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The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40 phox. While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40 phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40 phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40 phox revealed that it plays a critical role in the binding to p67 phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways. [ABSTRACT FROM AUTHOR]

Lipoxin B4 (LXB4) is metabolized to 20-hydroxy-LXB4 by rat liver microsomes. The ω-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, indicating involvement of a cytochrome P-450 (P-450). This is supported by inhibition of the reaction by antibodies raised against NADPH—P-450 reductase. The P-450 appears to be the one responsible for leukotriene B4 ω-hydroxylation, because leukotriene B4 inhibits the formation of 20-hydroxy-LXB4 and LXB4 blocks the leukotriene B4 ω-hydroxylase activity in microsomes. Incubation of 20-hydroxy-LXB4 with both rat liver cytosol and NAD leads to formation of a more polar metabolite on high-performance liquid chromatography. The metabolite is identified as 20-carboxy-LXB4 a novel metabolite of LXB4 based on analyses by ultraviolet spectrometry and by gas chromatography/mass spectrometry. The 20-carboxy-LXB4 forming activity is localized in cytosol, with an optimal pH of 8.5. The activity is dependent on NAD+, but NADP- can not replace NAD-. The reaction is inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by substrates of the enzyme such as ethanol and 20-hydroxy-leukotriene B4. Disulfiram, an inhibitor of aldehyde dehydrogenase, also blocks the 20-carboxy-LXB4 formation. These observations suggest that both alcohol dehydrogenase and aldehyde dehydrogenase participate in the oxidation of 20-hydroxy-LXB4 to 20-carboxy-LXB4. [ABSTRACT FROM AUTHOR]

Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD + and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD + and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for ω-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes ω-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1: 2. The affinity of the dehydrogenase for 20-OHLTB4 may be higher than that for 12-hydroxylaurate (Km = 70 µM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase. [ABSTRACT FROM AUTHOR]

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OHLTB4) by the 150 000 × 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-1 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 F2, or laurie 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 welt 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 c inhibited 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 LTB4 ω-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 LTB4 ω-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 ω-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, LTB4 ω-hydroxylase, is different from the ones reported to be involved in ω-hydroxylation reactions of prostaglandins and fatty acids. [ABSTRACT FROM AUTHOR]

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system, The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin—agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 ± 0.53 nmol/mg protein (mean ± SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0–7.5, The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 ± 3.5 μmol O2- · min-1 mg-1 membrane-derived protein (mean ± SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids. [ABSTRACT FROM AUTHOR]

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3-domain-containing protein p40 phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40 phox and its partner in phagocytes, p67phox. Both p40 phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40 phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40 phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40 phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40 phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox. [ABSTRACT FROM AUTHOR]