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, 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.
Cheng
Yajun
Y
Cell Biology B, Biomedical Center, B11, Lund University, S-22184 Lund, Sweden.
Nilsson
Ake
A
Tömquist
Elisabeth
E
Duan
Rui-Dong
RD
D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007422:Intestines / Q000201:enzymology*; D017830:Octoxynol / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism*; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D017830:Octoxynol; D013108:Sphingomyelin Phosphodiesterase