Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver.
A novel human liver cytochrome P-450 isozyme (P-450-AA), which catalyzes arachidonic acid epoxidation, has been purified to electrophoretic homogeneity from human liver. As judged spectrally, the newly described isozyme is low spin in the oxidized state, with a soret band at 415 nm and an increased maximum at 451 nm in the CO-difference spectrum. Cytochrome P-450-AA appeared homogeneous as judged by the appearance of a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 53,100. Although cytochrome P-450-AA had a relatively low specific content of 10.8 nmol/mg, it possessed a high activity of arachidonic acid epoxidation. The P-450-AA oxidized arachidonic acid in a reconstituted system into the four regioisomeric epoxyeicosatrienoic acids (EETs) (5, 6-, 8, 9-, 11, 12-, 14, 15-EETs) at a rate of 2,010 pmol/nmol/min, a rate which is 37-fold higher than that observed with the crude microsomal preparation. Moreover, the purified cytochrome P-450-AA catalyzed the de-ethylation of 7-ethoxyresorufin at the rate of 2970 pmol/nmol/min, whereas other cytochrome P-450-dependent reactions were carried out at 23-2,000-fold lower rates and ranged between 0.3-130 pmol/nmol/min. The amino acid composition is different from that of other cytochrome P-450 isozymes. The NH2-terminal sequence of 20-amino acid residues was compared to that of LM2 and PB2-B2, the phenobarbital-induced forms in rabbit and rats, respectively. Comparison was also made with two forms of human cytochrome P-450, HLc and HLd. There were 7/20 identical residues for P-450-AA and LM2 and 4/20 for P-450-AA and PB2-B2. There were 2/20 identical residues for P-450-AA and HLd, and no identical residues were found for HLc. We conclude that the biologically active EETs, are formed by a distinct and unique P-450 isozyme from human liver and that arachidonic acid can serve as a screen for detection of the novel P-450 isozyme.
D000328:Adult; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004852:Epoxy Compounds / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D013053:Spectrophotometry
D000596:Amino Acids; D001095:Arachidonic Acids; D004852:Epoxy Compounds; D007527:Isoenzymes; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System