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-lipoxyge nase of rat pineal gland also reacted with linoleic and cr-linolenic a cids at 35% and 101% the rate of arachidonate 12-oxygenation, respecti vely. Upon Western blot analysis using polyclonal antibody against: po rcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gl and 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 r at pineal gland and the identity of the 12-lipoxygenase cDNA was confi rmed by its expression in E. coli. The amino acid sequence of the enzy me was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75 305. The enzyme s howed 72% identity of amino acid sequence with porcine leukocyte 12-li poxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase. Taken together, the high reactivi ty with C-18 fatty acids, the immunoreactivity and the amino acid homo logy 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-lipoxygenas e mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence o f 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybrid ization of rat brain. Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.