Prostaglandin-sensitive adenylate cyclase of a murine macrophage-like cell line (P388D1): II. Isolation and partial characterization of PGE2-binding proteins.
Properties of prostaglandin (PG) E2 binding sites of a murine macrophage cell line (P388D1) were investigated. The specific binding of [3H]-PGE2 to intact P388D1 cells at 4 degrees C in the presence of cytochalasin D (10 micrograms/ml) approached saturation at concentration greater than 7.5 X 10(-9) M, and could be displaced most effectively by unlabeled PGE2 and less effectively by unlabeled PGI2. The Scatchard analysis of the binding data clearly indicated the heterogeneity with respect to the PGE2 binding affinity and showed the presence of about 3.9 fmol/10(8) cells of the high affinity sites (KD = 1.1 X 10(-9) M) and about 24 fmol/10(8) cells of the low affinity sites (KD = 2 X 10(-8) M). PGE2-binding proteins were isolated from the detergent lysate of the radiolabeled P388D1 cells by affinity chromatography on Sepharose 4B coupled to PGE2. The affinity-isolated materials were further purified by successive use of Sephadex G-100 gel filtration and isoelectric focusing in the presence of dithiothreitol (1 mM) and Triton X-100 (0.5%). The final step yielded about 0.25% of the original radioactivity, which sharply focused as a single peak at pH near 6.5. The electrofocused PGE2-binding proteins migrated as a single band with a m.w. of 95,000 during SDS-PAGE. The electrofocused PGE2-binding proteins bound specifically to [3H]-PGE2 but showed again the heterogeneity with respect to their affinity.
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification*; D015232:Dinoprostone; D007525:Isoelectric Focusing; D007700:Kinetics; D007941:Leukemia P388 / Q000378:metabolism*; D007942:Leukemia, Experimental / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice; D008970:Molecular Weight; D011458:Prostaglandins E / Q000378:metabolism* / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D011982:Receptors, Prostaglandin / Q000032:analysis / Q000187:drug effects / Q000302:isolation & purification*
D002352:Carrier Proteins; D008565:Membrane Proteins; D011458:Prostaglandins E; D011956:Receptors, Cell Surface; D011982:Receptors, Prostaglandin; D015232:Dinoprostone