Subcellular localization of arachidonate 12-lipoxygenase and morphological effect of its overexpression on murine keratinocytes.
Arachidonate 12-lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-lipoxygenase cDNA or a vector without 12-lipoxygenase cDNA, these structural changes were not observed.
Nakamura
M
M
Department of Biochemistry, Tokushima University School of Medicine, Kuramoto-cho, Tokushima 770, Japan.
Yamamoto
S
S
Ishimura
K
K
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000032:analysis* / Q000235:genetics; D001792:Blood Platelets / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D013347:Subcellular Fractions / Q000201:enzymology*; D014162:Transfection
D001092:Arachidonate 12-Lipoxygenase