Identification of subunits of the 650 kDa 12(S)-HETE binding complex in carcinoma cells

Abstract

Cytosol and nuclei of Lewis lung carcinoma (LLC) cells contain high affinity binding sites specific for the arachidonic acid metabolite 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). In this report we present evidence that the cytosolic 12(S)-HETE binding complex also occurs in human erythroleukemia (HEL) and promonocytic leukemia (U937) cells as well as in murine 3T3-L1 preadipocytes but not in intestinal epithelial cells (Int407). The cytosolic 650 kDa 12(S)-HETE-binding complex was found to consist of subunits; raising the ATP concentration in cytosol led to conversion of the 650 kDa complex to a 50 kDa binding component, presumably the actual 12(S)-HETE binding polypeptide. Lowering of the cytosolic concentration of ATP had the opposite effect, i.e., the amount of the 650 kDa complex increased. Another subunit of the 650 kDa complex was identified as heat shock protein 70 (hsp70) by Western blot analyses and coimmunoprecipitation. Hsp70 was present in substoichiometric amounts, in an approximate 1:6 ratio. The multimeric nature of the binding complex and the identification of hsp70 as a subunit suggest that there are similarities between the 12(S)-HETE binding protein and receptors of the steroid/thyroid hormone superfamily.—Herbertsson, H., T. Kühme, U. Evertsson, J. Wigren, and S. Hammarström. Identification of subunits of the 650 kDa 12(S)-HETE binding complex in carcinoma cells.

Keywords

• 50 kDa ligand-binding protein (a subunit of cytosolic 12(S)-HETE binding complex)
• 12-lipoxygenase
• Lewis lung carcinoma
• gel permeation chromatography
• heat shock protein (hsp) 70 monoclonal antibody
• immunoprecipitation