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

Journal Article · Wed Aug 01 04:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.15.5638· OSTI ID:6214246

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

---

1. Vanderbilt Univ., Nashville, TN (USA)

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.

• OSTI ID: 6214246
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 87:15; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

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