• 미생물학회지
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• 제32권3호
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• pp.222-231
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• 1994

Saccharomyces cerevisiae에서 얻은 purine nucleoside phosphorylase (PNP)의 반응 기작을 밝히기 위해 반응속도론적 분석이 수행되어졌다. 반응기작에 PNP${\cdot}$phosphate와 PNP${\cdot}$ribose 1-phosphate의 binary complex가 형성되는 것으로 추정되어진다. Initial velocity와 product inhibition study의 결과는 반응이 ordered bi, bi reaction으로 일어난다는 것과 일치하고 있다. 두 개의 기질중 무기인산이 효소에 먼저 붙고, 그 다음에 nucleoside, 그리고 base가 효소를 떠나는 첫 번째 생성물이고 마지막으로 ribose 1-phosphate가 생성되고 효소는 원래의 상태로 돌아간다. 반응속도론적 분석에 이해 제안된 작용기작은 sulfhydryl reagents인 p-chloromercuribenzoate(PCMB) and 5,5'-dithiobisnitrobenzoate (DTNB)에 의한 효소의 불활성화에 대한 기질으 보호작용의 결과와 일치하고 있다. PNP는 ribose 1-phosphate와 phosphate에 의해 보호되지만 nucleoside나 base에 의해서는 아무런 효과과 없다는 사실은 반응 순서가 효소에 무기인산이 먼저 붙는 ordered bi, bi 기작이라는 것을 지지하고 있다. PCMB 나 DTNB에 의해 불활성화된 PNP는 dithiothreitol(DTT)에 의해서는 활성이 완전히 회복되고 2-mercaptoethanol에 의해서는 77%의 활성이 회복된다는 사실은 효소의 불활성화가 가역적이라는 것을 시사하고 있다. PCMB에 의해 불활성화된 효소는 inosine이 변화하는 기질일때 정상효소보다 높은 $K_m$과 낮은 $V_m$ 값을 보여주고 이런 현상은 DTT 처리시 원래의 상태로 돌아온다. DTNB에 의해 불활성화된 효소는 PCMB 처리시와 비슷하게 정상효소보다 높은 $V_m$ 값을 보이지만 $V_m$ 값은 큰 변화가 없다. S. cerevisiae PNP에서 발견되는 높은 무기인산의 농도에서의 하위단위체간의 음성적 협동성이 PCMB나 DTNB를 처리한 PNP에서는 보이지 않았다.

• BMB Reports
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• 제30권2호
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• pp.113-118
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• 1997

Aspartase from Hafnia alvei was inactivated by N-ethylmaleimide (NEM) and 5,5' -Dithiobis-(2-znitrobenzoic acid) (DTNB) following pseudo-first order kinetics. Their apparent reaction orders were 0.83 and 0.50 for NEM and DTNB modifications, respectively, indicating that inactivation was due to a sulfhydryl group in the active site of aspartase and participation of the sulfhydryl group in an essential step in the catalytic reaction. When aspartase was modified by DTNB, the enzyme activity was restored by dithiothreitol treatment, indicating that cysteine residuetsl islarel possibly at or near the active site. The pH-dependence of the inactivation rate by NEM suggested that an amino acid residue having pK value of 8.3 was involved in the inactivation. When aspartase was incubated with NEM and L-aspartate together, L-aspartate markedly protected the enzyme from inactivation by NEM, but the other reagents used did not.

• BMB Reports
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• 제28권1호
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• pp.17-20
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• 1995

Thioredoxin reductase is a flavoprotein oxidoreductase catalyzing the reduction of a cystine disulfide in thioredoxin. Thioredoxin, in turn, can reduce disulfide bonds in other proteins and serves as a reducing agent in enzymatic reactions such as those of ribonucleotide reductase and methionine sulfoxide reductase. In this work thioredoxin reductase was found to directly reduce DTNB in the absence of thioredoxin. This new reactivity of E. coli thioredoxin reductase was produced by relatively high concentrations of univalent cations such as $Na^+$, $K^+$, $Li^+$, and ${NH_4}^+$, and it appeared with the oxidation of NADPH. These results indicate that E. coli thioredoxin reductase may be slightly modified by univalent cations, and the modified enzyme directly reacts with DTNB. This DTNB-reducing activity offers a new assay method for E. coli thioredoxin reductase.

• Journal of Microbiology and Biotechnology
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• 제22권10호
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• pp.1406-1411
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• 2012

5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was selected as an electron transfer mediator and was covalently immobilized onto high porosity carbon cloth to employ as a working electrode in an electrochemical $NAD^+$-regeneration process, which was coupled to an enzymatic reaction. The voltammetric behavior of DTNB attached to carbon cloth resembled that of DTNB in buffered aqueous solution, and the electrocatalytic anodic current grew continuously upon addition of NADH at different concentrations, indicating that DTNB is immobilized to carbon cloth effectively and the immobilized DTNB is active as a soluble one. The bioelectrocatalytic $NAD^+$ regeneration was coupled to the conversion of L-glutamate into ${\alpha}$-ketoglutarate by L-glutamate dehydrogenase within the same microreactor. The conversion at 3 mM monosodium glutamate was very rapid, up to 12 h, to result in 90%, and then slow up to 24 h, showing 94%, followed by slight decrease. Low conversion was shown when substrate concentration exceeding 4 mM was tested, suggesting that L-glutamate dehydrogenase is inhibited by ${\alpha}$-ketoglutarate. However, our electrochemical $NAD^+$ regeneration procedure looks advantageous over the enzymatic procedure using NADH oxidase, from the viewpoint of reaction time to completion.

• The Korean Journal of Physiology and Pharmacology
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• 제7권1호
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• pp.15-23
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• 2003

Cellular redox state is known to be perturbed during ischemia and that $Ca^{2+}$ and $K^2$ channels have been shown to have functional thiol groups. In this study, the properties of thiol redox modulation of the ATP-sensitive $K^2$ ($K_{ATP}$) channel were examined in rabbit ventricular myocytes. Rabbit ventricular myocytes were isolated using a Langendorff column for coronary perfusion and collagenase. Single-channel currents were measured in excised membrane patch configuration of patch-clamp technique. The thiol oxidizing agent 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB) inhibited the channel activity, and the inhibitory effect of DTNB was reversed by dithiothreitol (disulfide reducing agent; DTT). DTT itself did not have any effect on the channel activity. However, in the patches excised from the metabolically compromised cells, DTT increased the channel activity. DTT had no effect on the inhibitory action by ATP, showing that thiol oxidation was not involved in the blocking mechanism of ATP. There were no statistical difference in the single channel conductance for the oxidized and reduced states of the channel. Analysis of the open and closed time distributions showed that DTNB had no effect on open and closed time distributions shorter than 4 ms. On the other hand, DTNB decreased the life time of bursts and increased the interburst interval. N-ethylmaleimide (NEM), a substance that reacts with thiol groups of cystein residues in proteins, induced irreversible closure of the channel. The thiol oxidizing agents (DTNB, NEM) inhibited of the $K_{ATP}$ channel only, when added to the cytoplasmic side. The results suggested that metabolism-induced changes in the thiol redox can also modulate $K_{ATP}$ channel activity and that a modulatory site of thiol redox may be located on the cytoplasmic side of the $K_{ATP}$ channel in rabbit ventricular myocytes.

• BMB Reports
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• 제31권3호
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• pp.258-263
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• 1998

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to leucine, valine, and isoleucine. Tobacco ALS was expressed in E. coli and purified to homogeneity. The recombinant tobacco ALS was inactivated by thiol-specific reagents, N-ethylmaleimide (NEM) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Inactivation of the ALS by NEM followed pseudo-first order kinetics and was first order with respect to the modifier. The substrate pyruvate protected the enzyme against the inactivation by NEM and DTNB. Extrapolation to complete inactivation of the enzyme by DTNB showed modification of approximately 2 out of 4 total cysteinyl residues (or 2 cysteinyl and 1 cysteinyl residues), with approximately 1 residue protected by pyruvate. The tobacco ALS was also inactivated by the tryptophanspecific reagent, N-bromosuccinimide (NBS), and was similarly protected by pyruvate. The kinetics of the inactivation was first-order with respect to NBS. The present data suggest that cysteinyl and tryptophanyl residues play a key role in the catalytic function of the enzyme.

• BMB Reports
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• 제36권4호
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• pp.417-420
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• 2003

The present work describes a colorimetric microplate assay for lipase activity based on the reaction between 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) and the hydrolysis product of 2,3-dimercapto-1-propanol tributyrate (DMPTB). Reaction mixtures containing DTNB, DMPTB, and lipase were prepared in microplate wells, and the absorbance at 405nm was recorded after incubation at $37^{\circ}C$ for 30 min. A linear relationship was obtained in the range of 0.1-1 U of lipase activity by this method. The reaction conditions were also optimized for the range of 0.01-0.1 U or 1-10 U. When assaying crude tissue extracts, the reaction of DTNB with non-specific reducing agents created a major source of error. However, this error was corrected by the use of blank samples that did not contain DMPTB.

• 대한화학회지
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• 제50권5호
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• pp.362-368
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• 2006

포스포리파아제 D(PLD)의 8개의 시스테인 잔기들의 특성을 파악하기 위해 설프히드릴(SH)기와 반응하는 각종 화학물질들을 동원하였다. 5,5-다이티오비스(2-니트로벤조산) (DTNB)는 시스테인 잔기의 SH기를 적정하기 위해 이용하였으며, 412nm에서의 환원된 DTNB의 값으로부터 자연 상태의 PLD는 1몰 당 4개의 SH기가 있는 것으로 나타났으나, 8 M의 요소 등으로 3차원 구조를 교란 시킨 변성된 PLD는 8개의 SH기가 적정되었다. 이 결과로 시스테인 잔기의 반(4개)은 외부에 노출되어 있고 그 나머지 반은 내부에 가려져 있다고 추정할 수 있다. SH기 변형 시약인 p-클로로머큐리벤조산(PCMB), 요오드아세트산, 요오드아세트아미드, 그리고 N-에칠마레이미드 등은 모두 PLD를 비활성화 시켰다. 이들 중 다이티오스라이톨(DTT)로 처리했을 때 유일하게 PCMB에 의해 비활성화 된 PLD는 가역적으로 그 활성이 회복되었다. 다양한 작용기를 갖는 다이설파이드들을 이용한 노출된 SH기의 주위 환경을 검토한 결과 음전하나 전하를 띄지 않은 다이설파이드들이 양전하를 띈 시스타민 보다 더 효과적으로 PLD를 비활성화 시키는 것으로 나타났다. 그 이외 시스테인 잔기의 산화-환원 전환이 PLD 활성에 미치는 영향을 과산화수소를 이용하여 검토하였다. 과산화수소 산화에 의해 70% 이상 잃은 PLD 활성은 대부분 DTT에 의해 복원되었다. 이들 결과로부터 양배추 PLD의 시스테인 잔기들이 모두 SH기로 존재한다는 것을 반응을 통해 확인 할 수 있었으며, 또한 외부에 노출된 4개의SH기는 PLD 활성 조절에 지대한 영향을 미치고 있는 것으로 나타났다.

• Applied Biological Chemistry
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• 제40권4호
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• pp.277-282
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• 1997

아라비돕시스 탈리아나의 아세토락테이트 합성 효소 (ALS)를 그 유전자를 포함하고 있는 대장균 MF 2000/pTATX로부터 부분 정제하였다. 부분 정제된 이 효소를 가지고 여러 가지 변형 화학물질들 즉, 요오드아세트산, 요오드아세타마이드, N-에틸말레이미드 (NEM), 5,5'-디티오비스(2-니트로벤조산) (DTNB), 파라염화수은벤조산 (PCMB), 그리고 페닐글리옥살 등에 대한 민감성을 조사하였다. PCMB가 가장 민감하게 저해를 했으며, DTNB와 NEM이 그 뒤를 따랐다. 이 효소의 기질인 피루브산이 요오드아세트산에 의한 활성 저해를 보호하지 못하였으므로 기질의 결합에 시스테인의 관련이 없는 것 같이 보인다. 한편, 기질이 페닐글리옥살에 의한 효소의 활성 저해를 부분적으로 보호하는 것으로 보아 기질이 아르기닌기와 상호 작용함을 암시하고 있다. 부분 정제된 효소는 발린과 이소루신에 민감하게 방해를 받았으나 루신은 그렇지 않았다. 그러나, PCMB로 변형시킨 효소는 되먹임 방해를 더 강하게 받았다. 그 외 ALS에 대한 새로운 제초제 후보인 피리미디설퍼 벤조산 유도체의 저해 효과를 검토하였다.

• 대한화학회지
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• 제19권4호
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• pp.246-251
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• 1975

Dideptidyl carboxypeptidases와 angiotenisn-coverting enzyme의 새로운 기질불질로서 thiol ester 인 S-Hippuryl thioglycolyl glycine을 합성하였으며, 이 기질에 의한 간편하고도 예민한 효소 활성도의 정량방법을 제시하였다. 이 경우 효소반응 생성물인 thioglycolyl glycine은 반응계중에 첨가한 5,5-dithio-bis-(2-nitrobenzoic acid), DTNB와 쉽게 반응하여 410nm에서 강한 흡광스펙트럼을 갖는 5-thio-2-nitrobenzoic acid(${\varepsilon}M=1.36{\times}10^4$)을 형성함으로서 효소의 새로운 미량정량 방법으로 이용 가치가 크다고 본다.