碩士 / 國立高雄海洋科技大學 / 水產食品科學研究所 / 93 / Pentachlorophenol（PCP）had been used in the preservation of wood and the bactericide of cooling water tower. Larger quantities of other cholorophenols were also discharged in industrial wastewater as effluent. Those chlorophenols would contaminate both soil and water, and then cause adverse effects on aquatic ecosystem.
In order to understand the persistence of chlorophenols on the aquatic environment in Taiwan, the present study was carried out to resolve the monooxygenase system and reconstitue the purified cytochrome P450 and NADPH-cytochrome c reductase with phosphatidylcholine. The monooxygenase system was prepared from the liver of tilapia which were induced by injecting daily 6.25 mg/kg-body weight PCP for 6 time in 3 days interval.
In the PCP-induced liver microsomes, the specific content of cytochrome P450 and specific activity of NADPH-cytochrome c reductase were 0.72 nmo/mg-protein and 5.128 nmo/min/mg-protein, respectively. But, in the liver microsomes without PCP-induced, the specific content of cytochrome P450 and specific activity of NADPH-cytochrome c reductase were were 0.1 nmol/mg-protein and 5.99 nmol/min/mg-protein, respectively. The results showed that PCP-induced liver microsomes had higher content of cytochrome P450 than the liver microsomes whithout PCP-induced. However, it was not found that the difference of specific activity of NADPH- cytochrome c reductase between with and without PCP-induced liver microsomes.
PCP-induced liver microsomes were solubilized with sodium cholate and Triton X-100. The solubilized microsome was subjected to purification of cytochrome P450 and NADPH- cytochrome c reductase.
Octylamine-Sepharose 4B and hydroxylapatic columns were used for cytochrome P450 purification. After purification, the 70kD protein were collected. This protein contained 9.099 n mol/mg-protein of cytochrome P450, and it’s purity was raised to 12.6 fold.
DEAE-cellulose and 2’,5’-adenosine bisphosphate Sepharose 4B column were used for purification of NADPH-cytochrome c reductase. After purification the 78kD proteins were collected. The specific activity of this protein was 113.1 nmo/min/mg-protein, and it’s purity was raised to 22 fold.
A liposome, reconstituted monooxygenase system was constructed with purified cytochrome P450, NADPH-cytochrome c reductase and phosphotidylcholine. In reconstitution studies, the activities of p-nitroanisol-O-demethylase (NAD), aryl hydrocarbon hydroxylase (AHH), ethylmorphine-N-deethylase (EMD), aniline-4-hydroxylase (AH) were compared among the reconstituted monooxygenase system, PCP-induced microsome and liver microsomes without PCP-induced. The results show that the activities from enzymes were 36.0 nmol/min/mg（NAD）, 18.8 nmol/min/mg-protein（AHH）, 48.6 nmol/min/mg-protein（EMD）, 7,951 nmol/min/mg-protein（AH） in the reconstituted monooxygenase system. In PCP-induced liver microsomes, the activities of four enzyme were 27.4 n mol/min/mg-protein（NAD）, 5.9 nmol/min/mg-protein（AHH）, 10.6 nmol/min/mg-protein（EMD）, 2,356 nmol/min/mg-protein（AH）. In liver microsome, the activities of four enzyme were 3.1 nmol/min/mg-protein（NAD）, 0.44 nmol /min/mg-protein（AHH）, 0.7 nmol/min/mg-protein（EMD）, 0.67 nmol/min/mg-protein（AH）.
It was indicated that the reconstituted system had the higher catalytic activity than other liver microsomes. The reconstituted monooxygenase system in vitro will provide an ideal model for further studies, such as the xenobiotic metabolisms and the interaction between the monooxygenase and xenobiotics.
| Identifer | oai:union.ndltd.org:TW/093NKIMT084009 |
| Date | January 2004 |
| Creators | Der-Har Lee, 李大鶴 |
| Contributors | Juang-Lin Lian, 連壯林 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 106 |
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