Purification and characterization of a second type of neutral ceramidase from rat brain: A second more hydrophobic form of rat brain ceramidase
Purification and characterization of a second type of neutral ceramidase from rat brain: A second more hydrophobic form of rat brain ceramidase
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DOI：
10.1016/j.bbalip.2010.12.012
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发表时间：
2011-04-01
影响因子：
4.8
通讯作者：
Galadari, Sehamuddin
中科院分区：
文献类型：
--
作者：
Thayyullathil, Faisal;Chathoth, Shahanas;Galadari, Sehamuddin
神经酰胺酶(Ceramidase，CDase)是一种催化神经酰胺N-酰基连接(Cer)水解生成鞘氨醇和游离脂肪酸的酶。在这项研究中，我们报道了一种新的大鼠脑第二类中性神经酰胺酶(RBCDase II)的纯化和性质。大鼠脑膜Triton X-100蛋白提取液依次经Q-Sepharose柱、HiLoad16/60Superdex 200pg柱、肝素琼脂糖柱、苯基琼脂糖HP柱、Mono Q柱纯化。经MonoQ处理后，该酶的比活力比大鼠脑匀浆增加了近15.000倍。该酶的最适pH为7.5，表观分子量(110 KDa)大于纯化前的90 kDa，具有中性大鼠脑CDase(RBCDase I)的特性。脱糖实验表明，RBCDase LAND II在SDS-PAGE上的分子质量差异并不是N-葡聚糖异质性的线索。RBCDase II部分被钙离子激活，但被嘧啶核苷酸如IMP和UMP抑制。这一发现意义重大，因为它首次证明了核苷酸对CDase活性的影响。氧化型和还原型GSH对该酶均有抑制作用。考察了金属离子的影响，发现该酶对Hg2+和Fe3-非常敏感，而不受Mn2+的影响。EDTA在20 mM浓度时有一定的抑制作用。(C)2011年，由爱思唯尔出版。
Ceramidases (CDase) are enzymes that catalyze the hydrolysis of N-acyl linkage of ceramide (Cer) to generate sphingosine and free fatty acids. In this study we report the purification and characterization of a novel second type of neutral ceramidase from rat brain (RBCDase II). Triton X-100 protein extract from rat brain membrane was purified sequentially using Q-Sepharose, HiLoad16/60 Superdex 200 pg, heparin-Sepharose, phenyl-Sepharose HP, and Mono Q columns. After Mono Q the specific activity of the enzyme increased by similar to 15.000-fold over that of the rat brain homogenate. This enzyme has pH optima of 7.5, and it has a larger apparent molecular weight (110 kDa) than the previously purified (90 kDa) and characterized neutral rat brain CDase (RBCDase I). De-glycosylation experiments show that the differences in molecular mass of RBCDase land II on SDS-PAGE are not clue to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca2+ and is inhibited by pyrimidine mono nucleotides such as IMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on a CDase activity. The enzyme was also inhibited by both oxidized and reduced GSH. The effects of metal ions were examined, and we found that the enzyme is very sensitive to Hg2+ and Fe3-, while it is not affected by Mn2+. EDTA was somewhat inhibitory at a 20 mM concentration. (C) 2011 Published by Elsevier B.V.