Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase
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, precipi...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2002-02-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520301747 |
| _version_ | 1829513856188153856 |
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| author | Yajun Cheng Åke Nilsson Elisabeth Tömquist Rui-Dong Duan |
| author_facet | Yajun Cheng Åke Nilsson Elisabeth Tömquist Rui-Dong Duan |
| author_sort | Yajun Cheng |
| collection | DOAJ |
| description | 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 Vmax was 930 μmol/h/mg and Km 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°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.—Cheng, Y., Å. Nilsson, E. Tömquist, and R-D. Duan. Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase. |
| first_indexed | 2024-12-16T13:08:03Z |
| format | Article |
| id | doaj.art-8825ba49129948d08843cd1dbb1fd3f9 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-16T13:08:03Z |
| publishDate | 2002-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-8825ba49129948d08843cd1dbb1fd3f92022-12-21T22:30:41ZengElsevierJournal of Lipid Research0022-22752002-02-01432316324Purification, characterization, and expression of rat intestinal alkaline sphingomyelinaseYajun Cheng0Åke Nilsson1Elisabeth Tömquist2Rui-Dong Duan3Cell Biology B, Biomedical Center, B11, Lund University, S-22184 Lund, SwedenCell Biology B, Biomedical Center, B11, Lund University, S-22184 Lund, SwedenCell Biology B, Biomedical Center, B11, Lund University, S-22184 Lund, SwedenTo whom correspondence should be addressed.; Cell Biology B, Biomedical Center, B11, Lund University, S-22184 Lund, SwedenIntestinal 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 Vmax was 930 μmol/h/mg and Km 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°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.—Cheng, Y., Å. Nilsson, E. Tömquist, and R-D. Duan. Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase.http://www.sciencedirect.com/science/article/pii/S0022227520301747sphingomyelinbile saltdigestionglutathioneTriton X100 |
| spellingShingle | Yajun Cheng Åke Nilsson Elisabeth Tömquist Rui-Dong Duan Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase Journal of Lipid Research sphingomyelin bile salt digestion glutathione Triton X100 |
| title | Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase |
| title_full | Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase |
| title_fullStr | Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase |
| title_full_unstemmed | Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase |
| title_short | Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase |
| title_sort | purification characterization and expression of rat intestinal alkaline sphingomyelinase |
| topic | sphingomyelin bile salt digestion glutathione Triton X100 |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520301747 |
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