Cloning of the cDNA for human 12-lipoxygenase.
A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.
Izumi
T
T
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Hoshiko
S
S
Rådmark
O
O
Samuelsson
B
B
D000595:Amino Acid Sequence; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015183:Restriction Mapping
D003062:Codon; D015345:Oligonucleotide Probes; D004247:DNA; D001092:Arachidonate 12-Lipoxygenase