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Journal of Lipid Research, Vol. 43, 316-324, February 2002
Copyright © 2002 by Lipid Research, Inc.

Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase

Correspondence to: Rui-Dong Duan, To whom correspondence should be addressed., rui-dong.duan{at}med.lu.se (E-mail)

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.

Supplementary key words: sphingomyelin, bile salt, digestion, glutathione, Triton X100

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