Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils.
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 omega-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 omega-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-OH-LTB4 may be higher than that for 12-hydroxylaurate (Km = 70 microM), 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.
Gotoh
Y
Y
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Sumimoto
H
H
Minakami
S
S
D000429:Alcohol Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids; D007975:Leukotriene B4 / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000097:blood; D008861:Microsomes / Q000201:enzymology; D009243:NAD / Q000097:blood; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D006893:Hydroxyeicosatetraenoic Acids; D009243:NAD; D007975:Leukotriene B4; C031571:5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid; C031911:20-carboxyleukotriene B4; D000429:Alcohol Oxidoreductases; C058675:20-hydroxyleukotriene B4 dehydrogenase