Arachidonate 12-lipoxygenase of rat pineal glands: catalytic properties and primary structure deduced from its cDNA.
When a crude extract of rat pineal glands (the 1000 x g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product. The 12-lipoxygenase of rat pineal gland also reacted with linoleic and alpha-linolenic acids at 35% and 101% the rate of arachidonate 12-oxygenation, respectively. Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx. 74 kDa. A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-lipoxygenase cDNA was confirmed by its expression in E. coli. The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75,305. The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-lipoxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase. Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-lipoxygenase is more closely related to leukocyte 12-lipoxygenase than to platelet 12-lipoxygenase. Upon RNA blot analysis, by far the highest content of 12-lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence of 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain. Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.
Hada
T
T
Department of Biochemistry, Tokushima University, School of Medicine, Japan.
Hagiya
H
H
Suzuki
H
H
Arakawa
T
T
Nakamura
M
M
Matsuda
S
S
Yoshimoto
T
T
Yamamoto
S
S
Azekawa
T
T
Morita
Y
Y
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D018076:DNA, Complementary / Q000737:chemistry*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D010870:Pineal Gland / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D013552:Swine
D018076:DNA, Complementary; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D016718:Arachidonic Acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase