Boopathy R, Balasubramanian A S
Biochem J. 1986 Oct 15;239(2):371-7. doi: 10.1042/bj2390371.
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.
花生四烯酸环氧化酶（前列腺素合成酶；前列腺素内过氧化物合成酶；EC 1.14.99.1）从绵羊血小板中纯化得到。纯化过程包括第一步使用布洛芬-琼脂糖、苯基-琼脂糖或花生酸-琼脂糖进行疏水柱层析，随后进行金属螯合琼脂糖和血红素-琼脂糖亲和层析。通过SDS/聚丙烯酰胺凝胶电泳和银染观察，纯化后的酶（分子量约为65,000）呈均一状态。该酶是一种以甘露糖为中性糖的糖蛋白。血红素或血红蛋白对其活性至关重要。纯化后的酶能够结合血红素，在410 nm处呈现特征性吸收峰。经过金属螯合柱层析后的酶能够被[乙酰基-3H]阿司匹林乙酰化。标记后的乙酰化酶无法与血红素-琼脂糖结合，推测是由于与血红素结合的丝氨酸残基发生了乙酰化。当使乙酰化酶结合血红素时，它也未能在410 nm处显示其特征性吸收峰。
