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标题：Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase
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作者：Yajun Cheng ; Åke Nilsson ; Elisabeth Tömquist 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2002
卷号：43
期号：2
页码：316-324
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：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 μmol/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°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. J. Lipid Res. 2002. 43: 316–324.
关键词：sphingomyelin ; bile salt ; digestion ; glutathione ; Triton X100