Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity
Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity
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DOI：
10.1074/jbc.273.14.8467
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发表时间：
1998-04-03
影响因子：
4.8
通讯作者：
Shayman, JA
中科院分区：
文献类型：
--
作者：
Abe, A;Shayman, JA
一种神经酰胺代谢的新途径，1- o -酰基神经酰胺的形成，以前被报道过(Abe， A, Shayman， J. A., and Radin， N. S.（1996）。在这个途径中，磷脂酰乙醇胺或磷脂酰胆碱的sn-2位置的脂肪酸被转移到神经酰胺的1-羟基位置。从小牛脑线粒体后上清中，通过连续的步骤，包括硫酸铵分离，DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75， concanavalin a -琼脂糖，肝素- sepharose色谱法。在Sephadex G-75上凝胶过滤，酶的分子量为40 kDa，结合到魔豆蛋白a-琼脂糖柱上的酶用含有500 mM -甲基- d -甘露pyranoside的缓冲液洗脱。通过sds -聚丙烯酰胺凝胶电泳和酸性天然凝胶分离后的回收率，观察到转酰基酶活性与分子质量为40 kDa的染色蛋白之间的一致性，肝素- sepharose色谱的第二个活性峰代表纯化的193,000倍。这些结果与该酶是一种分子质量约为40 kDa的糖蛋白，具有单个多肽链一致。纯化后的酶在pH为4.5时具有最佳pH值，Ca2+和Mg2+二价阳离子增强了转酰基酶的活性，但对转酰基酶的活性不是必需的。在2 mM ATP或2 mM二硫代索糖醇存在下，酶活性既没有激活也没有失活，酶与1 mM n -乙基马来酰亚胺、1 mM苯基甲基磺酰氟或3.1 μ M溴烯醇内酯（一种有效的胞质Ca2+依赖性磷脂酶a抑制剂）进行预孵育(2)。在大于773 μ M Triton X-100的存在下，酶活性完全消失。在10-100 μ g/ml肝素的存在下，酶活性被部分抑制，在没有n -乙酰鞘氨醇的情况下，酶表现为磷脂酶a(2)。这些结果强烈表明，1- o -酰基神经酰胺合成酶既是一种转酰基酶，也是一种新型磷脂酶a(2)。
A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A, Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389), In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide, An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75, The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity, Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel, The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold, These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5, The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity, Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol, Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 mu M bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A(2), had no significant effect on the enzyme activity, The enzyme activity was completely abolished in the presence of greater than 773 mu M Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 mu g/ml heparin, In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A(2). These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A(2).