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 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.
Highlights
Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis
Phenylmethylsulfonyl fluoride (PMSF), benzamidine, anti-chicken IgY conjugated with alkaline phosphatase, cholate, taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), glycocholate (GC), glycodeoxycholate (GDC), glycochenodeoxycholate (GCDC), Triton X 100 (TX100), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), dithothreitol (DTT), ceramide, and oxidized and reduced glutathione were from Sigma Co
We found that the purified SMase had no activity against PC in assay buffer for alkaline SMase, but it could hydrolyze PC in a buffer which is optimal for PC-specific phospholipase C
Summary
Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. The enzyme from rat has been purified 1,589fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Unlike neutral SMase, intestinal alkaline SMase was not Mg2؉ dependent, not inhibited by EDTA, and not inhibited by glutathione. Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase. Unlike the widely distributed acid and neutral SMases, alkaline SMase is distributed in the intestinal mucosa, with high activity localized at the middle small intestine and low activity in the colon [9, 10]. The rat intestinal alkaline SMase has been purified and several properties characterized. An antibody has been developed, and expression of the enzyme in various organs was studied
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