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The ubiquitous hydroxylated fatty acids derived from arachidonic acid (HETEs) or linoleic acid (HODEs) exhibit diverse biological effects including chemotaxis, cell proliferation, and modulation of several enzymatic pathways, including the 5-lipoxygenase leading to the inflammatory leukotrienes. It was observed that 12(S)-and 15(S)-HETE and 13(S)-HODE (12-and 15-lipoxygenase-derived metabolites, respectively) inhibited the 5-lipoxygenase present in rat basophilic leukemia (RBL -1) cell homogenates whereas the 15(R) chiral enantiomer and the nonhydroxylated linoleic, oleic, and stearic acids were either less potent or ineffective. In examining the mechanism of this inhibition, the relative effectiveness of several fatty acids in displacing [ 3 H]15-HETE bound to cytosol preparations were compared and the results indicated that these (S) hydroxy fatty acids and 5(S)-HETE were significantly more potent than either the 15(R) enantiomer, 15(S)-HETE methyl ester, arachidonic acid, or prostaglandin F 2 ␣ . In order to identify the protein(s) that specifically binds HETEs, 15(S)-HETE biotin hydrazide was used as a probe to detect any HETEprotein complexes as this compound both inhibited the 5lipoxygenase and interfered with the binding of [ 3 H]15-HETE to cytosol preparations. SDS-PAGE analysis and chemiluminescent detection revealed that the major cytosolic proteins that bound this biotinylated probe had molecular masses of 43 and 51 kD. Fatty acid competition experiments indicated that the order of effectiveness in displacing this probe from these proteins was 13(S)-HODE Ͼ 5(S)-HETE Ϸ 15(S)-HETE Ͼ Ͼ stearic acid Ϸ arachidonic acid Ϸ 15(R)-HETE. Amino acid sequence analysis showed that the 43 kD protein was actin. These findings suggest the possibility that actin may play a major role in the biological effects of monohydroxylated metabolites derived from cellular 5-, 12-, and 15-lipoxygenases.-Kang, L-T., and J. Y. Vanderhoek. Mono (S) hydroxy fatty acids: novel ligands for cytosolic actin.