title: Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits.
abstract: In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis.
authors.affiliation: Institute of Biochemistry, University Clinics Charité, Hessische Str. 3-4, Humboldt-University Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Berger
authors.forename: K
authors.initials: K
authors.lastname: Schwarz
authors.forename: H
authors.initials: H
authors.lastname: Thiele
authors.forename: I
authors.initials: I
authors.lastname: Reimann
authors.forename: A
authors.initials: A
authors.lastname: Huth
authors.forename: S
authors.initials: S
authors.lastname: Borngräber
authors.forename: H
authors.initials: H
authors.lastname: Kühn
authors.forename: B J
authors.initials: BJ
authors.lastname: Thiele
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis* / Q000235:genetics; D001093:Arachidonate 15-Lipoxygenase / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D004247:DNA; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase