Purification and characterisation of prostaglandin endoperoxide synthetase from sheep vesicular glands.
The membrane-bound prostaglandin endoperoxide synthetase was purified until homogeneity, starting from sheep vesicular glands. The enzyme was obtained as a complex with Tween-20, containing 0.69 mg detergent per mg protein. No residual phospholipid could be detected. Prostaglandin endoperoxide synthetase appeared to be a glycoprotein, containing mannose and N-acetyl-glucosamine. No haemin or metal atoms were present. A molecular weight of 126 000 was found for the apoprotein by ultracentrifugation in 0.1% Tween solutions. The polypeptide chain without carbohydrate had a molecular weight of 69 000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pure enzyme displays both cyclooxygenase and peroxidase activity, thus converting arachidonic acid into prostaglandin H2. The isolated synthetase requires haemin, which possibly acts as an easily dissociable prosthetic group, and a suitable hydrogen donor to protect the enzyme from peroxide inactivation and which is consumed in stoichiometric amounts to reduce the intermediate hydroperoxy group.
D000117:Acetylglucosamine / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006427:Hemin / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology; D008297:Male; D008358:Mannose / Q000032:analysis; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification; D008970:Molecular Weight; D011451:Prostaglandin-Endoperoxide Synthases / Q000302:isolation & purification / Q000378:metabolism; D012669:Seminal Vesicles / Q000201:enzymology*; D012756:Sheep
D000596:Amino Acids; D006873:Hydroquinones; D006427:Hemin; D006899:Mixed Function Oxygenases; D011451:Prostaglandin-Endoperoxide Synthases; D008358:Mannose; D000117:Acetylglucosamine