Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.
Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.
D000107:Acetylation; D000818:Animals; D001241:Aspirin / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000378:metabolism*; D006454:Hemoglobins / Q000494:pharmacology; D066298:In Vitro Techniques; D011451:Prostaglandin-Endoperoxide Synthases / Q000097:blood* / Q000302:isolation & purification; D012756:Sheep
D002241:Carbohydrates; D006454:Hemoglobins; D006418:Heme; D006427:Hemin; D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin