Characterization of human neutrophil leukotriene B4ω‐hydroxylase as a system involving a unique cytochrome P‐450 and NADPH–cytochrome P‐450 reductase

Hideki SUMIMOTO, Koichiro TAKESHIGE, Shigeki MINAKAMI

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Abstract

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20‐hydroxyleukotriene B4 (20‐OH‐LTB4) by the 150000 × g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P‐450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P‐450. The addition of LTB4 to the microsomal fraction gave a type‐I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4, with the cytochrome P‐450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 μM, in agreement with the kinetically determined apparent Km, value for LTB4 (0.30 μM). Such a spectral change was not observed with prostaglandins A1, E1 and F, or lauric acid, none of which inhibited the LTB4, ω‐hydroxylation. The inhibition of the LTB4, ω‐hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen‐activating component of the LTB4, ω‐hydroxylase system is a cytochrome P‐450. Ferricytochrome cinhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH‐cytochrome‐P‐450 reductase inhibited both LTB4ω‐hydroxylase activity and the NADPH‐cytochrome‐c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH–cytochrome‐P‐450 reductase acts as an electron carrier in LTB4ω‐hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH–cytochrome‐c reductase activity but not the LTB4ω‐hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4‐hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P‐450 in human neutrophils, LTB4ω‐hydroxylase, is different from the ones reported to be involved in ω‐hydroxylation reactions of prostaglandins and fatty acids.

Original languageEnglish
Pages (from-to)315-324
Number of pages10
JournalEuropean Journal of Biochemistry
Volume172
Issue number2
DOIs
Publication statusPublished - Mar 1988

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Leukotriene B4
Leukotrienes
Cytochrome P-450 Enzyme System
Oxidoreductases
Neutrophils
Hydroxylation
Carbon Monoxide
Cytochromes b5
lauric acid
NADP
Human Activities
Liver
Rats
Dithionite
Light
Dinoprost
Molecular oxygen
Antibodies
Alprostadil
Electron Transport Complex IV

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Characterization of human neutrophil leukotriene B4ω‐hydroxylase as a system involving a unique cytochrome P‐450 and NADPH–cytochrome P‐450 reductase. / SUMIMOTO, Hideki; TAKESHIGE, Koichiro; MINAKAMI, Shigeki.

In: European Journal of Biochemistry, Vol. 172, No. 2, 03.1988, p. 315-324.

Research output: Contribution to journalArticle

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N2 - Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20‐hydroxyleukotriene B4 (20‐OH‐LTB4) by the 150000 × g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P‐450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P‐450. The addition of LTB4 to the microsomal fraction gave a type‐I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4, with the cytochrome P‐450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 μM, in agreement with the kinetically determined apparent Km, value for LTB4 (0.30 μM). Such a spectral change was not observed with prostaglandins A1, E1 and F2α, or lauric acid, none of which inhibited the LTB4, ω‐hydroxylation. The inhibition of the LTB4, ω‐hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen‐activating component of the LTB4, ω‐hydroxylase system is a cytochrome P‐450. Ferricytochrome cinhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH‐cytochrome‐P‐450 reductase inhibited both LTB4ω‐hydroxylase activity and the NADPH‐cytochrome‐c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH–cytochrome‐P‐450 reductase acts as an electron carrier in LTB4ω‐hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH–cytochrome‐c reductase activity but not the LTB4ω‐hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4‐hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P‐450 in human neutrophils, LTB4ω‐hydroxylase, is different from the ones reported to be involved in ω‐hydroxylation reactions of prostaglandins and fatty acids.

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