• BMB Reports
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• v.38 no.3
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• pp.366-369
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• 2005

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome $b_5$, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of $A_{610}$. MTT reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;20\;{\mu}M$, $k_{cat}\;=\;1,910\;min^{-1}$). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.

• BMB Reports
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• v.37 no.5
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• pp.629-633
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• 2004

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring $A_{520}$ reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was $4.09\;mM^{-1}\;cm^{-1}$. DPPH reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;28\;{\mu}M$, $K_{cat}\;=\;1690\;min^{-1}$). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive.

• Journal of the Korean Chemical Society
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• v.19 no.4
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• pp.246-251
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• 1975

A spectrophotometric assay technique is descriead for the measurement of free SH-groups in the enzyme reaction mixture. The method utilizes a new substrate, S-hippuryl-thioglycolyl-glycine(S-Hip-thioglycol-Gly) which is the basis for a convenient assay of angiotensin-converting enzyme and other dipeptidyl carboxypeptidases. This substrate contains an appropriately located thioester linkage that is hydrolyzed by the converting enzyme and other dipeptidyl carboxypeptidases. One of the products, thioglycolyl glycine, is readily measured by reaction with Ellman's reagent, 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, to produce 5-thio-2-nitrobenzoic acid which has a strong absorption band at 410 nm. The method is sensitive (${\varepsilon}M = 1.36{\times}10^4$ at 412 nm) and can be applied as a continuous recording with DTNB present in the enzymatic reaction mixture.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1092-1097
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• 2009

A novel exopolysaccharide named Ebosin was produced by Streptomyces sp. 139, with medicinal activity. Its biosynthesis gene cluster (ste) has been previously identified. For the functional study of the ste7 gene in Ebosin biosynthesis, it was disrupted with a double crossover via homologous recombination. The monosaccharide composition of EPS-7m produced by the mutant strain Streptomyces sp. 139 ($ste7^-$) was found altered from that of Ebosin, with fucose decreasing remarkably. For biochemical characterization of Ste7, the ste7 gene was cloned and expressed in Escherichia coli BL21. With a continuous coupled spectrophotometric assay, Ste7 was demonstrated to have the ability of catalyzing the transfer of fucose specifically from GDP-$\beta$-L-fucose to a fucose acceptor, the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 ($ste7^-$). Therefore, the ste7 gene has been identified to code for a fucosyltransferase, which plays an essential role in the formation of repeating sugars units during Ebosin biosynthesis.