Expression and characterization of cytochrome P450 2X1 in channel catfish (Ictalurus punctatus).
Previous studies in channel catfish identified a novel cDNA encoding the cytochrome P450 isoform, CYP2X1. To characterize the substrate specificity of CYP2X1, the 57 kDa protein was expressed in Sf9 cells. Microsomes from Sf9 cells transfected with CYP2X1 demonstrated a maximum carbon monoxide-reduced difference spectrum at 450 nm and catalyzed aminopyrine and benzphetamine demethylase activity with catalytic efficiency (Vmax/Km) values of 0.82 pmol/nmol P450/min and 4.39 pmol/nmol P450/min, respectively. However, enzymatic activity was not observed following incubation with p-nitrophenol, benzyloxyresorufin or pentoxyresorufin. Expression of CYP2X1 transcription was significantly elevated in the gills and liver relative to that detected in brain, kidney and heart. In the brain, liver and heart, intraperitoneal injections with clofibric acid, ethanol, pyridine and rifampin failed to alter expression of CYP2X1 mRNA. In kidney, pyridine significantly suppressed the expression of CYP2X1 transcription (p < or = 0.05). These results indicate CYP2X1 displays minimal catalytic activities consistent with other piscine CYP2 isoforms, and unique tissue expression and regulation patterns in juvenile channel catfish.
Mosadeghi
Sasan
S
Department of Environmental Sciences, University of California, Riverside, CA 92521-0144, USA.
Furnes
Bjarte
B
Matsuo
Aline Y O
AY
Schlenk
Daniel
D
D000632:Aminopyrine / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D001589:Benzphetamine / Q000378:metabolism; D002248:Carbon Monoxide; D002384:Catalysis; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D007059:Ictaluridae / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008861:Microsomes / Q000378:metabolism; D009841:Oligonucleotides; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera; D013379:Substrate Specificity
D009841:Oligonucleotides; D000632:Aminopyrine; D001589:Benzphetamine; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System