Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.
Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.
D002458:Cell Fractionation / Q000379:methods; D003577:Cytochrome P-450 Enzyme System; D000072470:Cytochrome P450 Family 4; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009245:NADH Dehydrogenase / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010100:Oxygen / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000072470:Cytochrome P450 Family 4; C040780:leukotriene B4 20-hydroxylase; D009245:NADH Dehydrogenase; D010100:Oxygen