Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase

Funk, C D; Furci, L; FitzGerald, G A

doi:10.1073/pnas.87.15.5638

Title: Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase

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Abstract

The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE), is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, the authors have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a M{sub r} 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-12lx) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes formore »« less

Authors:

Funk, C D; Furci, L; FitzGerald, G A ^[1]

Vanderbilt Univ., Nashville, TN (USA)

Publication Date:: Wed Aug 01 00:00:00 EDT 1990

OSTI Identifier:: 6214246

Resource Type:: Journal Article

Journal Name:: Proceedings of the National Academy of Sciences of the United States of America; (USA)

Additional Journal Information:: Journal Volume: 87:15; Journal ID: ISSN 0027-8424

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; ARACHIDONIC ACID; METABOLISM; OXYGENASES; AMINO ACID SEQUENCE; RECOMBINANT DNA; DNA SEQUENCING; BIOLOGICAL PATHWAYS; BLOOD PLATELETS; DNA-CLONING; LEUKEMIA; MESSENGER-RNA; PHOSPHORUS 32; TUMOR CELLS; ANIMAL CELLS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CARBOXYLIC ACIDS; CLONING; DAYS LIVING RADIOISOTOPES; DISEASES; DNA; DNA HYBRIDIZATION; ENZYMES; HEMIC DISEASES; HYBRIDIZATION; IMMUNE SYSTEM DISEASES; ISOTOPES; LIGHT NUCLEI; MATERIALS; MOLECULAR STRUCTURE; MONOCARBOXYLIC ACIDS; NEOPLASMS; NUCLEI; NUCLEIC ACIDS; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; OXIDOREDUCTASES; PHOSPHORUS ISOTOPES; RADIOISOTOPES; RNA; STRUCTURAL CHEMICAL ANALYSIS; 550201* - Biochemistry- Tracer Techniques

Citation Formats


                    Funk, C D, Furci, L, and FitzGerald, G A. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.  United States: N. p., 1990. 
        Web.  doi:10.1073/pnas.87.15.5638.

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                    Funk, C D, Furci, L, & FitzGerald, G A. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.  United States.  https://doi.org/10.1073/pnas.87.15.5638

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                    Funk, C D, Furci, L, and FitzGerald, G A. 1990.  
        "Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase".  United States.  https://doi.org/10.1073/pnas.87.15.5638.

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@article{osti_6214246,

  title        = {Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase},

  author       = {Funk, C D and Furci, L and FitzGerald, G A},

  abstractNote = {The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE), is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, the authors have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a M{sub r} 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-12lx) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.},

  doi          = {10.1073/pnas.87.15.5638},

  url          = {https://www.osti.gov/biblio/6214246},
  journal      = {Proceedings of the National Academy of Sciences of the United States of America; (USA)},

  issn         = {0027-8424},

  number       = ,

  volume       = 87:15,

  place        = {United States},

  year         = {Wed Aug 01 00:00:00 EDT 1990},

  month        = {Wed Aug 01 00:00:00 EDT 1990}

}

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