibited Kv1.5 currents at +50 mV in a concentration-dependent manner, with an $IC_{50}$ of $54.7{\pm}8.2\;{\mu}M$ and a Hill coefficient of $1.1{\pm}0.2$. Pretreatment of Kv1.5 with protein tyrosine kinase inhibitors ($10\;{\mu}M$ lavendustin A and $100\;{\mu}M$ AG1296) and a tyrosine phosphatase inhibitor ($500\;{\mu}M$ sodium orthovanadate) did not block the inhibitory effect of genistein. The inhibition of Kv1.5 by genistein showed voltage-independence over the full activation voltage range positive to 0 mV. The activation (at +50 mV) kinetics was significantly delayed by genistein: time constant for an activation of $1.4{\pm}0.2$ msec under control conditions and $10.0{\pm}1.5$ msec in the presence of $60\;{\mu}M$ genistein. Genistein also slowed the deactivation of the tail currents, resulting in a crossover phenomenon: a time constant of $11.4{\pm}1.3$ msec and $40.0{\pm}4.2$ msec under control conditions and in the presence of $60\;{\mu}M$ genistein, respectively. Inhibition was reversed by the application of repetitive depolarizing pulses, especially during the early part of the activating pulse. These results suggest that genistein directly inhibits Kv1.5 channels, independent of phosphotyrosine-signaling pathway.