title: Arachidonate 12-lipoxygenase of platelet-type in human epidermal cells.
abstract: A homogenate of epidermal cells isolated from human skin converted arachidonic acid to 12S-hydroxy-5, 8,10,14-eicosatetraenoic acid and 15-hydroxy-5, 8,11,13-eicosatetraenoic acid as the main lipoxygenase products. The production of these hydroxy acids was not stimulated by the addition of 1 mM NADPH required for cytochrome P-450 reaction, but inhibited by 65-75% with 40 microM nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor. In addition to these lipoxygenase products, the epidermal cell homogenate converted arachidonic acid to prostaglandin E2 together with minor amounts of prostaglandins D2 and F2a and 12-hydroxy-5,8,10-heptadecatrienoic acid. Thromboxane B2 was not detected. This finding rules out the possible contamination of platelet 12-lipoxygenase in the epidermal cells. After subcellular fractionation of the epidermal cell homogenate, the 12-lipoxygenase activity was found in the 164,000 x g supernatant, the 164,000 x g pellet, and the 10,000 x g pellet. The cytosolic enzyme and the enzymes solubilized from the two pellets produced 12S-hydroperoxy-5,8,10,14-eicosatetraenoic acid as the primary product in contrast to cytochrome P-450 which produces primarily hydroxy acids. The 12-lipoxygenase in the 164,000 x g supernatant and the solubilized enzymes from the 164,000 x g pellet and 10,000 x g pellet were precipitable by antibodies raised against human platelet 12-lipoxygenase, but not by antibodies against porcine leukocyte 12-lipoxygenase. The immunoprecipitated 12-lipoxygenase from each fraction was almost inactive with linoleic acid as substrate, characteristic of 12-lipoxygenase of platelet-type. Furthermore, 12-lipoxygenase mRNA in the epidermal cells could be reverse-transcribed and amplified by polymerase chain reaction with the primers specific for human platelet 12-lipoxygenase cDNA, but not with those for porcine leukocyte 12-lipoxygenase cDNA. Thus, the 12-lipoxygenase of human epidermal cells is similar to human platelet 12-lipoxygenase in terms of immunogenicity, catalytic property, and primary structure, and distinct from leukocyte 12-lipoxygenase.
authors.affiliation: Department of Biochemistry, Tokushima University, School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Takahashi
authors.forename: G R
authors.initials: GR
authors.lastname: Reddy
authors.forename: N
authors.initials: N
authors.lastname: Ueda
authors.forename: S
authors.initials: S
authors.lastname: Yamamoto
authors.forename: S
authors.initials: S
authors.lastname: Arase
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001483:Base Sequence; D001792:Blood Platelets; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004247:DNA; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012995:Solubility
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D004247:DNA; D001092:Arachidonate 12-Lipoxygenase