Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme

Boopathy, R. ; Balasubramanian, A. S. (1986) Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme Biochemical Journal, 239 (2). pp. 371-377. ISSN 0264-6021

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Official URL: http://www.biochemj.org/bj/239/0371/bj2390371_brow...

Abstract

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.

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ID Code:91611
Deposited On:22 May 2012 12:17
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