Cytochrome P450 Essay

1954 Words May 21st, 2012 8 Pages

Cytochrome P450s (CYPs) are monooxygenase proteins that catalyze the metabolism of exogenous and endogenous substrates. CYPs function as enzymes and are found in all kingdoms of life. The P in P450 refers to pigment and 450nm refers to the wavelength of CYPs in solution exposed to carbon monoxide (CO). CYPs belong to the superfamily of proteins containing a heme (iron) cofactor active site. The heme active site is tethered to the CYP protein via thiolate ligand derived from a cysteine residue.The cysteine and accompanying residues in the C-terminal are highly conserved and contain the PROSITE consensus sequence [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C - [LIVMFAP] - [GAD] [2-8]. The heme is used to oxidize
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Generally, AhR is located in the cytosol in a heterotetrameric protein complex in its inactive state; however, upon activation by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), AhR is translocated to the nucleus.

CYP2S1 regulation by AhR
In a 2010 Toxicology journal [18], studies of CYP2S1 mRNA and protein expression conclude that the aryl hydrocarbon receptor (AhR) is involved in the transcriptional regulation of CYP2S1 in rats. Using reverse transcriptase polymerase chain reaction (RT-PCR) and an immunoblot assay, mRNA and protein expression was compared and quantified. Male and female rats were treated with an AhR agonists; 3-methylcholanthrene (3-MC) at a dose of 25mg/kg each day for a total of 3 days or with a single injection of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) at doses of 0, 1, 5, 10, 50, 100mug/kg [18]. After treatment with 3-MC, an increase CYP2S1 levels were seen in lung, stomach, jejunum and ileum. They also saw an increase of CYP2S1 levels in lung, liver and kidney after treatment with TCDD [18].
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Methods: RT-PCR and western blot analysis
RT-PCR is a procedure that uses reverse transcriptase to make complimentary DNA and PCR to amplify it. RNA is reverse transcribed into cDNA using a reverse transcriptase enzyme, forward/reverse primers about 20 nucleotides in length, dNTPs and RNase inhibitor. In a reaction, the temperature is raised to 70°C

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