Date

2002

Author

Demirdöğen Can, Birsen

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The flavin-containing monooxygenases (also called flavin-monooxygenases and abbreviated to FMO; E.C.I. 14.13.8) catalyze the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. FMOs have been found in various organisms from bacteria to humans and five mammalian FMO genes have been identified, FM01-FM05. Sheep liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO enzymes. The average specific activity of sheep liver microsomal FMO was found to be 3.8 ±1.5 nmol/min/mg protein (Mean ± SE, n=7) when no detergent was added to the reaction 111medium, and 6.1 + 1.4 nmol/min/mg protein (Mean ± SE, n=6) in the presence of 0.1 % Triton X-100 in the reaction mixture. The rate of reaction was linear up to 500 |j.g of sheep liver microsomal protein and for 27 minutes of incubation period. The maximum FMO enzyme activity was detected at 37 °C, at pH 8.0. Effects of detergents; Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations. The apparent Vmax and Km values of sheep liver microsomal FMO for methimazole substrate were found as 12.3 nmol/min/mg protein and 0.133 mM, respectively. Thermostability of sheep liver FMO was studied at four different temperatures; 24 °C, 37 °C, 50 °C, and 65 °C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 °C, 1.5 hours at 50 ° C, 2 days at 37 °C, and 20 days at 24 ° C. Effects of two metal ions, Hg+2 and Mg+2, on sheep liver FMO activity was studied using HgCh and MgCİ2. Sheep liver microsomal FMO activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 10.7 and 12.3 nmol NADPH oxidized/min/mg protein, respectively. Effects of imipramine, chlorpromazine, and n-octylamine on methimazole oxidation activity of sheep liver microsomal FMO was studied and found that the two drug substrates and n-octylamine inhibited FMO-catalyzed methimazole oxidation activity when they are used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO 3 in sheep liver, sheep lung, and bovine liver microsomes. The immunoblot analysis suggested that FM03 expression is higher in sheep lung, compared to sheep and bovine liver.

Subject Keywords

FMO, Flavin-containing monooxygenase, Methimazole, Imipramine, Sheep, Liver, Microsome, Characterization

URI

https://hdl.handle.net/11511/12405

Collections

Graduate School of Natural and Applied Sciences, Thesis