Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin.
Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin.
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N-乙酰咪唑对前列腺素内过氧化物合酶的乙酰化：与阿司匹林乙酰化的比较。
DOI：
10.1021/bi00155a002
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Marnett,LJ
中科院分区：
文献类型：
--
作者：
Wells,I;Marnett,LJ
摘要：用超过100或1000倍的n -乙酰咪唑（NAI）处理前列腺素内过氧化物（PGH）合成酶载脂蛋白会导致环加氧酶和过氧化物活性的时间依赖性失活。在NAI孵育之前用血红素重组载脂蛋白基本上保护了酶的失活。乙酰水杨酸（阿司匹林）或(w -2-氟-a-甲基-4-联苯乙酸（氟比洛芬）可抑制环氧化酶活性，但不改变NAI对过氧化物酶失活的时间过程。用羟胺处理nai灭活的apoPGH合成酶导致环加氧酶和过氧化物酶活性的大量再生。PGH合成酶与[3H-acefy/] NAI孵育后的放射性定量显示掺入1.7 ~ 0.9个乙酰基/70-kDa亚基。在非变性条件下，用胰蛋白酶裂解乙酰化蛋白，然后进行高效液相色谱分析，结果表明，尽管在38-kDa片段中也检测到明显的放射性，但大部分放射性被纳入了33-kDa片段。对乙酰化蛋白的胰糜肽图谱显示，许多潜在的乙酰化位点分布在蛋白质的广泛不同区域。载脂蛋白和全酶的凝色图没有明显的差异，这表明导致催化活性丧失的加合物对色谱条件不稳定。经NAI或阿司匹林乙酰化的PGH合成酶的不同生化特性表明阿司匹林对Ser530特异性的主要决定因素是水杨酸段与PGH合成酶蛋白这一区域的结合。前列腺素内过氧化物(PGH)* 1合成酶（EC 1.14）99.1)催化花生四烯酸级联分支的前两个步骤，导致前列腺素、血栓素和前列环素（Smith & Marnett, 1991）。其环氧合酶活性将花生四烯酸氧化为PGG2，其过氧化物酶活性在还原底物存在下将PGG2还原为PGH2 （Hamberg et al., 1974; nuteren & Hazelhof, 1973）。PGH合成酶是70 kda亚基的同二聚体，每个亚基结合一个血红素组（Van Der Ouderaa et al., 1977; Ruf et al., 1984）。血红素是两个环氧基都需要的
Revised Manuscript Received July 7, 1992 abstract: Treatment of prostaglandin endoperoxide (PGH) synthase apoprotein with a 100-or 1000-fold excess of N-acetylimidazole (NAI) led to time-dependent inactivation of both cyclooxygenase and peroxide activities. Reconstitution of apoprotein with heme prior to incubation with NAI substantially protected the enzyme from inactivation. Pretreatment of the protein with either acetylsalicylic acid (aspirin) or (ą)-2-fluoro-a-methyl-4-biphenylacetic acid (flurbiprofen), which inhibit cyclooxygenase activity, did not alter the time course of peroxidase inactivation by NAI. Treatment of NAI-inactivated apoPGH synthase with hydroxylamine led to substantial regeneration of both cyclooxygenase and peroxidase activities. Quantitation of radioactivity following incubation of PGH synthase with [3H-acefy/] NAI indicated incorporation of 1.7 ą 0.9 acetyl groups/70-kDa subunit. Cleavage of acetylated protein with trypsin under nondenaturing conditions followed by high-performance liquid chromatography analysis demonstrated that most of the radioactivity was incorporated into the 33-kDa fragment although significant radioactivity was also detectable in the 38-kDa fragment. Chymotryptic peptide mapping of acetylated protein revealed numerous potential sites of acetylation distributed in widely divergent regions of the protein. No apparent differences were observed between the chymotryptic maps of apo-and holoenzyme, suggesting that the adduct responsible for loss of catalytic activity is unstable to the chromatographic conditions. Thedifferent biochemical properties of PGH synthase acetylated by NAI or aspirin suggest that a major determinant of the specificity of aspirin for Ser530 is binding of the salicylate moiety to this region of the PGH synthase protein.Prostaglandin endoperoxide (PGH)* 1 synthase (EC 1.14. 99.1) catalyzes the first two steps of the branch of the arachidonic acid cascade leading to prostaglandins, thromboxane, and prostacyclin (Smith & Marnett, 1991). Its cyclooxygenase activity oxidizes arachidonic acid into PGG2, and its peroxidase activity reduces PGG2 into PGH2 in the presence of a reducing substrate (Hamberg et al., 1974; Nugteren & Hazelhof, 1973). PGH synthase isa homodimer of 70-kDa subunits that binds one heme group per subunit (Van Der Ouderaa et al., 1977; Ruf et al., 1984). Heme is required for both the cyclooxy-