Rat intestinal ceramidase: purification, properties, and physiological relevance.
Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.
Olsson
Maria
M
Dept. of Medicine, Gastroenterology and Nutrition Laboratory, BMC B11, Lund Univ. Hospital, S-22184 Lunds Universitet, Sweden.
Duan
Rui-Dong
RD
Ohlsson
Lena
L
Nilsson
Ake
A
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology; D002210:Caprylates / Q000378:metabolism; D002250:Carbon Radioisotopes; D002412:Cations / Q000494:pharmacology; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism*; D002918:Chymotrypsin / Q000494:pharmacology; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D051381:Rats; D013110:Sphingosine / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D001647:Bile Acids and Salts; D002210:Caprylates; D002250:Carbon Radioisotopes; D002412:Cations; D002518:Ceramides; D005229:Fatty Acids, Monounsaturated; C008757:palmitoleic acid; D002918:Chymotrypsin; D014357:Trypsin; D000581:Amidohydrolases; C526748:Asah2 protein, rat; D055572:Ceramidases; D055576:Neutral Ceramidase; D013110:Sphingosine