Subcellular localization of the PGE2 synthesis activity in mouse resident peritoneal macrophages.
The aim of this work was to establish, on a quantitative basis, the subcellular distribution of the enzyme system that converts arachidonic acid into prostaglandin (PG) E2 in mouse resident peritoneal (MRP) macrophages. Kinetic studies were conducted on cell-free extracts derived from cells cultivated for 1 d, using [1-14C]arachidonic acid as substrate and measuring the label in PGE2 after extraction and thin layer chromatography. The activity was synergistically enhanced by L-adrenaline and reduced glutathione, inhibited by indomethacin, and linearly related to the concentration of the cell-free extract. It was labile at 0 degrees C in the medium used for homogenization and fractionation of the cells (half-life less than 2 h). Addition of catalase (0.15 mg/ml) to the suspension medium increased the initial activity (by congruent to 70%) and the stability (half-life congruent to 6 h) of the enzyme in cytoplasmic extracts. It enabled us to establish the density distribution after isopycnic centrifugation in a linear gradient of sucrose. The sample centrifuged consisted of untreated cytoplasmic extracts, or cytoplasmic extracts treated with digitonin and Na pyrophosphate. Comparison of the centrifugation behavior of PGE2 synthesis activity with that of various enzymes used as reference for the major subcellular entities has revealed that PGE2 synthesis fairly fits the density profile of sulfatase C in each case. The conclusion is that at least the rate-limiting reaction in the conversion of arachidonic acid into PGE2 is catalyzed by an enzyme associated with the endoplasmic reticulum.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001202:Ascitic Fluid / Q000276:immunology; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D015232:Dinoprostone; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004789:Enzyme Activation; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D008264:Macrophages / Q000145:classification / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D008815:Mice, Inbred Strains; D011458:Prostaglandins E / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000201:enzymology
D001095:Arachidonic Acids; D011458:Prostaglandins E; D016718:Arachidonic Acid; D002374:Catalase; D005978:Glutathione; D015232:Dinoprostone