A novel enzyme that catalyzes the esterification of N-acetylsphingosine - Metabolism of C-2-ceramides
A novel enzyme that catalyzes the esterification of N-acetylsphingosine - Metabolism of C-2-ceramides
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DOI：
10.1074/jbc.271.24.14383
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发表时间：
1996-06-14
影响因子：
4.8
通讯作者：
Radin, NS
中科院分区：
文献类型：
--
作者：
Abe, A;Shayman, JA;Radin, NS
在Madin-Darby犬肾细胞和小鼠组织匀浆中发现了一种独特的转酰酶，可催化短链神经酰胺N-乙酰鞘氨醇的酯化反应。它酯化了神经酰胺碳-1位的羟基。该酶的最适pH为4.2，在pH 4.5时对N-乙酰鞘氨醇的Km为9.4 μ M。转酰酶活性不依赖于游离脂肪酸或酰基辅酶A，而是利用磷脂酰乙醇胺或磷脂酰胆碱的α-酰基。匀浆中的转酰酶活性存在于100，000 x g上清液中，并且从膜部分提取的脂质可以用作酰基的供体。当由二油酰磷脂酰胆碱：1-棕榈酰-2-[C-14]花生四烯酸-磷脂酰乙醇胺：硫脂（70：0.2：30）组成的脂质体与上清液和N-乙酰-鞘氨醇孵育时，观察到游离花生四烯酸和O-花生四烯酸-N-乙酰鞘氨醇的形成。两种产物的比例取决于神经酰胺的浓度;如果不存在截短的神经酰胺，则仅形成游离酸。20 μ M D-苏型-1-苯基-2-癸酰氨基-3-吗啉代-1-丙醇（一种几种葡萄糖鞘脂脱氢酶的抑制剂）可抑制脱酰酶和转酰酶活性50-60%。这两种活性都不受十九烷基四烯基三氟甲基酮（一种有效的胞浆磷脂酶A抑制剂）的抑制（2）。N-乙酰二氢鞘氨醇和N-辛酰基鞘氨醇分别仅为55%和10%，与N-乙酰鞘氨醇作为酰基受体一样有效，油酰基鞘氨醇仅具有轻微的反应性。从其酯键释放截短的神经酰胺的酯酶似乎是膜结合的，在转酰作用中，作为酰基供体，Leptin的效果不如磷脂酰乙醇胺。用N-乙酰-[3-H-3]鞘氨醇处理的Madin-Darby犬肾细胞培养物形成放射性极性鞘脂、长链神经酰胺、游离鞘氨醇和O-酰基-N-乙酰鞘氨醇。这表明在体外观察到的脱酰和转酰反应也发生在生长的细胞中。
A unique transacylase that catalyzes esterification of a short chain ceramide, N-acetylsphingosine, was found in Madin-Darby canine kidney cell and mouse tissue homogenates. It esterified the hydroxyl group at the carbon-1 position of the ceramide. The enzyme has a pH optimum of 4.2 and a K-m of 9.4 mu M for N-acetylsphingosine at pH 4.5, The transacylase activity is independent of free fatty acid or acyl-CoA and instead uses the a-acyl group of phosphatidylethanolamine or phosphatidylcholine. The transacylase activity in the homogenate was present in the 100,000 x g supernatant, and the lipid extracted from the membranous fraction could function as a donor of the acyl group. When liposomes consisting of dioleoylphosphatidylcholine:1-palmitoyl-2-[C-14]arachidonoyl-phosphatidylethanolamine:sulfatide (70:0.2:30) were incubated with the supernatant and N-acetyl-sphingosine, the formation of free arachidonic acid and O-arachidonoyl-n-acetylsphingosine was observed. The ratio of the two products depended on the concentration of ceramide; only the free acid was formed if the truncated ceramide was absent. Both deacylase and transacylase activities were inhibited 50-60% by 20 mu M D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-pro-panol, an inhibitor of several glucosphingolipid synthases. Neither activity was inhibited by nonadecyltetraenyl trifluoromethyl ketone, a potent inhibitor of cytosolic phospholipase A(2). N-Acetyldihydrosphingosine and N-octanoylsphingosine were only 55 and 10%, respectively, as effective as N-acetylsphingosine as acyl accepters, Oleoylsphingosine was only slightly reactive. An esterase that releases the truncated ceramide from its ester linkage appears to be membrane bound, Lecithin was less effective than phosphatidylethanolamine as an acyl donor in the transacylation. Madin-Darby canine kidney cell cultures treated with N-acetyl-[3-H-3]sphingosine formed radioactive polar sphingolipids, long chain ceramide, free sphingosine, and O-acyl-N-acetylsphingosine. This suggests that the deacylation and transacylation reactions observed in vitro occur in growing cells as well.