• Title/Summary/Keyword: cytochrome b

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Purification and Comparison of NADH-Cytochrome $b_5$ Reductase from Mitochondrial Outer Membrane of Bovine Heart and Turnip

  • 이재양;김영호;이상직
    • Bulletin of the Korean Chemical Society
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    • v.19 no.2
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    • pp.160-164
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    • 1998
  • The NADH-cytochrome b5 reductase (NCBR), a mitochondrial external electron carrier, was purified from bovine heart and turnip and their properties were examined. The mitochondrial outer membranes separated were subjected to NCBR isolation through DEAE-Cellulose ion exchange, DEAE-Sephadex gel chromatography, and hydroxyapatite adsorption chromatography. These processes yielded the purification folds of 88 and 42 and the recovery percentages of 0.2%, 5.67% for turnip and bovine heart, respectively. The molecular weight of the NCBR from the two sources was estimated to be 35,000 using SDS polyacrylamide gel electrophoresis. The Michaelis constant Km and maximum velocity Vmax were determined by measuring the NADH-ferricyanide redox system as well as the NADPH-ferricyanide redox system. The kinetics showed that both NCBRs had higher affinities for NADH than artificial electron-acceptor substrate ferricyanide. Although NADPH had a lower affinity for the enzymes than NADH, this study showed the 2'-phosphate dinucleotide could be used as a substrate.

Chemopreventive activity of Prunella Herba Vulgaris L. Aqua-acupuncture Solution (댑싸리하고초(夏枯草) 약침액(藥鍼液)의 암예방 활성)

  • Park Shin-Hwa;Lim Jong-Kook
    • Korean Journal of Acupuncture
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    • v.18 no.1
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    • pp.11-20
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    • 2001
  • Cancer chemoprevention refer to the use of natural or synthetic substances to prevent the initiational and promtional events that occur during the process of carcinogenesis. The effect of Prunella Herba Vulgaris L. Aqua-acupuncture Solution (PVAS) and Prunella Herba Vulgaris L. Water-extracted Solution (PVWS) on the induction of phase II detoxification enzyme (quinone reductase, Glutathione S-transferase) and inhibition of phase I enzyme (cytochrome P4501A1) and benzo[a]pyrene-DNA adduct formation was examined. PVAS is potent inducers of quinone reductase activity. Glutathione levels were increased with PVAS, in cultured murine hepatoma Hepa1c1c7 cells. In addition glutathione S-transferase levels were increased with PVAS. However, there was 45.2% inhibition in the activity of cytochrome P4501A1 enzyme with the treatment of PVAS, $5{\times}$. At concentration of $1{\times}$ and $3{\times}$ of PVAS, the binding of $[^3H]B[a]P$ metabolites to DNA of NCTC-clone 1469 cell was inhibited by 25.3%, 45.0%, respectively. These results suggest that PVAS has chemopreventive potential by inducing quinone reductase and glutathione S-transferase activities, increasing GSH levels, inhibiting the activity of cytochrome P4501A1 and benzo[a]pyrene-DNA adduct formation.

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Identification of a Cytochrome P450 Hydroxylase Gene Involved in Rifamycin Biosynthesis by Amycolatopsis mediterranei S699

  • Lee, Sang-Kil;Choi, Cha-Yong;Ahn, Jong-Seog;Cho, Jae-Yong;Park, Cheon-Seok;Yoon, Yeo-Joon
    • Journal of Microbiology and Biotechnology
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    • v.14 no.2
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    • pp.356-365
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    • 2004
  • In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orj0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orj0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orj0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orj0 using this novel vector. These consecutive results verified that the arf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.

A Study on KASP Analysis Based on SNP to Rapidly Identify Caviar-Producing Sturgeon Species (캐비어를 생산하는 철갑상어의 신속 종판별을 위한 SNP 기반 KASP 분석에 관한 연구)

  • Sun Hee Lee;Bo Reum Park;Hyung Il Kim;Sooyeul Cho;Kyung-Hun Son
    • Journal of Food Hygiene and Safety
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    • v.39 no.3
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    • pp.209-220
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    • 2024
  • Cavier is graded as Beluga, Osetra, and Sevruga based on the species of sturgeon (Acipenser sinensis). In this study, we developed an analytical method for determining the grade of black caviar using DNA barcodes and KASP markers. To identify the sturgeon species, ten black caviar samples were collected, and a reference DNA barcode library was developed using five genes (namely, 16S ribosomal RNA, cytochrome b, cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, and NADH dehydrogenase subunit 5 genes). To develop the KASP markers, we selected 11 markers that could distinguish between the five grades of black caviar. As a result, specific markers for each of the targeted caviars were clustered into FAM-positive sections. DNA barcoding and the KASP assay revealed that one Beluga, six Osetra, and three Sevruga were identified among the ten caviar samples. Moreover, we found that the sturgeon species were mislabeled in two products. Here, we aimed to develop a KASP assay based on SNP that allows rapid and easy identification of caviar grade. These methods are expected to contribute to preventing the distribution of illegal products.

Suicidal gene therapy with rabbit cytochrome P450 4B1/2-aminoanthracene or 4-ipomeanol system in human colon cancer cell

  • Jang, Su Jin;Kang, Joo Hyun;Moon, Byung Seok;Lee, Yong Jin;Kim, Kwang Il;Lee, Tae Sup;Choe, Jae Gol;Lim, Sang Moo
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.1 no.2
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    • pp.118-122
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    • 2015
  • Suicidal gene therapy is based on the transduction of tumor cells with "suicide" genes encoding for prodrug-activating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4-ipomeanol (4-IPO) or 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate.In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/2-AA or 4-IPO system were evaluated in HT-29 (human colon cancer cell). pcDNA-CYP4B1 vector was transfected into HT-29 by lipofection and stable transfectant was selected by treatment of hygromycin ($500{\mu}g/mL$) for 3 weeks. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed for confirmation of CYP4B1 expression in CYP4B1 gene transduced cell. The cytotoxic effects of CYP4B1 transduced cell were determined using dye-exclusion assay after treatment of 2-AA or 4-IPO for 96 hrs. Dye-exclusion assay showed that $IC_{50}$ of HT-29 and CYP4B1 transduced HT-29 was 0.01 mM and 0.003 mM after 4-IPO or 2-AA treatment at 96 hrs exposure, respectively. In conclusion, CYP4B1 based prodrug gene therapy probably have the potential for treatment of colorectal adenocarcinoma.