Uroporphyrinogen oxidation catalyzed by human cytochromes P450.
Porphyria cutanea tarda is associated with excess hepatic production of uroporphyrin. Oxidation of uroporphyrinogen to uroporphyrin was previously demonstrated to be specifically catalyzed by cytochrome P450 (CYP) 1A2. Here, we investigated the ability of human CYP1A2 to catalyze uroporphyrinogen oxidation (UROX). UROX activity in human liver microsomes was maximally only 10% of the activity in microsomes from livers of untreated mice. There was a poor correlation of UROX activity with methoxyresorufin demethylation, an activity catalyzed predominantly by CYP1A2 and strongly correlated with immunodetectable CYP1A2. With CYP forms expressed in HepG2 cells, the methoxyresorufin demethylation and (ethoxyresorufin deethylation) activities of murine and human CYP1A2 forms were similar, but UROX activity catalyzed by human CYP1A2 was only 15-20% of the activity catalyzed by murine CYP1A2. Human CYP1A1, CYP1A2, and CYP3A4 expressed in lymphoblastoid cells all catalyzed UROX. In insect cells, CYP1A2 was more active in catalyzing UROX than was CYP1A1, CYP2E, CYP3A4, or CYP3A5. Human CYP1A2 expressed in Escherichia coli as a fusion protein with rat CYP oxidoreductase also catalyzed UROX. Reconstituted human CYP1A2 and CYP3A4 were active in catalyzing UROX, with reconstituted CYP1A2 having the highest specific activity obtained in this study. From inhibitor studies, it was concluded that some of the UROX activity in the insect cell microsomes was attributable to expressed CYP and some to an unidentified source. These results indicate that human CYP1A2 is active in catalyzing UROX but has lower activity than the murine orthologue. The results also indicate that most of the UROX activity found in human liver microsomes is not due to CYP1A2.
Sinclair
P R
PR
Department of Biochemistry, Dartmouth Medical School, USA. psinc@Dartmouth.edu
Gorman
N
N
Tsyrlov
I B
IB
Fuhr
U
U
Walton
H S
HS
Sinclair
J F
JF
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014577:Uroporphyrinogens / Q000378:metabolism*
D008081:Liposomes; D011993:Recombinant Fusion Proteins; D014577:Uroporphyrinogens; D003577:Cytochrome P-450 Enzyme System