Advanced

Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase.

Cheng, Yajun LU ; Nilsson, Åke LU ; Tömquist, Elisabeth and Duan, Rui-Dong LU (2002) In Journal of Lipid Research 43(2). p.316-324
Abstract
Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the V(max) was 930 micromol/h/mg and K(m) was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more... (More)
Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the V(max) was 930 micromol/h/mg and K(m) was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more effective than glycine conjugated ones, and the most effective bile salts were taurocholate and taurochenodeoxycholate. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X100 (TX100) had no stimulatory effects. Unlike neutral SMase, intestinal alkaline SMase was not Mg2+ dependent, not inhibited by EDTA, and not inhibited by glutathione. The enzyme was stable during incubation with temperatures up to 50 degree C and in pHs from 7 to 10. Trypsin and chymotrypsin had no effects on its activity, and 10 mM dithiothreitol reduced its activity by 25%. A specific antibody against the enzyme was developed, and Western blot showed that the enzyme was expressed in the intestine but not in other organs. In conclusion, we purified a potentially important SMase in the intestine with several properties different from neutral SMase. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Animal, Bile Acids and Salts : pharmacology, Cattle, Enzyme Activation : drug effects, Hydrogen-Ion Concentration, Hydrolysis, Intestines : enzymology, Octoxynol : pharmacology, Phosphatidylcholines : metabolism, Rats, Rats Sprague-Dawley, Sphingomyelin Phosphodiesterase : isolation & purification : metabolism, Sphingomyelins : metabolism, Support Non-U.S. Gov't, Substrate Specificity
in
Journal of Lipid Research
volume
43
issue
2
pages
316 - 324
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000174102900016
  • pmid:11861674
  • scopus:0036191897
ISSN
1539-7262
language
English
LU publication?
yes
id
9198abea-c45a-4b98-a924-a632b868d434 (old id 106794)
alternative location
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11861674&dopt=Abstract
date added to LUP
2007-06-27 15:25:28
date last changed
2017-07-30 03:44:28
@article{9198abea-c45a-4b98-a924-a632b868d434,
  abstract     = {Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the V(max) was 930 micromol/h/mg and K(m) was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more effective than glycine conjugated ones, and the most effective bile salts were taurocholate and taurochenodeoxycholate. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X100 (TX100) had no stimulatory effects. Unlike neutral SMase, intestinal alkaline SMase was not Mg2+ dependent, not inhibited by EDTA, and not inhibited by glutathione. The enzyme was stable during incubation with temperatures up to 50 degree C and in pHs from 7 to 10. Trypsin and chymotrypsin had no effects on its activity, and 10 mM dithiothreitol reduced its activity by 25%. A specific antibody against the enzyme was developed, and Western blot showed that the enzyme was expressed in the intestine but not in other organs. In conclusion, we purified a potentially important SMase in the intestine with several properties different from neutral SMase.},
  author       = {Cheng, Yajun and Nilsson, Åke and Tömquist, Elisabeth and Duan, Rui-Dong},
  issn         = {1539-7262},
  keyword      = {Animal,Bile Acids and Salts : pharmacology,Cattle,Enzyme Activation : drug effects,Hydrogen-Ion Concentration,Hydrolysis,Intestines : enzymology,Octoxynol : pharmacology,Phosphatidylcholines : metabolism,Rats,Rats Sprague-Dawley,Sphingomyelin Phosphodiesterase : isolation & purification : metabolism,Sphingomyelins : metabolism,Support Non-U.S. Gov't,Substrate Specificity},
  language     = {eng},
  number       = {2},
  pages        = {316--324},
  publisher    = {American Society for Biochemistry and Molecular Biology},
  series       = {Journal of Lipid Research},
  title        = {Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase.},
  volume       = {43},
  year         = {2002},
}