• Title/Summary/Keyword: Enzyme inhibition

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Establishment of New Method for the Assay of Glutamate-cysteine Ligase Activity in Crude Liver Extracts

  • Kwon Young-Hye;Stipanuk Martha H.
    • Toxicological Research
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    • v.22 no.1
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    • pp.39-45
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    • 2006
  • As the antioxidant and free radical scavenger, glutathione (GSH) participates in the preservation of cellular redox status and defense against reactive oxygen species and xenobiotics. Glutamate-cysteine ligase (GCL; also known as ${\gamma}$-glutamylcysteine synthetase, EC 6.3.2.2) is the rate limiting enzyme in GSH synthesis. In the present study, the accurate method for determination of GCL activity in crude liver extracts was developed by measuring both ${\gamma}$-glutamylcysteine and GSH from cysteine in the presence of glutamate, glycine and an ATP-generating system. We added glycine to promote the conversion of ${\gamma}$-glutamylcysteine to GSH, and to minimize the possibility of ${\gamma}$-glutamylcysteine metabolism to cysteine and oxoproline by ${\gamma}$-glutamylcyclotransferase. We established optimal conditions and substrate concentrations for the enzyme assay, and verified that inhibition of GCL by GSH did not interfere with this assay. Therefore, this assay of hepatic GCL under optimal conditions could provide a more accurate measurement of this enzyme activity in the crude liver extracts.

Effect of Alum on the Activity of Raw Starch-Digesting Enzyme Produced by Bacillus sp. (Bacillus sp.가 생산하는 전분 분해효소의 활성에 미치는 Alum첨가의 영향)

  • Lee, Shin-Young;Lee, Sang-Gui;Kang, Tae-Su;Lee, Myong-Yul
    • Korean Journal of Food Science and Technology
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    • v.27 no.5
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    • pp.773-775
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    • 1995
  • The effect of alum$(Al{\cdot}K(SO_4)_2{\cdot}12H_{2}O)$ on the activity of raw starch-digesting enzyme produced by alkali-tolerant Bacillus sp. was investigated. In adding alum of 0.5%(w/w), activities of raw starch-digesting enzyme on the gelatinized and raw rice starches have not been inhibited. In case of adding alum of 5%(w/w), competitive and uncompetitive inhibition were observed for the gelatinized and raw rice starches, respectively. The inhibitory effect on the raw starch was much higher than that on the gelatinized starch.

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Substitution of Gly-224 Residue to Ile in Yeast Alcohol Dehydro-genase and Enzyme Reaction Mechamism

  • Lee, Kang-Man;Ryu, Ji-Won
    • Archives of Pharmacal Research
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    • v.16 no.3
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    • pp.231-236
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    • 1993
  • Gly-224 residue of yeast alcohol dehydrogenase was mutated by site-directed mufagenesis to isoleucine, which is the corresponding amino acid residue of horse liver alcohol dehydrogenase. The mutated gene on M13 vector was subcloned in YEp13 and used to transform Saccharomyces cerevisiae 302-21 #2 strain, and the expressed protein was purified. The tumover numbers of mutant enzyme for ethanol and acetaldehyde were decreased copared to wild-type enzyme. The results of product inhibition studies indicated that the reaction mechanism was changed to Iso Theorell-Chance from Ordered Bi Bi. We supposed that Gly-224 was related to the enzyme reaction mechanism.

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The Kinetic Characteristics of K228G Mutant Horse Liver Alcohol Dehydrogenase

  • Cho, Sun-Hyoung;Ryu, Ji-Won;Lee, Kang-Man
    • Archives of Pharmacal Research
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    • v.22 no.1
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    • pp.13-17
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    • 1999
  • The kinetic constants and the reaction mechanism of the K228G mutant horse liver alcohol dehyrogenase isoenzyme E (HLADH-E) were compared to the wild-type enzyme. All the Km and Ki constants of the mutant enzyme for NAD+, ethanol, acetaldehyde and NADH were larger than those of the wild-type enzyme. The dissociation constants for the NADH and $NAD^{+}$ (Kiq and Kia) were greatly increased by 130-and 460-fold, respectively. The product inhibition patterns suggested that the reaction mechanism of the mutant enzyme was changed to Random Bi Bi. These results could attribute to the increase in the dissociation rate of coenzyme with the substitution at Lys-228 residue.

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Effect of the Constituents of Angelicae dahuricae Radix on Hepatic Drug Metabolizing Enzyme Activity (백지근(白芷根) 성분(成分)이 간(肝)의 약물대사효소활성(藥物代謝酵素活性)에 미치는 효과(效果))

  • Shin, Kuk-Hyun;Kim, Ok-Nam;Woo, Won-Sick
    • Korean Journal of Pharmacognosy
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    • v.19 no.1
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    • pp.19-27
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    • 1988
  • The hexane and ether extracts from the roots of Angelica dahurica caused a significant inhibition of hepatic drug-metabolizing enzyme (DME) activity. Through systematic fractionation by $SiO_2\;column$ and vacuum liquid chromatography monitoring by bioassays, three furanocoumarins, phellopterin, byakangelicin and tert-O-methylbyakangelicin were isolated as active principles. These components have biphasic responses, both inhibitory and inducing effects on DME system. Tert-O-methyl byakangelicin was found to have the strongest enzyme inhibitory potency.

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Potentiometric Homogeneous Enzyme-Linked Binding Assays for Riboflavin and Riboflavin Binding Protein

  • 김진목;김혜진;김미정;이동주;한상현;차근식
    • Bulletin of the Korean Chemical Society
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    • v.17 no.11
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    • pp.1018-1022
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    • 1996
  • Adenosine deaminase (ADA) has been utilized as the label in devising a potentiometric homogeneous assay for riboflavin and riboflavin binding protein (RBP). The proposed homogeneous assay method employs an ADA-biotin conjugate as the signal generator and an avidin-riboflavin conjugate as the signal modulator in the solution phase. The catalytic activity of the ADA-biotin conjugate is inhibited in the presence of an excess amount of the avidin-riboflavin conjugate, and the observed inhibition is reversed in an amount proportional to the concentration of RBP added. When the analyte riboflavin is added to this mixture of ADA-biotin, avidin-riboflavin and RBP, the activity of the enzyme conjugate is re-inhibited in an amount proportional to the concentration of riboflavin. Since the enzyme label used in this system is ADA, an ammonia-producing enzyme, a potentiometric rather than photometric detection scheme is used to monitor the enzymatic activity in the assay.

Effects of Psoralen and Angelicin on Hepatic Drug-Metabolizing Enzyme Activities

  • Shin, Kuk-Hyun;Woo, Won-Sick
    • Archives of Pharmacal Research
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    • v.11 no.2
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    • pp.122-126
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    • 1988
  • The effects of psoralen and angelicin on hepatic microsomal drug-metabolizing enzyme (DME) activities were investigated to elucidate the mode of the interaction of furanocoumarins with DME system. A single administration (30 mg/kg,i. p.) of both coumarins to mice cased a significant prolonagation of hexobarbital-induced hypnosis as well as an increase in strychnine toxicity. The inhibitory potencies of both coumarins as measured by rat hepatic microsomal aminopyrine N-demethylase and hexobarbital hydroxylase activities in vitro were considerably weaker than those of other furanocoumarins which possess a side chain moiety. Both coumarins were found to have significant inducing effects of DME system, with repeated treatments of them. The activities of an angular coumarin were stronger than those of a linear coumarin.

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Comparison of Enalapril Maleate Tablets on Bioavailability and the Time Course of Inhibition of Plasma Angiotensin-Converting Enzyme (Enalapril Maleate 정제의 동등성에 관한 연구 ; 약동학적 성상 및 혈장 ACE 활성도 억제 효과)

  • Jang, In-Jin;Jang, Byung-Soo;Shin, Sang-Goo;Shin, Jae-Gook;Rho, Il-Kun;Lee, Kyeong-Hun;Park, Chan-Woong
    • The Korean Journal of Pharmacology
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    • v.26 no.2
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    • pp.219-226
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    • 1990
  • Enalapril maleate tablets of two different producers were tested for bioequivalence. Enalapril is rapidly metabolized to an active metabolite, enalaprilat which inhibits angiotensin-converting enzyme (ACE). The pharmacokinetics of enalapril maleate and the time course of inhibition of plasma ACE activity after administration of the drugs were studied. Two single doses of 10mg each of enalapril maleate were administered orally to twelve male volunteers in a balanced, randomized, two-way crossover investigation. Plasma enalaprilat concentrations were determined over a 23-hour after the dose by enzyme inhibition assay and enalapril by the same method following in vitro hydrolysis. Urinary recoveries of enalapril and enalaprilat were determined for the calculation of renal clearance. Plasma ACE activity was determined by an enzyme assay. Peak plasma levels of enalapril were observed about 1 hour after the doses, and practically all enalapril had disappeared from plasma within 6 hour. Peak enalapril concentrations of both formulations were almost identical ($Vasotec^{\circledR}$, 61.38 ng/ml; $Beartec^{\circledR}$, 64.27 ng/ml). The values of the pharmacokinetic parameters of enalaprilat computed for $Vasotec^{\circledR}$ and $Beartec^{\circledR}$ tablets are presented in that order; area under the curve=330.63:320.96 $ng{\cdot}hr/ml$; peak concentration=38.63:39.43 ng/ml; time to peak=3.83:4.08 hour; elimination half-life=3.95:3.92 hours. No statistically significant difference was detected when area under the curve and all other parameters were compared. Using criteria of 95% confidence interval for the comparison of these parameters, only the upper limits of area under the curve and time to peak of enalapril were over 120%. All the parameters of enalaprilat were acceptable. Percent inhibition of plasma ACE to plasma enalaprilat concentration showed the sigmoid concentration-inhibition relationship. Time courses of plasma ACE inhibition after the administration of both formulations were quite similar. The formulations were found to be equivalent when compared on the premise that no significant difference was detected when pharmacokientic parameters and inhibition of ACE activity were compared, based on the confidence limits analysis.

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Antioxidant and ACE Inhibitory Activities of Soybean Hydrolysates: Effect of Enzyme and Degree of Hydrolysis

  • Lee, Ji-Soo;Yoo, Mi-Ae;Koo, Seung-Hyun;Baek, Hyung-Hee;Lee, Hyeon-Gyu
    • Food Science and Biotechnology
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    • v.17 no.4
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    • pp.873-877
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    • 2008
  • Native soy protein isolate (SPI) was hydrolyzed with 4 different proteolytic enzymes, including bromelain, papain, Neutrase, and Flavourzyme. SPI hydrolysates with the degree of hydrolysis (DH) in range of 6 to 15% were prepared by each enzyme. The angiotensin 1 converting enzyme (ACE) inhibitory and the antioxidant activities of the SPI hydrolysates, such as superoxide dismutase-like activity and inhibition of the linoleic acid autoxidation, were evaluated. Overall, as the DH increased, all evaluated bioactivities of the SPI hydrolysates significantly increased. The significantly highest ACE inhibitory and antioxidant activities were found in hydrolysates made with papain and bromelain, respectively. SPI hydrolysates by Flavourzyme showed the significantly lowest activity in all tested bioactivities. The results suggested that ACE inhibitory and antioxidant activities of SPI hydrolysates were determined by the DH and by the enzyme used.

Properties of Penicillin Amidohydrolase Immobilized on Nylon Fiber

  • B. L. Seng;Iw-Han Cho;J. S. Rhee;Dewey D. Y. Ryu
    • Bulletin of the Korean Chemical Society
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    • v.1 no.1
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    • pp.10-17
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    • 1980
  • Penicillin amidohydrolase was partially purified from the fermented broth of Bacillus megaterium, and was immobilized on nylon fiber. The surface area of nylon fiber was increased by roughening it with fine sand and activated by acid treatment. The free amino groups on the nylon fiber exposed by such treatment were then utilized to immobilize the penicillin amidase. Enzymatic properties of penicillin amidohydrolase immobilized on the nylon fiber by covalent bonding and cross linking with glutaraldehyde were studied and compared with those of soluble enzyme. The optimal pH and temperature profile of immobilized enzyme showed only slightly broader peaks, and the values of kinetic constants, $K_m$, $K_{ia}$, and $K_{ip}$, of the immobilized enzyme are only slightly greater than those of the soluble enzyme. These results suggest that the mass transfer effect on the reaction rate for the penicillin amidase immobilized on nylon fiber is not so significant as the enzyme immobilized on some other support material like bentonite. The experimental results of batch reaction agreed well with the results of computer simulation for both the immobilized and soluble enzyme systems, confirming the validity of the rate equation derived which was based on the combined double inhibition by two reaction products.