Molecular cloning and enzymatic characterization of sheep CYP2J

Abstract

1. Cytochrome P450 (CYP) 2Js have been studied in various mammals, but not in sheep, as an animal model used to test veterinary drug metabolism.

2. Sheep CYP2J was cloned from liver messenger RNA (mRNA) by RACE. The cDNA, after modification at its N- and C-terminals, was expressed in Escherichia coli and the sheep CYP2J protein, purified by chromatography, was 80% homologous to human and monkey CYP2J2.

3. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that CYP2J mRNA was expressed in liver, cortex, respiratory and olfactory mucosa, heart, bronchi, lung, spleen, small intestine and kidney.

4. The purified enzyme was catalytically active towards aminopyrine, all-trans-retinoic acid, and particularly arachidonic acid forming 20-HETE, 19-HETE, and 18-HETE (about 86% of the total) and 14,15-, 11,12-, 8,9-, and 5,6-EETs (cis-epoxyeicosatrienoic acids; about 14% of total), with a regioselectivity similar to that shown by the mammalian CYP2J2s.

Keywords::

• CYP2J
• sheep
• CYP2J expression

Acknowledgements

The authors acknowledge Dr Silvia Burchielli of Fondazione Toscana “G. Monasterio” for experimental support.