Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain
Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain
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DOI：
10.1074/jbc.274.39.27948
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发表时间：
1999-09-24
影响因子：
4.8
通讯作者：
Hannun, YA
中科院分区：
文献类型：
--
作者：
El Bawab, S;Bielawska, A;Hannun, YA
我们已经将膜结合神经酰胺酶纯化22，300倍至表观均一性。纯化方案包括Triton X-100膜提取，然后进行Q-琼脂糖凝胶、蓝色琼脂糖凝胶、苯基-琼脂糖凝胶和MonoS柱色谱。纯化的酶显示出的表观分子量为90 kDa的SDS-聚丙烯酰胺凝胶电泳在还原条件下和95 kDa的Superose 12上的色谱法估计。使用C-16-神经酰胺作为底物，该酶表现出广泛的pH值在中性至碱性范围内的最佳。开发了混合胶束测定，并使用Triton X-100/神经酰胺混合胶束，该酶表现出经典的Michaelis-Menten动力学，K-m为1.29 mol %，V-max为4.4 μ mol/min/mg。当二氢神经酰胺被用作底物时，这些值分别为3.84摩尔%和1.2 μ mol/min/mg，表明该酶优先水解神经酰胺。纯化的神经酰胺酶的活性不需要阳离子，它被还原剂抑制。磷脂酰胆碱和鞘磷脂对酶活性没有影响，而磷脂酸和磷脂酰丝氨酸刺激活性的3倍。鞘氨醇作为竞争性抑制剂，IC_（50）为5-10 μ M。这些结果表明，纯化的酶是一种新的神经酰胺酶。
We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C-16-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K-m of 1.29 mol % and a V-max of 4.4 mu mol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 mu mol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC50 of 5-10 mu M. These results indicate that the purified enzyme is a novel ceramidase.