title: Purification and characterization of a second type of neutral ceramidase from rat brain: a second more hydrophobic form of rat brain ceramidase.
abstract: 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 200pg, 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 (110kDa) than the previously purified (90kDa) 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 Ca(2+) 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 Hg(2+) and Fe(3+), while it is not affected by Mn(2+). EDTA was somewhat inhibitory at a 20mM concentration.
authors.affiliation: Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Science, UEA University, PO Box 17666, Al- Ain, UAE.
authors.forename: Faisal
authors.initials: F
authors.lastname: Thayyullathil
authors.forename: Shahanas
authors.initials: S
authors.lastname: Chathoth
authors.forename: Abdulkader
authors.initials: A
authors.lastname: Hago
authors.forename: Mahendra
authors.initials: M
authors.lastname: Patel
authors.forename: Zdzislaw M
authors.initials: ZM
authors.lastname: Szulc
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Hannun
authors.forename: Sehamuddin
authors.initials: S
authors.lastname: Galadari
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes; D055576:Neutral Ceramidase / Q000302:isolation & purification* / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats; D019163:Reducing Agents / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D013107:Sphingolipids / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D002412:Cations; D007527:Isoenzymes; D010743:Phospholipids; D019163:Reducing Agents; D013107:Sphingolipids; C037405:heparin-sepharose; C020927:phenyl-sepharose; D012685:Sepharose; D006026:Glycoside Hydrolases; D055576:Neutral Ceramidase