Rabbit lung prostaglandin ω-hydroxylase (P450 4A4) was expressed in Escherichia coli using the isopropyl β-d-thiogalactopyranoside (IPTG) inducible expression vector pCWori+, containing the full-length cDNA encoding the P450 4A4. The first seven codons were changed to reflect E, coli codon bias [a modification of the method of Barnes et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5597–5601]; only the second residue of P450 4A4 was altered (Ser to Ala), while the remaining mutations were silent. This strategy was adopted in order to minimize changes in the structure of the expressed enzyme. Induction by IPTG of the apoprotein peaked after 6 h, and by including the heme precursor δ-aminolevulinic acid, enzymatic activity peaked 12 h after addition of IPTG. The isolated membrane fraction, free of cell debris, contained 12–15 nmol of P450/L of media. The expressed enzyme was purified to electrophoretic homogeneity, and kinetic and spectrophotometric data indicate that this expressed, purified enzyme is equivalent to the enzyme purified from rabbit lung. The Km for PGE1 was determined to be 3.0 µM, which is the same as that obtained for the enzyme purified from lung [Williams et al. (1984) J. Biol. Chem. 259, 14600–14608]. The CO-reduced difference spectrum of purified P450 4A4 exhibited a λmax at 450 nm, and the absolute absorbance spectrum of the pyridine hemochromogen revealed a typical b type heme. To characterize P450 4A4 further, the catalytic activities with prostaglandin E1 (PGE1), arachidonate, 15-hydroxyeicosatetraenoic acid (15-HETE), and palmitate were investigated. PGE1 and arachidonate ω-hydroxylation activities were highly dependent on the concentration of NADPH-cytochrome P450 oxidoreductase, with maximal activities being achieved at a 10–20-fold excess of reductase. Interestingly, activities with arachidonate, palmitate, and 15-HETE, but not with PGE1, were found to be highly dependent on the amount of l-α-dilauroylphosphatidylcholine (DLPC) in the reaction mixtures. Using optimal amounts of DLPC, initial velocity kinetic experiments were performed. Surprisingly, in spite of structural dissimilarity among these substrates, the Km values did not differ significantly. Cytochrome b5 had basically no effect on the Km values but doubled Vmax values for PGE1, palmitate, and 15-HETE and tripled the Vmax for arachidonate. The Vmax for arachidonate was found to be the highest, 37 pmol min−1 pmol−1, and for PGE1 the lowest, 8.4 pmol min−1 pmol−1, determined in the presence of cytochrome b5. The Vmax/Km values were determined to be 22, 10, 6.1, and 2.8 for arachidonate, palmitate, 15-HETE, and PGE1, respectively, in the presence of cytochrome b5. These results demonstrate that P450 4A4 utilizes arachidonate efficiently and suggest that this enzyme is contributing to the physiological co-hydroxylation of arachidonate.
ASJC Scopus subject areas