TY - JOUR
T1 - Purification and characterization of a second type of neutral ceramidase from rat brain
T2 - A second more hydrophobic form of rat brain ceramidase
AU - Thayyullathil, Faisal
AU - Chathoth, Shahanas
AU - Hago, Abdulkader
AU - Patel, Mahendra
AU - Szulc, Zdzislaw M.
AU - Hannun, Yusuf
AU - Galadari, Sehamuddin
N1 - Funding Information:
We wish to thank the lipidomics core facility at the Medical University of South Carolina, USA, for their kind provision of the substrate and other rare sphingolipids. This work was financially supported by grant from The Emirates Foundation (13 - 2008/075), and in parts, from The Terry Fox Foundation for Cancer Research , and The Terry Fox Foundation for medical Research.
PY - 2011/4
Y1 - 2011/4
N2 - 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 ~ 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 I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca2+ and is inhibited by pyrimidine mono nucleotides such as TMP 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.
AB - 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 ~ 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 I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca2+ and is inhibited by pyrimidine mono nucleotides such as TMP 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.
KW - Ceramidase
KW - Ceramide
KW - Chromatography
KW - Glycosidase F
KW - Sphingosine
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U2 - 10.1016/j.bbalip.2010.12.012
DO - 10.1016/j.bbalip.2010.12.012
M3 - Article
C2 - 21224012
AN - SCOPUS:79551617173
SN - 1388-1981
VL - 1811
SP - 242
EP - 252
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
IS - 4
ER -