Ito M, Kurita T, Kita K
Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1995 Oct 13;270(41):24370-4. doi: 10.1074/jbc.270.41.24370.
We describe a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids. The enzyme was purified about 3000-fold with 5% recovery from the culture filtrate of a newly isolated bacterium (Pseudomonas sp. TK4) by ammonium sulfate precipitation followed by several steps of high performance liquid chromatography. The purified enzyme preparation was completely free of exoglycosidases, sphingomyelinase, and proteases, and showed a single protein band corresponding to a molecular mass of 52 kDa on SDS-polyacrylamide slab gel electrophoresis after staining with Coomassie Brilliant Blue. The enzyme shows quite wide specificity, i.e. it hydrolyzes both neutral and acidic glycosphingolipids, and simple glycosphingolipid cerebrosides to polysialogangliosides such as GQ1b. Furthermore the enzyme also hydrolyzes sphingomyelin to produce the respective lyso form. However, the enzyme shows hardly any activity on ceramides, indicating that it is completely different from the ceramidase (EC 3.5.1.23) reported previously. This enzyme, which is tentatively named sphingolipid ceramide N-deacylase, should greatly facilitate the further study of sphingolipids as well as lysosphingolipids.
我们描述了一种新型酶，它能水解各种鞘脂的神经酰胺中脂肪酸与鞘氨醇碱基之间的N-酰基键。通过硫酸铵沉淀，随后经过几步高效液相色谱法，从新分离的细菌（假单胞菌属TK4）的培养滤液中纯化得到该酶，纯化倍数约为3000倍，回收率为5%。纯化后的酶制剂完全不含外切糖苷酶、鞘磷脂酶和蛋白酶，用考马斯亮蓝染色后，在SDS-聚丙烯酰胺平板凝胶电泳上显示出一条对应分子量为52 kDa的单一蛋白带。该酶具有相当广泛的特异性，即它能水解中性和酸性糖鞘脂，以及从简单的糖鞘脂脑苷脂到多唾液酸神经节苷脂如GQ1b。此外，该酶还能水解鞘磷脂产生相应的溶血形式。然而，该酶对神经酰胺几乎没有任何活性，这表明它与先前报道的神经酰胺酶（EC 3.5.1.23）完全不同。这种酶暂命名为鞘脂神经酰胺N-脱酰基酶，它将极大地促进对鞘脂以及溶血鞘脂的进一步研究。
