• 농약과학회지
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• 제5권3호
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• pp.41-46
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• 2001

분지아미노산 생합성 과정에 관여하는 첫 번째 효소인 acetolactate synthase (ALS)를 대상으로 수행할 수 있도록 고효율 검색방법(High Throughput Screening, HTS)을 개발하였고, 이를 이용하여 식물특이적 효소 저해제로 알려진 107개의 기존 화합물 중에서 새로운 ALS 저해 화합물을 선발하였다. 기존의 방법과 비교할 경우 한사람이 1회 수행한다고 하면 8 배 효율이지만 연속적으로 수행한다고 할 경우 1/10 이하의 양, 동일한 재료의 적용, 측정 결과의 계산, enzyme kinetics 등을 감안하면 최소 100 배 이상의 효과를 얻을 수 있다. 새로운 ALS 저해제로 탐색된 화학물질은, ammooxyacetic acid, azelaic acid, citric acid, cyanuric fluoride, glyoxylic acid, itaconic acid, malonic acid, niclosamid, oxalic acid, 2-oxoglutaric acid, suramin 등이었다. 앞으로 이들을 기본 구조로 하여 신규 ALS 저해 제초제의 개발을 위한 유도체의 합성에 이용되었으면 한다.

• BMB Reports
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• 제34권3호
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• pp.194-200
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• 2001

Secreted arginase from Evernia prunastri thallus has been purified 616-fold from the incubation medium. Purified arginase was resolved as only one peak in a capillary electrophoresis with a pI value of 5.35. The protein contained high amounts of acidic amino acids, such as Asx and Glx, and a relatively high quantity of Ser and Gly. The molecular mass of native, purified arginase was estimated as about 26 kDa by SE-HPLC. Substrate saturated kinetic showed a typical Michaelis-Menten relationship with a K_m value of 3.3 mM L-arginine. Atranorin behaved as a mixed activator of the enzyme (apparent $K_m$ = 0.96 mM); whereas evernic and usnic acid were revealed as non competitive inhibitors (apparent $K_m$ values were 3.16 mM and 3.05 mM, respectively). Kinetics of atranorin binding indicated that saturation was reached from 0.18 ${\mu}mol$ of the total atranorin and the occurrence of multiple sites for the ligand. This agrees with a possible aggregation of several enzyme subunits during the interaction process. A value of binding sites of about 12 was obtained. The binding of evernic acid was saturated from 23 nmol of total phenol. The number of binding sites was about 5. The loss of the binding ability of evernic acid could be interpreted as a single negative cooperatively. Usnic acid behaves in a similar way to evernic acid, although the binding saturation occurs at $0.14\;{\mu}moles$ of the ligand. This binding appears to be unspecific, and has 28 usnic acid binding sites to the protein.

• Archives of Pharmacal Research
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• 제28권10호
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• pp.1196-1202
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• 2005

Omeprazole (OMP) is a proton pump inhibitor used as an oral treatment for acid-related gastrointestinal disorders. In the liver, it is primarily metabolized by cytochrome P-450 (CYP450) isoenzymes such as CYP2C19 and CYP3A4. 5-Hyroxyomeprazole (5-OHOMP) and omeprazole sulfone (OMP-SFN) are the two major metabolites of OMP in human. Cimetidine (CMT) inhibits the breakdown of drugs metabolized by CYP450 and reduces, the clearance of coad-ministered drug resulted from both the CMT binding to CYP450 and the decreased hepatic blood flow due to CMT. Phenobarbital (PB) induces drug metabolism in laboratory animals and human. PB induction mainly involves mammalian CYP forms in gene families 2B and 3A. PB has been widely used as a prototype inducer for biochemical investigations of drug metabolism and the enzymes catalyzing this metabolism, as well as for genetic, pharmacological, and toxicological investigations. In order to investigate the influence of CMT and PB on the metabolite kinetics of OMP, we intravenously administered OMP (30 mg/kg) to rats intraperitoneally pretreated with normal saline (5 mL/kg), CMT (100 mg/kg) or PB (75 mg/kg) once a day for four days, and compared the pharmacokinetic parameters of OMP. The systemic clearance ($CL_{t}$) of OMP was significantly (p<0.05) decreased in CMT-pretreated rats and significantly (p<0.05) increased in PB-pretreated rats. These results indicate that CMT inhibits the OMP metabolism due to both decreased hepatic blood flow and inhibited enzyme activity of CYP2C19 and 3A4 and that PB increases the OMP metabolism due to stimulation of the liver blood flow and/or bile flow, due not to induction of the enzyme activity of CYP3A4.

• BMB Reports
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• 제40권1호
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• pp.100-106
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• 2007

The wound-inducible lipoxygenase obtained from maize is one of the nontraditional lipoxygenases that possess dual positional specificity. In this paper, we provide our results on the determination and comparison of the kinetic constants of the maize lipoxygenase, with or without detergents in the steady state, and characterization of the dependence of the kinetic lag phase or initial burst, on pH, substrate, and detergent in the pre-steady state of the lipoxygenase reaction. The oxidation of linoleic acid showed a typical lag phase in the pre-steady state of the lipoxygenase reaction at pH 7.5 in the presence of 0.25% Tween-20 detergent. The reciprocal correlation between the induction period and the enzyme level indicated that this lag phenomenon was attributable to the slow oxidative activation of Fe (II) to Fe (III) at the active site of the enzyme as observed in other lipoxygenase reactions. Contrary to the lagging phenomenon observed at pH 7.5 in the presence of Tween-20, a unique initial burst was observed at pH 6.2 in the absence of detergents. To our knowledge, the initial burst in the oxidation of linoleic acid at pH 6.2 is the first observation in the lipoxygenase reaction. Kinetic constants (Km and kcat values) were largely dependent on the presence of detergent. An inverse correlation of the initial burst period with enzyme levels and interpretations on kinetic constants suggested that the observed initial burst in the oxidation of linoleic acid could be due to the availability of free fatty acids as substrates for binding with the lipoxygenase enzyme.

• BMB Reports
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• 제33권2호
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• pp.172-178
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• 2000

The lipoxygenase was purified 35 fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoreses and Sepharose 6B column chromatography. The purified enzyme with 2 M $(NH_4)_2SO_4$ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at $-20^{\circ}C$. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenase purified from the red potato were found to be pH 9.0. and $30^{\circ}C$, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were $48\;{\mu}M$ and $0.03\;{\mu}M$ per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10mM EDTA, and 1 mM $NaN_3$), but was inhibited by several divalent cations, such as $Cu^{++}$, $Co^{++}$ and $Ni^{++}$. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward's reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) processed in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.

• 한국수산과학회지
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• 제31권6호
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• pp.829-834
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• 1998

옥수수 전분가수분해물의 용해도는 D.E. 값이 높을수록 증가하였고, 10 이상에서는 거의 차이가 없었다. $T_g^{'}$ 값은 D.E. 값이 증가함에 따라 감소하는 직선적인 관계를 나타내었다. Alkaline phosphatase의 동결계에서 거동은 glass cynamic mechanism, 즉 효소의 가수분해 속도는 첨가된 물질의 $T_g^{'}$ 이하의 온도에서 지연되거나 억제되었다. 옥수수전분 가수분해물은 동결계에서 유리전이온도를 높임으로서 동결계가 유리상태로 되고 따라서 점도는 높아지고 단백질은 분자간 접촉의 기회가 줄어들어 단백질이 보호된다는 cryostabilization mechanism으로 설명 가능하였다.

• Reproductive and Developmental Biology
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• 제41권1호
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• pp.1-6
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• 2017

In all mammalian species, progesterone is essential to both the preparation for, and maintenance of, pregnancy. The $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) enzyme predominantly converts progesterone into its biologically inactive form $20{\alpha}$-hydroxyprogesterone, thereby regulating its activity. Thus, to directly assess sexual maturation in the MediKinetics $micropig^{(R)}$, we analyzed the concentration of the steroid hormones progesterone and estradiol during the estrous cycle. Our results show that the progesterone level exhibited by the analyzed $micorpig^{(R)}$ was low at the beginning of the estrous cycle, and then abruptly increased to $30.32{\pm}10.0ng/mL$ and $46.37{\pm}11.0ng/mL$ by days 9 and 11 of the cycle, respectively. It reached the highest level $55.87{\pm}3.5ng/mL$ on day 13 of the estrous cycle, before decreasing to $46.58{\pm}13.1ng/mL$ and $10.0{\pm}7.6ng/mL$ by days 15 and 17 of the cycle, respectively. In contrast, the estradiol level was shown to be highest ($27.13{\pm}11.2ng/mL$) at the initiation of the estrous cycle, after which point it decreased to $13.29{\pm}6.5ng/mL$ and $10.94{\pm}5.9ng/mL$ by days 4 and 5 of the estrous cycle, respectively. By day 17 of the estrous cycle, the estradiol level decreased to $4.13{\pm}7.6ng/mL$. We anticipate that these results will provide useful information to enable the study of human ovulation and reproductive physiology using the MediKinetics $micoripig^{(R)}$ as a model system. We recommend further investigation to elucidate the functional mechanisms underlying the regulation of sexual maturation in the MediKinetics $micropig^{(R)}$.

• 생태와환경
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• 제33권4호통권92호
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• pp.380-386
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• 2000

순수배양한 조류와 자연군집의 식물플랑크톤을 대상으로 유기인삼염해분해효소의 kinectic parameter를 비교하였고, 용존무기인의 농도와 각 parameter와의 관계를 알아보았다. 기질과의 친화력을 나타내는 AP$_{ase}$의 K$_{m}$은 조류의 종마다 매우 상이한 값을 보였다. Chlorella sp.를 제외한 녹조류가 다른 종에 비해 큰 K$_{m}$을 보였고, 규조류와 Chlorella sp.에서 작은 K$_{m}$을 보였다. 순수배양한 남조류 Anabaena flos-aquae에서 extracelluar free enzyme 의 K$_{m}$이 cell-bound enzyme의 K$_{m}$보다 작았다. 자연군집인 소양호에서는 남조류 Anabaena sp. 가 우점하였던 여름의 식물플랑크톤의 K$_{m}$이 규조류가 우점하였던 봄의 군집보다 컸다. AP$_{ase}$ 활성도의 V$_{max}$는 수중의 DIP 농도보다는 식물플랑크톤의 세포내 인의 함량에 직접적인 영향을 받는 것으로 나타났다. 소양호에서 가을부터 봄까지 extracelluar free enzyme의 활성도가 총 AP$_{ase}$ 활성도의 36${\sim}$97%의 기여를 하였는데, 이는 이 시기에 extracelluar free enzyme이 호수의 인순환에서 매우 중요한 요인으로 작용한다는 것을 시사한다. 그리고 extracelluar free enzyme과 총 AP$_{ase}$의 K$_{m}$이 거의 비슷하였는데, 이는 extracelluar free enzyme이 조류로부터 기인되었기 때문으로 사료된다.

• Applied Biological Chemistry
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• 제35권1호
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• pp.23-29
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• 1992

한국산 파인애플로부터 bromelain을 추출하여 황산암모늄염석, DEAE-cellulose ion-exchange chromatography, gel filtration을 이용하여 약 21배 정제하였고, 정제효소는 전기영동상 단일밴드를 나타내었으며, 분자량은 약 22,000이었다. 정제된 bromelain은 glycine과 serine이 가장 많이 함유되어 있었으며 최적작용 pH와 온도는 6.0, $60^{\circ}C$였다. $pH\;5{\sim}7,\;50^{\circ}C$이하에서 안정성을 보였으며 $Mn^{2+}$에 의해 활성이 촉진되었고, $Mg^{2+},\;Fe^{2+}$ 등의 금속이온에 의해 급격한 활성저해가 관찰되었다. 정제효소는 p-chloromercuribenzoic acid에 의해 저해되어 SH효소로 추측되었으며, Km과 Vmax값는 각각 $5.747{\times}10^{-4}M,\;131.58\;{\mu}g/min$ 이었다. 돈육 조직에 효소를 처리하였을 때 효소 농도가 증가함에 따라 육단백질이 가수분해되어 유리되는 수용성 질소량과 아미노태 질소량이 증가되어 연육효과가 있음이 확인되었다.

• Restorative Dentistry and Endodontics
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• 제47권4호
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• pp.42.1-42.11
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• 2022

Objective: This study investigated the effects of various concentrations of sodium hypochlorite (NaOCl) on human whole-blood clotting kinetics, the structure of the blood clots formed, and transforming growth factor (TGF)-β1 release. Materials and Methods: Human whole blood was collected from 5 healthy volunteers and divided into 4 groups: CG (control, 0.5 mL of blood), BN_0.5 (0.5 mL of blood with 0.5 mL of 0.5% NaOCl), BN₃ (0.5 mL of blood with 0.5 mL of 3% NaOCl), and BN_5.25 (0.5 mL of blood with 0.5 mL of 5.25% NaOCl). The effects of NaOCl on clotting kinetics, structure of fibrin and cells, and release of TGF-β1 were assessed using thromboelastography (TEG), scanning electron microscopy (SEM), and enzyme-linked immunosobent assay, respectively. Statistical analysis was conducted using the Kruskal Wallis and Mann-Whitney U tests, followed by the post hoc Dunn test. A p value < 0.05 indicated statistical significance. Results: The blood samples in BN_0.5 and BN₃ did not clot, whereas the TEG of BN_5.25 showed altered clot formation. Samples from the CG and BN₃ groups could only be processed with SEM, which showed that the latter lacked fibrin formation and branching of fibers, as well as clumping of red blood cells with surface roughening and distortion. TGF-β1 release was significantly highest in BN₃ when all groups were compared to CG (p < 0.05). Conclusions: Each concentration of NaOCl affected the release of TGF-β1 from blood clots and altered the clotting mechanism of blood by affecting clotting kinetics and cell structure.