Purification, localization, and expression of human intestinal alkaline sphingomyelinase

Sphingomyelin (SM) metabolism in the gut may have an impact on colon cancer development. In this study, we purified alkaline sphingomyelinase (alk-SMase) from human intestinal content, and studied its location in the mucosa, expression in colon cancer, and function on colon cancer cells. The enzyme was purified by a series of chromatographies. The molecular mass of the enzyme is 60 kDa, optimal pH is 8.5, and isoelectric point is 6.6. Under optimal conditions, 1 mg of the enzyme hydrolyzed 11 mM SM per hour. The properties of the enzyme are similar to those of rat intestinal alk-SMase but not to those of bacterial neutral SMase. Immunogold electronmicroscopy identified the enzyme on the microvillar membrane in endosome-like structures and in the Golgi complexes of human enterocytes. The expression and the activity of the enzyme were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. The enzyme inhibited DNA biosynthesis and cell proliferation dose dependently and caused a reduction of SM in HT29 cells.Intestinal alk-SMase is localized in the enterocytes, down-regulated in human colon cancer, and may have antiproliferative effects on colon cancer cells. Sphingomyelin (SM) metabolism in the gut may have an impact on colon cancer development. In this study, we purified alkaline sphingomyelinase (alk-SMase) from human intestinal content, and studied its location in the mucosa, expression in colon cancer, and function on colon cancer cells. The enzyme was purified by a series of chromatographies. The molecular mass of the enzyme is 60 kDa, optimal pH is 8.5, and isoelectric point is 6.6. Under optimal conditions, 1 mg of the enzyme hydrolyzed 11 mM SM per hour. The properties of the enzyme are similar to those of rat intestinal alk-SMase but not to those of bacterial neutral SMase. Immunogold electronmicroscopy identified the enzyme on the microvillar membrane in endosome-like structures and in the Golgi complexes of human enterocytes. The expression and the activity of the enzyme were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. The enzyme inhibited DNA biosynthesis and cell proliferation dose dependently and caused a reduction of SM in HT29 cells. Intestinal alk-SMase is localized in the enterocytes, down-regulated in human colon cancer, and may have antiproliferative effects on colon cancer cells. Metabolism of sphingomyelin (SM) generates the lipid messengers ceramide, sphingosine, and sphingosine-1-phosphate, which may have antiproliferative and apoptotic effects in many cell types (1Hannun Y.A. Linardic C.M. Sphingolipid breakdown products: anti-proliferative and tumor-suppressor lipids.Biochim. Biophys. Acta. 1993; 1154: 223-236Google Scholar, 2Merrill A.H. Schmelz E.M. Wang E. Dillehay D.L. Rice L.G. 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Supplementing SM and other derivatives of ceramide in the diet inhibited the formation of aberrant crypt foci, i.e., the earliest sign of tumor development, in mice treated with 1,2-dimethylhydrazine (6Dillehay D.L. Webb S.K. Schmelz E-M. Merrill A.H. Dietary sphingomyelin inhibits 1,2-dimethylhydrazine-induced colon cancer in CF1 mice.J. Nutr. 1994; 124: 615-620Google Scholar, 7Schmelz E.M. Dillehay D.L. Webb S.K. Reiter A. Adams J. Merrill A.H. Sphingomyelin consumption suppresses aberrant colonic crypt foci and increases the proportion of adenomas versus adenocarcinomas in CF1 mice treated with 1,2-dimethylhydrazine: implications for dietary sphingolipids and colon carcinogenesis.Cancer Res. 1996; 56: 4936-4941Google Scholar, 8Schmelz E.M. Bushnev A.S. Dillehay D.L. Liotta D.C. Merrill A.H. Suppression of aberrant colonic crypt foci by synthetic sphingomyelins with saturated or unsaturated sphingoid base backbones.Nutr. Cancer. 1997; 28: 81-85Google Scholar). In cultures of human colon cancer cells, ceramide and sphingosine were found to arrest the cell cycle at G2/M phase and caused accumulation of cells in the S phase (9Ahn E.H. Schroeder J.J. Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells.Exp. Biol. Med. 2002; 227: 345-353Google Scholar). It was recently reported that ceramide and sphingosine reduced the levels of cytosolic and nuclear β-catenin, indicating an effect on the APC/β-catenin system (10Schmelz E.M. Roberts P.C. Kustin E.M. Lemonnier L.A. Sullards M.C. Dillehay D.L. Merrill A.H. Modulation of intracellular beta-catenin localization and intestinal tumorigenesis in vivo and in vitro by sphingolipids.Cancer Res. 2001; 61: 6723-6729Google Scholar). These findings link the effects of sphingolipids in the colon to a major signaling pathway responsible for the initial tumor formation in colon. Three decades ago, Nilsson identified a sphingomyelinase (SMase) activity with optimum alkaline pH in the intestinal tract of rats, pigs, and humans (11Nilsson Å The presence of sphingomyelin- and ceramide-cleaving enzymes in the small intestinal tract.Biochim. Biophys. Acta. 1969; 176: 339-347Google Scholar). Differing from other types of SMase, such as acid and neutral SMase, which have been purified and cloned (12Quintern L.E. Schuchman E.H. Levran O. Suck M. Ferlinz K. Reinke H. Sandhoff K. Desnick R.J. Isolation of cDNA clones encoding human acid sphingomyelinase: occurrence of alternatively processed transcripts.EMBO J. 1989; 8: 2469-2473Google Scholar, 13Chatterjee S. Han H. Rollins S. Cleveland T. Molecular cloning, characterization, and expression of a novel human neutral sphingomyelinase.J. Biol. Chem. 1999; 274: 37407-37412Google Scholar), the alkaline SMase (alk-SMase) activity was only found in the intestinal tract of many species and additionally in human bile (14Duan R-D. Hertervig E. Nyberg L. Tauge T. Sternby B. Lillienau J. Farooqi A. Nilsson Å. Distribution of alkaline sphingomyelinase activity in human beings and animals.Dig. Dis. Sci. 1996; 41: 1801-1806Google Scholar, 15Nyberg L. Duan R-D. Axelsson J. Nilsson Å. Identification of an alkaline sphingomyelinase activity in human bile.Biochim. Biophys. Acta. 1996; 1300: 42-48Google Scholar, 16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar). The enzyme was present in human meconium, in the intestine of germ-free mice, and in sterile human bile, indicating that it did not originate from intestinal bacteria (11Nilsson Å The presence of sphingomyelin- and ceramide-cleaving enzymes in the small intestinal tract.Biochim. Biophys. Acta. 1969; 176: 339-347Google Scholar, 14Duan R-D. Hertervig E. Nyberg L. Tauge T. Sternby B. Lillienau J. Farooqi A. Nilsson Å. Distribution of alkaline sphingomyelinase activity in human beings and animals.Dig. Dis. Sci. 1996; 41: 1801-1806Google Scholar). We recently purified and characterized rat intestinal alk-SMase and showed that the enzyme was only expressed in the intestine and not in other organs in rats (16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar). Studies of the longitudinal distribution in the gut demonstrated the highest activity of the enzyme in the jejunum and ileum, where most of the digestion of dietary SM to ceramide occurs (17Nyberg L. Nilsson Å. Lundgren P. Duan R-D. Localization and capacity of sphingomyelin digestion in the rat intestinal tract.J. Nutr. Biochem. 1997; 8: 112-118Google Scholar). We previously found a significant reduction of the enzyme activity in human colorectal adenocarcinomas and in the mucosa of patients with familial adenomatous polyposis (FAP) (18Hertervig E. Nilsson Å. Nyberg L. Duan R-D. Alkaline sphingomyelinase activity is decreased in human colorectal carcinoma.Cancer. 1996; 79: 448-453Google Scholar, 19Hertervig E. Nilsson Å. Bjo¨rk J. Hultkrantz R. Duan R-D. Familial adenomatous polyposis is associated with a marked decrease in alkaline sphingomyelinase activity: a key factor to the unrestrained cell proliferation.Br. J. Cancer. 1999; 81: 232-236Google Scholar). In addition, in patients with longstanding extensive ulcerative colitis that is associated with an increased risk of colon cancer, alk-SMase activity was decreased (20Sjo¨qvist U. Hertervig E. Nilsson Å. Duan R-D. Ost A. Tribukait B. Lofberg R. Chronic colitis is associated with a reduction of mucosal alkaline sphingomyelinase activity.Inflamm. Bowel Dis. 2002; 8: 258-263Google Scholar). Furthermore, ursodeoxycholate, a bile salt known to have anticarcinogenic effects in colon, was found to increase the colonic alk-SMase activity (21Cheng Y. Tauschel H-T. Nilsson Å. Duan R-D. Administration of ursodeoxycholic acid increases the activities of alkaline sphingomyelinase and caspase-3 in rat colon.Scand. J. Gastroenterol. 1999; 34: 915-920Google Scholar). Thus, several pieces of evidence suggest that metabolites generated by alk-SMase may influence cellular growth and differentiation in the gut. In addition, SM metabolism in the gut may affect intestinal lipoprotein metabolism and sterol absorption. It has been demonstrated that hydrolysis of membrane SM in cultured intestinal cells influenced both sterol absorption (22Chen H. Born E. Mathur S.N. Johlin F.C. Field F.J. Sphingomyelin content of intestinal cell membranes regulates cholesterol absorption. Evidence for pancreatic and intestinal cell sphingomyelinase activity.Biochem. J. 1992; 286: 771-777Google Scholar, 23Field F.J. Born E. Murthy S. Mathur S.N. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of plasma membrane cholesterol to the endoplasmic reticulum.J. Lipid Res. 1998; 39: 333-343Google Scholar) and lipoprotein secretion (24Field F.J. Chen H. Born E. Dixon B. Mathur S. Release of ceramide after membrane sphingomyelin hydrolysis decreases the basolateral secretion of triacylglycerol and apolipoprotein B in cultured human intestinal cells.J. Clin. Invest. 1993; 92: 2609-2619Google Scholar), and that ceramide hydrolysis could affect the size and composition of lipoproteins secreted (25Kirby R.J. Zheng S. Tso P. Howles P.N. Hui D.Y. Bile salt-stimulated carboxyl ester lipase influences lipoprotein assembly and secretion in intestine: a process mediated via ceramide hydrolysis.J. Biol. Chem. 2002; 277: 4104-4109Google Scholar). In the gut lumen, the physical interaction between sterols and SM influences the course of both sterol absorption and SM hydrolysis (26Nyberg L. Duan R-D. Nilsson Å. A mutual inhibitory effect on absorption of sphingomyelin and cholesterol.J. Nutr. Biochem. 2000; 11: 244-249Google Scholar, 27Eckhardt E.R. Wang D.Q. Donovan J.M. Carey M.C. Dietary sphingomyelin suppresses intestinal cholesterol absorption by decreasing thermodynamic activity of cholesterol monomers.Gastroenterology. 2002; 122: 948-956Google Scholar) In the present study, we purified human intestinal alk-SMase and located the enzyme in the intestinal mucosa using a polyclonal antiserum. The expressions of the enzyme in normal and colonic cancer tissue were compared and the potential effects of the enzyme on proliferation and apoptosis of human colon cancer cells were studied. Human small intestinal contents were collected from ileostomy stomas of six individuals who had undergone a colectomy due to ulcerative colitis more than 3 months earlier. Before sample collection, the patients had been fasted overnight. Samples from human colonic tumors and surrounding normal tissues were obtained from patients undergoing resection at the Department of Surgery, Malmo¨ University Hospital. Human colonic biopsy samples were taken during colonoscopic examinations at the Gastroenterology Division, Lund University Hospital. All studies involving humans had been approved by the Human Ethics Committee, Lund University. Bovine milk SM was provided by Dr. Lena Nyberg at Skane Dairy Co. (Malmo¨, Sweden) and labeled with [N-14C-CH3]choline by the methods of Stoffel (28Stoffel W. Chemical synthesis of choline-labeled lacithins and sphingomyelin.Methods Enzymol. 1975; 36: 533-541Google Scholar). The specific activity of [14C]SM was 56 μCi/mg. DEAE Sepharose, Sephadex G25 (PD10) column, and Phenyl Sepharose 6 FF were obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Uno Q anion exchange chromatography cartridge, prepacked SE gel chromatography column, Biologic HR protein purification system, and isoelectrofocusing instrument Rotofor were obtained from Bio-Rad (Sundberg, Sweden). YM 30 filtration membranes with 30 kDa molecular mass cut-off were purchased from Amicon (Beverly, MA). HT-29 cells were obtained from American Tissue Culture Collection (Rockville, MD). The cell death detection kit and the cell proliferation reagent 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazoliol]1,3-benzene disulfonate (WST-1) were purchased from Roche Diagnostics GmbH (Mannheim, Germany). [3H]thymidine and [3H]choline chloride were bought from DuPont Corporation (Cambridge, MA). The human intestinal contents were freeze dried and dissolved in ice-cold 0.15 M NaCl containing 1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride (PMSF). The nonsoluble materials were removed by centrifugation at 8,000 rpm for 15 min at 4°C. The proteins were precipitated by acetone as previously described (16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar) and dissolved in 20 mM Tris-HCl buffer, pH 8.2, containing 0.075 M NaCl, 1 mM benzamidine, and 1 mM PMSF (DEAE buffer). The sample was subjected to a series of chromatographies by a means very similar to that used for purification of rat alk-SMase (16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar). Briefly, the sample was first loaded on a DEAE Sepharose column that had been equilibrated with DEAE buffer. After loading, the column was eluted with the same buffer and the fractions containing nonretained proteins were collected. The column was then eluted with the same buffer but contained 0.5 M NaCl to obtain the retained proteins. The intestinal alk-SMase activity was in the fractions containing nonretained proteins. These fractions were pooled, supplemented with ammonium sulfate to 1.0 M, and applied to a column packed with Phenyl Sepharose for hydrophobic interaction chromatography (HIC). The column was eluted with a gradient of ammonium sulfate from 1.0 to 0 M in 20 mM Tris-Maleate buffer (pH 7.0). After HIC, the fractions containing alk-SMase activity were pooled and desalted, followed by loading on a Uno Q column for high-affinity anion exchange chromatography. The column was eluted with a gradient of NaCl from 0 to 0.25 M in 20 mM Tris buffer (pH 7.0). The fractions with high enzyme activity were pooled, concentrated by ultra filtration, and loaded on an SE column, followed by elution with 20 mM Tris, 0.15 M NaCl (pH 8.2). The fractions with high alk-SMase activity were subjected to native isoelectric focusing electrophoresis with pH ranging from 3 to 10. The protein concentrations during chromatographies were monitored by a UV detector or quantified by a kit from Bio Rad with bovine albumin as a standard. The purity of the enzyme was visualized by 10% SDS polyacrylamide gel electrophoresis (PAGE) and the gel was stained by silver staining. Alk-SMase activity was determined by two methods according to Duan and Nilsson (29Duan R.D. Nilsson Å. Enzymes hydrolysing sphingolipids in gastrointestinal tract.Methods Enzymol. 1999; 311: 276-286Google Scholar). For monitoring the migration of the activity during the purification procedure, 2 μl of samples from fractions collected were added to the tubes followed by adding 50 mM Tris-HCl buffer (pH 9.0) containing 0.15 M NaCl, 2 mM EDTA, 10 mM taurocholate (TC), 0.1 mM SM, and 0.80 μM [14C]SM (∼8,000 dpm) to a final volume of 100 μl. After incubation at 37°C for 30 min, the reaction was terminated by adding 0.4 ml of chloroform-methanol (2:1, v/v) followed by centrifugation at 10,000 rpm for 10 s. An aliquot (100 μl) of the upper phase containing the cleaved phosphocholine was analyzed for radioactivity by liquid scintillation. The activity was expressed as dpm in the upper phase after 30 min incubation. For assaying the enzyme activity in HT 29 cells, tissue samples, and in characterization studies, 5 μl of samples were added in 50 mM Tri-HCl buffer containing 0.15 M NaCl, 2 mM EDTA, and 10 mM TC (pH 9.0) (assay buffer) to a final volume of 80 μl. The reaction was started by adding 80 pmol [14C]SM (∼8,000 dpm) in a 20 μl assay buffer and incubating at 37°C for 30 min. The reaction was terminated as above, and the activity was calculated and expressed as nmol substrate hydrolyzed by 1 mg sample protein in 1 h (nmol/h/mg). The hydrolytic capacity of the alk-SMase was determined by adding 5 ng enzyme in 100 μl assay buffer containing different amounts of SM ranging from 5 μg to 640 μg together with 100 pmol of [14C]SM. Percentage of hydrolysis of the substrate was calculated from the ratio of dpm in the upper phase to the total dpm added in the system. The mass of SM that had been hydrolyzed in 1 h was calculated from the percentage hydrolysis. The Lineweaver-Burk plot was used to determine the Vmax of the enzymatic reaction under the assay condition. The purified alk-SMase was subjected to 10% SDS-PAGE and stained by 0.1% Coomassie blue. The enzyme band was excised and homogenized and the homogenates were used to immunize two rabbits once a month for 3 months by subcutaneous injections. Bleeding started 2 weeks after the second injection. The immune procedure was performed by the company Agri Sera AB (Va¨nna¨s, Sweden). Human normal ileum biopsy samples were fixed in 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4°C. After a rinse with 0.1 M phosphate buffer (pH 7.2), the small intestinal samples were infused with 2.3 M sucrose for 30 min and then mounted on top of a metal pin and frozen in liquid nitrogen. Ultracryosections (∼50 nm) were cut in an RMC 6,000 XL ultracryomicrotome and collected with a sucrose droplet and attached to formvar and carbon-coated nickel grid. Immunogold labeling was performed according to Hansen et al. (30Hansen G.H. Immerdal L. Thorsen E. Niels-Christiansen L-L. Nystrom B.T. Demant E.J.F. Danielsen E.M. Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes.J. Biol. Chem. 2001; 276: 32338-32344Google Scholar) using either the rabbit anti-human SMase serum or the preimmune serum as a control. The ultracryosections were finally examined in a Zeiss EM900 electron microscope and electronmicrographs were obtained using a Mega View II CCD camera and an Image analysis system. The tissue samples were homogenized in 50 mM Tris buffer containing 2 mM EDTA, 10 mM TC, 1 mM PMSF, 1 mM benzamidine, and 0.5 mM DTT (pH 7.4), followed by sonication for 10 s. After centrifugation at 13,000 rpm for 10 min at 4°C, 50 μg proteins of each sample were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane by electrophoresis. The membrane was blocked in 20 mM Tris buffer containing 0.15 M NaCl and 5% fat free dried milk for 2 h and probed with antiserum (1:500) for 2 h. After washing, the membrane was incubated with biotinylated goat anti-rabbit antibody for 2 h and then with a complex of equal amounts of streptavidin and biotinylated-alkaline phosphatase for 3 h. After color development, the density of the protein bands was quantified by Scion Image software program. HT29 cells were cultured in RPMI-1640 medium with l-glutamine containing 100 IU/ml penicillin, 10 μg/ml streptomycin, and 10% (v/v) heat inactivated fetal calf serum. The purified alk-SMase was diluted to a concentration with an activity being 200 nmol/h/ml. HT29 cells seeded in 96 well plate were incubated with the alk-SMase in different doses for 24 h. WST-1reagent was added in each well and cell proliferation was determined as previously described (31Liu J-J. Nilsson Å. Oredsson S. Badmaev V. Zhao W-Z. Duan R-D. Boswellic acids trigger apoptosis via a pathway dependent on caspase-8 activation but independent of Fas/Fas ligand interaction in colon cancer HT29 cells.Carcinogenesis. 2002; 23: 2087-2093Google Scholar). For assaying DNA replication, the cells were treated with alk-SMase for 24 h. [3H]thymidine at a dose of 0.5 μCi/well was added and the incorporation of [3H]thymidine in DNA was determined as described (31Liu J-J. Nilsson Å. Oredsson S. Badmaev V. Zhao W-Z. Duan R-D. Boswellic acids trigger apoptosis via a pathway dependent on caspase-8 activation but independent of Fas/Fas ligand interaction in colon cancer HT29 cells.Carcinogenesis. 2002; 23: 2087-2093Google Scholar). Apoptosis was determined by ELISA using a cell death detection ELISA kit, which is based on the quantification of mono- and oligonucleosomes in the cytoplasm. The cells were treated with alk-SMase for 24 h and lysed by the lysis buffer provided with the kit. The cytoplasmic histone-associated DNA fragments were quantified, and the specific enrichment was determined as described previously (31Liu J-J. Nilsson Å. Oredsson S. Badmaev V. Zhao W-Z. Duan R-D. Boswellic acids trigger apoptosis via a pathway dependent on caspase-8 activation but independent of Fas/Fas ligand interaction in colon cancer HT29 cells.Carcinogenesis. 2002; 23: 2087-2093Google Scholar). Determination of cellular SM levels was performed according to Hedlund et al. (32Hedlund M. Svensson M. Nilsson Å. Duan R-D. Svanborg C. Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli.J. Exp. Med. 1996; 183: 1037-1044Google Scholar). In brief, the cells were preincubated with [3H]choline for 48 h at a concentration of 0.5 μCi/ml and then treated with alk-SMase as described above. The total lipids were extracted according to Bligh and Dyer (33Bligh E.H. Dyer W.J. A rapid method for total lipid extraction and purification.Can J. Biochem. Physiol. 1959; 37: 911-918Google Scholar) and applied on Silica gel plate (60 F, 0.25 mm) for thin layer chromatography. The plates were developed by chloroform-methanol-25% ammonium hydroxide (65:25:4, v/v/v) and the lipid bands were visualized by iodine vapor. The SM bands were scraped according to the internal standard and the radioactivity in bands was measured by liquid scintillation counting. In the initial chromatographic separation on DEAE-Sepharose column, two portions with alk-SMase activity were identified (Fig. 1). The activity in the first portion (Portion A) was not retained on the column and eluted with the loading buffer (20 mM Tris buffer pH 8.2 with 0.075 M NaCl). A second and smaller peak of alk-SMase (Portion B) was eluted by increasing NaCl concentration to 0.5 M in the same buffer. According to our previous studies, portion A contains intestinal alk-SMase, whereas portion B is likely represent bile alk-SMase (16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar, 34Duan R-D. Nilsson Å. Purification of a newly identified alkaline sphingomyelinase in human bile and effects of bile salts and phosphatidylcholine on enzyme activity.Hepatology. 1997; 26: 823-830Google Scholar). Portion A was saved and subjected to Phenyl Sepharose HIC (Fig. 2A), Uno Q high affinity anion chromatography (Fig. 2B), and SE chromatography (Fig. 2C). The fractions containing alk-SMase were marked by arrows in each panel. The alk-SMase activity in SE gel chromatography was found in fraction 28 to 33 with peak activity in fraction 30 and 31. The proteins in these fractions were visualized by 10% SDS-PAGE (Fig. 3A). A band at about 60 kDa (marked by arrow) was identified as correlated with the enzyme activity. To further confirm that this 60 kDa protein is alk-SMase, fractions 30 and 31 were combined and subjected to native electric focusing electrophoresis. A total of 20 fractions were collected. The highest alk-SMase activity was found in the fraction with pH 6.6. SDS-PAGE confirmed that the fraction contained a single protein with a mass of 60 kDa (Fig. 3B), which was identical to those shown in Fig. 3A.Fig. 2Profiles of chromatographies. The portion A from DEAE chromatography was subjected to Phenyl Sepharose hydrophobic interaction chromatography (HIC) (A), Uno Q anion exchange chromatography (B), and SE gel chromatography (C). Ammonium sulfate gradient in HIC was from 1.0 to 0 M, and NaCl gradient in anion exchange chromatography was from 0 to 0.25 M. Protein concentrations were monitored by UV detector. The alk-SMase activities were determined and the fractions with high activity are indicated by the arrows.View Large Image Figure ViewerDownload (PPT)Fig. 3SDS-PAGE. A: Twenty microliters from each fraction after SE chromatography was subjected to 10% SDS-PAGE and the gel was stained by silver staining. The fraction numbers are shown above the panel and SMase activities below it. The bands indicated by an arrow are the ones correlated with SMase activity. B: Fraction 30 and 31 from SE chromatography were combined and subjected to electrofocusing. The fractions with alk-SMase activity after electrofocusing were resolved by 10% SDS-PAGE and the gel was stained by silver staining. The pH values of each fraction are shown at the top of the panel and SMase activity at the bottom. The bands indicated by an arrow are purified alk-SMase.View Large Image Figure ViewerDownload (PPT) The optimal pH, divalent ion dependency, bile salt dependency, glutathione inhibition, and substrate specificity of alk-SMase were examined according to methods described previously (16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar). The pH dependence was similar to that of rat intestinal alk-SMase. Maximal activity was obtained at pH 8.5 and the activity at pH 7.5 was about 68% of the maximal activity. Like the rat enzyme, human alk-SMase did not require Ca2+ and Mg2+ and was not inhibited by EDTA. In addition, the activity at alkaline pH without divalent cations remained significantly higher than that at pH 7.5 with Mg2+ present. In analogy with rat alk-SMase, the enzyme activity was specifically stimulated by TC and taurochenodeoxycholate, but inhibited by other detergents such as CHAPS and Triton X-100. Glutathione, which inhibits neutral SMase, had no significant effects on human alk-SMase. Although the major product generated by the enzyme was ceramide, the enzyme had weak activity against phosphatidylcholine at neutral pH in the presence of Ca2+. Because these properties described above are very similar to those for rat intestinal alk-SMase reported previously (16Cheng Y. Nilsson Å. To¨mquist E. Duan R-D. Purification, characterization and expression of rat intestinal alkaline sphingomyelinase.J. Lipid Res. 2002; 43: 316-324Google Scholar), detailed data are not presented, but the properties for rat and human alk-SMase are summarized in Table 1.TABLE 1Comparison of properties of intestinal alkaline SMases in rat and humanRatHumanMolecular mass (kDa) 58 60Optimal pH 9.0 8.5Isoelectric point 6.2 6.6Vmax (mmol/h/mg) 0.93 11.0Stimulation by bile saltTC, TCDCTC, TCDCGlutathione inhibition no noMg2+ dependence no noEffect of EDTA no noCross immunoreaction yes yesActivity against PC yes yesPC, phosphatidylcholine; SMase, sphingomyelinase; TC, taurocholate; TCDC, taurochenodeoxycholate. The comparison was made based on the results in this paper and those in our previous investigation (16). The pI value in rat alkaline SMase is from our unpublished data. Open table in a new tab PC, phosphatidylcholine; SMase, sphingomyelinase; TC, taurocholate; TCDC, taurochenodeoxycholate. The comparison was made based on the results in this paper and those in our previous investigation (16). The pI value in rat alkaline SMase is from our unpublished data. To determine the hydrolytic capacity of the enzyme, the activity of alk-SMase (5 ng) was assayed in the presence of different amounts of SM in 100 μl assay buffer (Fig. 4A). The Vmax was determined by a Lineweaver-Burk plot (Fig. 4B). One milligram of the enzyme is able to hydrolyze about 11 mmol SM in 1 h. By immunogold labeling, alk-SMase was foun