Novel membrane target proteins for lipoxygenase-derived mono(S)hydroxy fatty acids

Hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HOD Es) are major bioactive lipids formed via the lipoxygenase oxygenation of a rachidonic and linoleic acid, respectively. These metabolites appear to be involved in various cellular actions including cell proliferation, migratio n and regulation of enzyme activities such as phospholipases and kinases. I n view of the diversity of biological effects of these hydroxy fatty acids, it seems likely that multiple mechanisms are involved. Previous reports sh owed that 15(S)-HETE inhibited the 5-lipoxygenase in rat basophilic leukemi a (RBL-1) cell homogenates and established the presence of specific cellula r HETE binding sites in these and other cells. The present study used 15(S) -HETE biotin hydrazide and 15(S)-HETE biotin pentyl amide as probes to iden tify membrane target proteins present in RBL-1 cells that specifically inte ract with HETEs and HODEs. Two membrane-associated proteins, with apparent molecular weights of 43 and 58 kDa, were identified that specifically inter act with these probes and competition experiments indicated that 13(S)-HODE and 15(S)-HETE were the most effective competitors for the hydrazide probe , followed in decreasing effectiveness by S(S)-HETE, arachidonic acid, 15(R )-HETE, stearic acid and 12(S)-HHT, a cyclooxygenase product. The two prote ins were isolated and microsequencing analysis established their identities as actin and the cc-subunit of mitochondrial ATP synthase, respectively. I n vitro binding studies confirmed that purified actin is a potential 15-HET E binding protein. Subcellular cytosolic fractions exhibited fewer protein- probe complexes than membrane fractions. The association of HETEs and HODEs with these cytoskeletal and mitochondrial proteins, respectively, represen ts a new development in the potential actions of these hydroxy fatty acids. (C) 1999 Elsevier Science B.V. All rights reserved.