Abe A, Shayman J A
Division of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1998 Apr 3;273(14):8467-74. doi: 10.1074/jbc.273.14.8467.
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 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.
先前报道了一种新的神经酰胺代谢途径，即1-O-酰基神经酰胺的形成（阿部，A.，谢曼，J. A.，和拉丹，N. S.（1996年）《生物化学杂志》271，14383 - 14389）。在该途径中，磷脂酰乙醇胺或磷脂酰胆碱sn-2位的脂肪酸转移至神经酰胺的1-羟基位置。通过连续步骤，包括硫酸铵分级分离、DEAE-葡聚糖凝胶、苯基-琼脂糖、S-琼脂糖、葡聚糖G-75、伴刀豆球蛋白A-琼脂糖和肝素-琼脂糖层析，从小牛脑线粒体后上清液中纯化出一种催化N-乙酰鞘氨醇酯化的酶。通过葡聚糖G-75凝胶过滤测定该酶的分子量为40 kDa。结合在伴刀豆球蛋白A-琼脂糖柱上的酶用含500 mM α-甲基-D-甘露吡喃糖苷的缓冲液洗脱。通过肝素-琼脂糖层析进一步纯化得到两个酶活性峰。通过SDS-聚丙烯酰胺凝胶电泳以及在酸性天然凝胶上分离后的回收测定，观察到转酰基酶活性与分子量为40 kDa的染色蛋白相吻合。肝素-琼脂糖层析的第二个活性峰代表纯化了193,000倍。这些结果与该酶是一种分子量约为40 kDa、具有单条多肽链的糖蛋白一致。纯化后的酶在pH 4.5时具有最佳pH值。二价阳离子Ca2+和Mg2+增强了转酰基酶活性，但并非其活性所必需。在2 mM ATP或2 mM二硫苏糖醇存在下，未观察到酶活性的激活或失活。用l mM N-乙基马来酰亚胺、1 mM苯甲基磺酰氟或一种有效的胞质Ca2+非依赖性磷脂酶A2抑制剂3.1 μM溴烯醇内酯对该酶进行预孵育，对酶活性没有显著影响。在大于773 μM Triton X-100存在下，酶活性完全丧失。在10 - 100 μg/ml肝素存在下，观察到酶活性部分受到抑制。在没有N-乙酰鞘氨醇的情况下，该酶表现为磷脂酶A2。这些结果强烈表明1-O-酰基神经酰胺合酶既是一种转酰基酶，也是一种新型磷脂酶A2。
