Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme.
The fatty acid cyclooxygenase (ec 1.14.99.1) that produces the prostaglandin and thromboxane precursor, 15-hydroperoxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid (PGG2), has been purified from sheep vesicular glands to a specific activity of 46,000 units/mg of protein by combining detergent solubilization, (NH4)SO4 fractionation, chromatography on DEAE-cellulose and Flurbiprofen-Sepharose, isoelectric focusing, and gel filtration. The final enzyme preparation exhibited only one band of 70,000 molecular weight following sodium dodecyl sulfate gel electrophoresis and staining with Coomassie blue. Treatment of the purified oxygenase with [3H] acetylsalicylic acid yielded a radioactive product which co-electrophoresed with the protein of 70,000 molecular weight. Thus, the isolated protein appeared to be the same one which, in crude preparations, selectively binds acetyl groups in association with prostaglandin synthetic activity. Incubation of the purified oxygenase with [1-14C] arachidonic acid in the presence of stannous chloride yielded only 9 alpha, 11 alpha, 15-trihydroxy-prosta-5,13-dienoic acid (PGF2alpha). Without stannous chloride, a mixture of radioactive products was observed which was characteristic of nonenzymic breakdown of PGG2. Thus, the isolated enzyme catalyzed the insertion of both oxygen molecules required for the formation of prostaglandins and thromboxanes from polyunsaturated fatty acid substrates. The aerobic absorption spectrum of the isolated oxygenase showed a faint peak at 412 nm indicative of heme. The iron content indicated that a significant amount of nonheme iron was present. The purified oxygenase was activated by added hemin, which was readily bound to the protein. The subsequently isolated heme-protein complex showed a major absorption peak at 407 nm.
D000818:Animals; D001051:Apoenzymes / Q000032:analysis; D001665:Binding Sites; D002846:Chromatography, Affinity; D007501:Iron / Q000032:analysis; D008297:Male; D006899:Mixed Function Oxygenases / Q000302:isolation & purification; D008970:Molecular Weight; D011451:Prostaglandin-Endoperoxide Synthases / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012669:Seminal Vesicles / Q000201:enzymology; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D001051:Apoenzymes; D007501:Iron; D006899:Mixed Function Oxygenases; D011451:Prostaglandin-Endoperoxide Synthases