J Biol Chem, Vol. 273, Issue 51, 34472-34479, December 18, 1998

Purification and Characterization of a Membrane Bound Neutral pH Optimum Magnesium-dependent and Phosphatidylserine-stimulated Sphingomyelinase from Rat Brain^*

Bin Liu^§, Daniel F. Hassler^¶, Gary K. Smith^¶, Kurt Weaver, and Yusuf A. Hannun^**

From the  Departments of Medicine and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710 and the ^¶ Department of Molecular Biochemistry and  Molecular Sciences, GlaxoWellcome, Research Triangle Park, North Carolina 27709

	ABSTRACT

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Sphingomyelin hydrolysis and ceramide generation catalyzed by sphingomyelinases (SMase) are key components of the signaling pathways in cytokine- and stress-induced cellular responses. In this study, we report the partial purification and characterization of the membrane bound, neutral pH optimal, and magnesium-dependent SMase (N-SMase) from rat brain. Proteins from Triton X-100 extract of brain membrane were purified sequentially with DEAE-Sephacel, heparin-Sepharose, ceramic hydroxyapatite, Mono Q, phenyl-Superose, and Superose 12 column chromatography. After eight purification steps, the specific activity of the enzyme increased by 3030-fold over the brain homogenate. The enzyme hydrolyzed sphingomyelin but not phosphatidylcholine and its activity was dependent upon magnesium with an optimal pH of 7.5 and a native pI of 5.2. Delipidation of the enzyme through chromatographic purification or by extraction with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid followed by gel filtration revealed that the enzyme became increasingly dependent on phosphatidylserine (PS). Up to 20-fold stimulation was observed with PS whereas other lipids examined were either ineffective or only mildly stimulatory. The K_m of the enzyme for substrate sphingomyelin (3.4 mol %) was not affected by PS. The highly purified enzyme was inhibited by glutathione with a >95% inhibition observed with 3 mM glutathione and with a Hill number calculated at approximately 8. The significance of these results to the regulation of N-SMase is discussed.

	INTRODUCTION

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Sphingomyelinase (SMase)¹ catalyzes the hydrolysis of membrane sphingomyelin to generate ceramide and phosphorylcholine. Multiple forms of SMases have been described and they include the lysosomal acidic SMase (A-SMase), the cytosolic Zn²⁺-dependent acidic SMase, the membrane neutral magenesium-dependent SMase (N-SMase), the cytosolic magnesium-independent N-SMase, and the alkaline SMase (1). Activation of SMases, especially the A-SMase and N-SMase, in cells in response to growth factors, cytokines, chemotherapeutic agents, irradiation, nutrient removal, and stress conditions are believed to be involved in regulation of cell growth, differentiation, cell cycle arrest, and programmed cell death (1-4).

Evidence has started to suggest that various SMases may participate in defined yet different cellular functions. Studies have shown that A-SMase activation is part of the signaling pathway for radiation-induced apoptosis and CD28-mediated T-cell maturation (5), although the involvement of A-SMase in apoptosis and/or differentiation induced by both tumor necrosis factor- (TNF) and Fas (6-9) is controversial. The purification and cloning of the A-SMase (10) has led to the attribution of the sphingolipid storage disorder, Niemann-Pick disease, to specific mutations in A-SMase (11), the identification of several A-SMase activator proteins (12), the generation of gene knockout animal models (13), and the confirmation of genetic origin of the Zn²⁺-dependent acidic SMase (14). In the past few years, increased attention has been focused on the neutral magnesium-dependent SMase (N-SMase) for its role in mediating a variety of cellular processes including differentiation, cell cycle arrest, and programmed cell death (apoptosis) through the generation of ceramide (1, 2). Although the existence of N-SMase has been known for three decades (15-18), lack of information on its biochemical characteristics and especially cellular regulation has significantly handicapped efforts to delineate pathways involving this enzyme. Therefore, it is evident that a great need exists for its purification and biochemical characterization. In this study, we report a significant purification of N-SMase from rat brain membranes, biochemical characteristics of the purified enzyme, its activation by phosphatidylserine, and inhibition by glutathione (GSH).

	EXPERIMENTAL PROCEDURES

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Materials

Frozen stripped rat brains were purchased from Pel Freez Biologicals (Rogers, AK) and stored at 80 °C. All chromatographic media were obtained from Pharmacia (Piscataway, NJ) unless indicated otherwise. Ceramic hydroxyapatite was from American International Chemical (Natick, MA). Triton X-100 was purchased from Boehringer-Mannheim. N-[methyl-¹⁴C]Sphingomyelin was synthesized as previously described (19). Bovine brain sphingomyelin was from Avanti Polar Lipids (Alabaster, AL). Bovine brain L--phosphatidyl-L-serine was from Sigma. Other lipids were from Sigma or Avanti Polar Lipids.

Methods

Preparation of Detergent Extract of Rat Brain Membrane Proteins-- The entire purification process was performed at 4 °C. Seven rat brains were thawed and the cerebella and brain stem, which are particularly enriched in acid SMase (20), were removed. The remainder of the brain tissues (7 g) were minced and homogenized with a motor-driven Teflon pestle glass homogenizer at highest speed in 5 volumes of homogenizing buffer (50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 mM EGTA, 1 mM sodium orthovanadate, 10 mM -glycerolphosphate, 1 mM sodium fluoride, 1 mM sodium molybdate, 5 mM dithiothreitol, 5 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A). The brain tissues were homogenized for 3 rounds of 10 passes each, and the crude homogenate was centrifuged for 15 min at 1,000 × g. The supernatant was designated as the postnuclear supernatant (PNS). The pellet was re-homogenized in 2 volumes of homogenizing buffer and the supernatants were combined. To remove peripheral membrane proteins, the PNS was incubated for 30 min with 0.5 M NaCl and 5 mM EDTA (final concentrations) and the mixture was then centrifuged for 90 min at 105,000 × g. The resulting pellet was dissolved in 50 ml of 1% Triton X-100 in homogenizing buffer and the detergent/membrane mixture was rocked for 60 min. Afterward, the mixture was centrifuged for 90 min at 105,000 × g. The supernatant was designated as the Triton X-100 solubilized membrane proteins and was used for column chromatography.

DEAE Sephacel-- A DEAE-Sephacel column (Pharmacia HR 10/10, 8 ml) was connected to a Pharmacia Automated FPLC system and equilibrated in buffer A consisting of 20 mM Tris-HCl, pH 7.4, 0.005% Triton X-100, 1 mM EDTA, 1 mM EGTA, and an inhibitor mixture (1 mM -glycerolphosphate, 0.2 mM sodium fluoride, 0.2 mM sodium molybdate, 0.5 mM dithiothreitol, 10 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A). The Triton X-100-solublized membrane proteins were diluted with 1 volume of buffer A and loaded onto the DEAE column. After sample loading, the column was washed with 100 ml of buffer A followed by a 15-ml linear gradient of 0-100% buffer B (20 mM Tris-HCl, pH 7.4, 1 M NaCl, 0.005% Triton X-100, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture) and then maintained for 100 ml at 100% buffer B. The column was finally eluted with a linear gradient from 0 to 100% of buffer C (20 mM Tris-HCl, pH 7.4, 1 M NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture) over 100 ml. The flow rate was 0.5 ml/ml, and fractions of 5 ml were collected. Fractions containing N-SMase activities eluted by the Triton X-100 gradient were pooled and buffer exchanged to buffer A using PD-10 columns (Pharmacia) according to the manufacturer's instructions.

Heparin-Sepharose CL-6B-- Desalted DEAE fractions were loaded onto a heparin-Sepharose CL-6B column (Pharmacia HR16/10, 20 ml) equilibrated in buffer D (20 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture). After sample loading, column was washed with 100 ml of buffer D and then eluted with a 60-ml linear gradient of 0-100% buffer E (20 mM Tris-HCl, pH 7.4, 2 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture). The flow rate was 0.25 ml/min and fractions of 4 ml were collected into tubes containing 0.4 ml of 75% glycerol. The flow-through fractions containing N-SMase activity were pooled and concentrated to 5 ml using the Macrosep concentrators (10-kDa cutoff, Pall Filtron, Northborough, MA).

Hydroxyapatite-- The concentrated heparin fractions were buffer-exchanged using the PD-10 columns to buffer E (10 mM potassium phosphate, pH 7.2, 0.1% Triton X-100, 10% glycerol, and the inhibitor mixture) and loaded onto a ceramic hydroxyapatite column (Pharmacia HR 10/10 column, 8 ml) equilibrated in buffer E. After sample loading, the column was washed with 40 ml of buffer E followed by a shallow gradient of 0-25% of buffer F (500 mM potassium phosphate, pH 7.2, 0.1% Triton X-100, 10% glycerol, and the inhibitor mixture) over 140 ml. The column was then eluted by stepping to 100% buffer F over 4 ml and maintaining at 100% buffer F for 40 ml. The flow rate was 0.25 ml/min. Fractions (4 ml) were collected into tubes preloaded with 8 µl each of 0.5 M EDTA and EGTA (1 mM final concentration). Fractions with N-SMase activity were pooled and concentrated to 10 ml with the Macrosep concentrators (10K).

Mono Q-- The concentrated hydroxyapatite fractions were loaded onto a 1-ml Mono Q column (Pharmacia) equilibrated in buffer G (20 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10% glycerol, and the inhibitor mixture). After sample loading, the column was washed with 10 ml of buffer G followed by a linear gradient of 0-50% of buffer H (20 mM Tris-HCl, pH 7.4, 1 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10% glycerol, and the inhibitor mixture) over 40 ml. Afterward, the gradient was stepped to 100% buffer H over 2 ml and maintained at 100% buffer H for 10 ml. The flow rate was 0.2 ml/min and fractions of 1 ml were collected. Fractions containing N-SMase activity were pooled and concentrated to 5 ml with the Macrosep concentrators (10K).

Phenyl-Superose-- The concentrated Mono Q fractions were adjusted to 2 M NaCl with 5 M NaCl (in 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA), diluted with 1 volume of buffer I (20 mM Tris-HCl, pH 7.4, 2 M NaCl, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture) and then loaded onto a 1-ml phenyl-Superose column (Pharmacia) equilibrated in buffer I. After sample loading, the column was washed with 15 ml of buffer I followed by a linear gradient of 0-100% buffer J (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture) over 15 ml and maintained at 100% buffer J for 20 ml. Afterward, the column was eluted with a linear gradient of 0-100% buffer K (20 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture) over 30 ml. The flow rate was 0.25 ml/min, and 1-ml fractions were collected into tubes containing 0.1 ml of 75% glycerol and 1 µl of leupeptin (5 mg/ml).

Superose 12-- The phenyl-Superose fractions-containing N-SMase activity were pooled and concentrated with the Microsep concentrators (10K) to 0.2 ml and then loaded onto a Superose-12 column (HR 10/30, Pharmacia) equilibrated in buffer L (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and the inhibitor mixture). The column was eluted at 0.2 ml/min with 30 ml of buffer L. Fractions (1 ml) were collected into tubes containing 0.1 ml of 75% glycerol and 1 µl of leupeptin (5 mg/ml).

Discontinuous Glycerol Gradient-- To prepare the gradient, stock solutions of 8-27.8% glycerol (2.2% increment) were prepared from a solution containing 30% glycerol in 20 mM Tris-HCl, pH 7.4, 0.05% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM dithiothreitol, and 1 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A. To a 12-ml polyallomer tube (Sorvall), 1 ml each of the stock solutions was overlaid, starting with the highest concentration from the bottom of the tube. Multiple tubes of the gradients were used for each round of purification and 150 µl of concentrated sample from Superose 12 column was loaded onto each tube. After centrifuging at 200,000 × g for 40 h at 4 °C, fractions of 250 µl each were removed and assayed for SMase activity.

Isoelectric Focusing (IEF) of N-SMase-- Preparative IEF was performed on a 100-ml Raisin RF-3 apparatus. The electrolytes were 0.1 M NaOH and 0.1 M phosphoric acid. The ampholyte solution (105 ml), containing 1.3 ml each of Ampholine 3.5-5 (Pharmacia) and Ampholine 5-7 (1% total solids), 2 mM DTT, 10% glycerol (w/v), and 0.1% (v/v) Triton X-100, was prefocused at 1500 volts prior to injection of the N-SMase sample near the center of pH gradient. The sample was focused at 1000 volts until 30 min after the current stabilized, after which the voltage was set at 500 for another 30 min. After reading the pH of every third fraction, 0.5-1 ml of 1 M Tris-HCl, pH 8.0, was mixed with each fraction. Fractions were stored at 80 °C until analysis for N-SMase activity.

SMase Activity Assay-- The activities of both N-SMase and A-SMase were determined using radiolabeled substrate in a mixed micelle assay system as described (19, 21). For N-SMase, the reaction mixture contained enzyme preparation in 100 mM Tris-HCl, pH 7.4, 5 nmol of [¹⁴C]sphingomyelin (100,000 dpm), 5 nmol of PS, 5 mM DTT, 0.1% Triton X-100 (1.54 mM), and 5 mM magnesium chloride in a final volume of 100 µl. A-SMase activity was measured in a 100-µl reaction mixture consisting of enzyme preparation in 100 mM sodium acetate, pH 5.0, 5 nmol of [¹⁴C]sphingomyelin (100,000 dpm), and 0.1% Triton X-100. Typically, [¹⁴C]sphingomyelin and bovine brain sphingomyelin were placed in a 13 × 100-mm borosilicate glass tube and dried under nitrogen. To the dried substrate was added the N-SMase assay buffer (200 mM Tris-HCl, pH 7.4, 0.2% Triton X-100, and 10 mM magnesium chloride) or the A-SMase assay buffer (200 mM sodium acetate, pH 5.0, and 0.2% Triton X-100). Mixed micelle solution was prepared by sonicating the tube for 1 min in a bath sonicator followed by incubation for 1 min at 37 °C. The substrate solution was always prepared within 10 min before use. Enzyme preparation was loaded into 12 × 75-mm borosilicate glass tubes kept in an ice-water bath and diluted to 50 µl with 20 mM Tris-HCl, pH 7.4 (for N-SMase), or 20 mM sodium acetate, pH 5.0 (for A-SMase). Blank tubes contained no enzyme preparation. The enzyme reaction was initiated by addition of 50 µl of substrate solution and the tubes were placed in a 37 °C water bath. The reaction was stopped by the addition of 1.5 ml of chloroform:methanol (2:1, v/v) and 0.2 ml of water. After vortexing and phase separation by centrifugation, 0.2 ml of the upper aqueous phase was removed and mixed with 5 ml of scintillation solution for radioactivity counting. All reactions were carried out with quantities of enzyme preparations and for the length of incubation time that gave less than 10% hydrolysis of substrate. The reaction was linear with incubation time up to 120 min. In instances where enzyme preparations carried over various amounts of Triton X-100, the amount of the detergent added to the substrate preparation was adjusted so that the final concentration of Triton X-100 in reaction mixture was maintained at 0.1%. To examine the effects of other lipids, substrate was mixed with these lipids before drying down with nitrogen. One unit of activity is defined as micromoles of SM hydrolyzed per hour.

SDS-PAGE-- Proteins in fractions were precipitated with deoxycholate:trichloroacetic acid (22). The protein pellet was dissolved in 15 µl of 2 × SDS sample buffer, titrated to neutral pH with 1 M Tris-HCl, pH 8.0, and boiled for 5 min. The proteins were separated on a 4-15% SDS-PAGE gels (Norvex) The gels were stained with Pro-Blue reagents (Integrated Separation Systems, Natick, MA), silver stained using a kit from Daichi, and dried with the Gel-Dry solutions from Novex following the manufacturer's instructions.

Protein Assay-- Protein concentration was determined with the BCA protein assay reagents from Pierce (Rockford, IL) following deoxycholate:trichloroacetic acid precipitation as described above.

All chromatography steps were performed at least 10 times. Other results are representative of at least three independent experiments.

	RESULTS

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Detergent Extraction-- Prior to chromatographic purification, the extraction efficiency and subcellular distribution of rat brain N-SMase were examined. Table I summarizes the results using one rat brain. About 84% of the N-SMase activity found in the whole homogenate was recovered in the 1,000 × g supernatant (PNS). Little N-SMase activity was found in the cytosolic fraction and the majority of N-SMase (87.3%) in PNS was associated with the membrane fraction. The membrane-associated N-SMase was resistant to chaotropic agents since incubation of the PNS with 0.5 M NaCl and 5 mM EDTA removed 12.4% of protein with little loss of N-SMase activity (2.8%). Incubation of PNS with up to 2 M NaCl or KCl did not result in additional loss of N-SMase activity. To determine the extraction efficiency and stability of extracted N-SMase, we used CHAPS, Nonidet P-40, sodium deoxycholate, and Triton X-100. Deoxycholate strongly inhibited N-SMase activity (apparent IC₅₀ = 0.075%), and a greater extraction efficiency was obtained with CHAPS than with Nonidet P-40. Above all, Triton X-100 was as effective as CHAPS in extracting N-SMase from membranes, but N-SMase extracted with Triton X-100 was more stable (14 days, 4 °C) than that extracted by CHAPS. Therefore, we decided to use Triton X-100 for the extraction and subsequent purification. Inclusion of protease inhibitors, phosphatase inhibitors (6), and reducing agents (21) in the homogenization and column purification buffers served to preserve and stabilize the N-SMase activity.

Purification-- Triton X-100 membrane protein extract was applied to a DEAE-Sephacel column. This step served to effectively remove the acidic SMase (21) in addition to gain purification of the N-SMase. The main portion of acid SMase (64%) did not bind to the column while all the N-SMase bound to the DEAE column. Elution with 1 M NaCl and 0.005% Triton X-100 removed the bound A-SMase together with 29% of the bound N-SMase. The bulk of N-SMase (71%) was eluted by Triton X-100 (Table II) with little contamination of A-SMase (<0.5%).

Next, the Triton X-100 eluted N-SMase was applied to a heparin-Sepharose column and the majority (78%) of N-SMase did not bind to the heparin column while the majority of the proteins did (Fig. 1). The flow-through from heparin column which contained the most active N-SMase fractions was concentrated and then buffer exchanged to 10 mM potassium phosphate buffer and applied to a ceramic hydroxyapatite column. The enzyme bound to the hydroxyapatite column, and the majority of the N-SMase activity was eluted between 40 and 100 mM phosphate (Fig. 2). We observed a 60% loss of total activity after the hydroxyapatite column. Further characterization revealed that the interaction of N-SMase with phosphate salt may result in inactivation of the enzyme and may interfere with the N-SMase activity assay (apparent IC₅₀ = 75 mM). Despite the loss of activity, we decided not to omit the hydroxyapatite column because inclusion of this column in our overall scheme gave us significantly more pure enzyme after the Mono Q column.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Heparin-Sepharose chromatography of N-SMase from rat brain. The N-SMase activity eluted by 1 M NaCl and Triton X-100 from DEAE-Sephacel column was desalted and applied to the column which was then eluted with a linear gradient of 0-1 M NaCl as described under "Methods."

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Ceramic hydroxyapatite chromatography of N-SMase. The flow-through N-SMase activity of the heparin-Sepharose column was desalted, buffer-exchanged, and applied to the column which was eluted with a linear gradient of 10-150 mM phosphate as described under "Methods."

The active fractions from the hydroxyapatite column were loaded onto a Mono Q column. The main peak of N-SMase activity now eluted between 150 and 350 mM NaCl without the requirement of Triton X-100 or 1 M NaCl (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Mono Q chromatography of N-SMase. The phosphate gradient-eluted N-SMase activity of the hydroxyapatite column was applied to the column which was eluted with a linear gradient of 0-0.5 M NaCl as described under "Methods."

The total activity of N-SMase recovered from the Mono Q column was slightly higher than that from the hydroxyapatite column, probably due to the inhibitory effects of phosphate. The activity from the Mono Q column was applied to a phenyl-Superose column, and the main peak of N-SMase activity was eluted when the concentration of NaCl in the buffer was decreased from 2 M to nearly 0 M (Fig. 4). The residual N-SMase activity, which remained bound to the column, was eluted with a Triton X-100 gradient (Fig. 4). Next, the N-SMase activity was loaded onto a sizing column (Superose 12) and the enzyme eluted as a single peak with an estimated mass around 150 kDa (Fig. 5).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Phenyl-Superose chromatography of N-SMase. The NaCl-eluted N-SMase activity of the Mono Q column was concentrated, adjusted to 2 M NaCl, and applied to the column which was eluted by dropping the NaCl concentration to, and maintained at, 0 M NaCl as described under "Methods."

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Superose 12 gel filtration chromatography of N-SMase. The peak N-SMase activity of the phenyl-Superose column was concentrated and applied to the column which was eluted isocratically as described under "Methods." The arrows depict the elution volume of molecular standards which were -amylase (200 kDa), alcohol dehydrogenase (150 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa).

The chromatographic process up to the Superose 12 column resulted in 3030-fold purification of the N-SMase from rat brain (Table II). In order to further purify the N-SMase, we employed the glycerol gradient technique which has proven quite powerful in resolving integral membrane proteins (23). The N-SMase peak from a Superose 12 run was loaded on top of a discontinuous glycerol gradient (8-27.8%, 10 ml). After centrifugation at 200,000 × g for 40 h, fractions of 0.25 ml were obtained and assayed for N-SMase activity and protein distribution by SDS-PAGE. The N-SMase activity migrated as a relatively tight band along the glycerol gradient (Fig. 6A) while proteins were more randomly distributed across the entirety of the gradient (Fig. 6B). It should be noted that a significant loss of N-SMase activity was observed (>70%).

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Separation of N-SMase by discontinuous glycerol sedimentation gradient. N-SMase from a Superose 12 column separation was concentrated and applied to the top of a discontinuous glycerol gradient (8-27.8%). After centrifugation, fractions were collected and assayed for N-SMase activity (A). Proteins in peak fractions were analyzed by a 4-20% gradient SDS-PAGE gel which was stained by Pro-blue and silver staining as described under "Methods" (B).

When the peak fractions (15-32) were analyzed for protein distribution by SDS-PAGE followed by silver stain, it was evident that proteins of different molecular mass were well separated by the glycerol gradient (Fig. 6B). Several bands migrated in the range of 55-90 kDa and correlated with the activity of N-SMase.

Effect of Lipids on N-SMase Activity-- To test the effects of lipids on N-SMase, peak fractions from the Superose 12 column-purified N-SMase were used. As shown in Fig. 7A, the activity of N-SMase was significantly stimulated by phosphatidylserine (PS). In the presence of 2.5 mol % sphingomyelin (SM), PS at 1 mol % gave rise to a 60% stimulation of N-SMase activity. At 2.5 mol % PS, a 5.7-fold stimulation was observed, and higher amounts of PS increased N-SMase activity such that an 18-fold stimulation was observed at 12.5 mol % PS. At 2.5 mol %, phosphatidic acid and phosphatidylinositol increased the N-SMase activity by 4.6- and 3.7-fold, respectively (Fig. 7A). However, increasing amounts of these two phospholipids up to 15 mol % did not result in further stimulation of the enzyme activity (Fig. 7A). Phosphatidylcholine (PC) and phosphatidylethanolamine were also less effective than PS, and maximal stimulation of only 2.5- and 2.2-fold were observed at 15 mol % (Fig. 7A). Lyso-PC gave rise to a 2.3-fold stimulation at 2.5 mol % and a maximal stimulation of 4-fold was observed at 10 mol % (Fig. 7A). Arachidonic acid, which has been reported to stimulate the N-SMase activity from HL-60 cells (24), stimulated the N-SMase activity by 3.7-4.8-fold at between 5 and 15 mol % (Fig. 7A).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effect of lipids on N-SMase activity. A, N-SMase from Mono Q peak fractions was assayed for activity in the presence of sphingomyelin (2.5 mol %), 5 mM MgCl2, and the indicated amounts of PS, phosphatidic acid (PA), phosphatidylethanolamine (PE), PC, phosphatidylinositol (PI), arachidonic acid (AA), or lyso-phosphatidylserine (lyso-PS). B, double-reciprocal plot for effects of PS on substrate (SM) dependence of N-SMase. The molarity of Triton X-100 was 1.54 mM.

No synergistic effects were observed for stimulation of N-SMase activity between PS and phosphatidic acid, PC, phosphatidylethanolamine, PI, or lyso-PC (data not shown). Diolein and short chain C₂- and C₆-ceramide had no effects on N-SMase activity by themselves or on the PS-stimulated N-SMase activity (data not shown). Interestingly, the length and/or degree of saturation of the side chains in PS affected its ability to stimulate N-SMase activity. When the N-SMase preparation purified by a sizing column from CHAPS extract was used, dioleoyl-PS was significantly more potent than the PS mixture, and dipalmitoyl-PS was barely effective in stimulation of N-SMase (data not shown). Analyzing the effects of PS on the substrate dependence of N-SMase, we found that PS did not alter the K_m of N-SMase for SM (Fig. 7B).

We noted that crude enzyme preparations were hardly responsive to PS. Therefore, we suspected that the amount of co-existing lipids, especially PS, in the Triton X-100 extracted N-SMase preparations from lipid-rich rat brain tissues might affect the responsiveness of N-SMase to PS stimulation. When the effects of PS were systematically studied at all steps of purification, increasing degree of PS stimulation was observed with enzyme from initial to later steps. Examining the lipid content of N-SMase from various purification steps revealed that N-SMase was highly lipidated when extracted from rat brain membranes by Triton X-100 and became increasingly delipidated upon further purification. Deliberate removal of the lipids associated with N-SMase in the Triton X-100 extract by solubilizing rat brain membrane fraction with 6% CHAPS and passing the CHAPS extract through a sizing column (Sephacryl S-300) rendered the enzyme very responsive to PS stimulation (data not shown).

Substrate Properties of N-SMase-- In the absence of PS, the K_m for sphingomyelin was 3.4 mol % (Fig. 7B). The enzyme hydrolyzed sphingomyelin as efficiently as dihydrosphingomyelin (data not shown). It showed no activity in hydrolyzing phosphatidylcholine. As shown in Fig. 8, when N-SMase enzyme preparation was incubated with 2.5-20 mol % (38.5-308 µM) of PC for 2 h, no cleaved radioactive product was detected, whereas significant hydrolysis of PC (154 µM) was seen with phospholipase C. The same SMase preparation was efficient in hydrolyzing SM (10 mol %, 154 µM, Fig. 8). In addition to not being utilized as a substrate, PC, along with several other phospholipids, exhibited no inhibitory effects on N-SMase-mediated hydrolysis of SM (Fig. 7A).

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Comparison of N-SMase and phospholipase C on hydrolysis of phosphatidylcholine. Indicated amounts of dipalmitoyl phosphatidylcholine (38.5-308 µM) mixed with tracer choline-[methyl-14H]L-dipalmitoyl phosphatidylcholine (NEN Life Science Products Inc., 0.5 µCi, 0.01 nmol for each tube) or [methyl-14C]sphingomyelin (SM, 154 µM) was incubated for 2 h with N-SMase or Bacillus cereus phospholipase C (PLC, Behringer-Mannheim) in a SMase mixed micelle assay system as described under "Methods."

Effects of Cations-- The activity of the neutral magnesium-dependent SMase was absolutely dependent upon the availability of Mg²⁺ in the assay. The K_m for Mg²⁺ was 1.25 mM. Manganese at up to 2.5 mM was more effective than Mg²⁺ and as effective as Mg²⁺ at 5 mM. Higher concentrations of Mn²⁺ were slightly inhibitory (Fig. 9A). Cu²⁺, Ca²⁺, or Ni²⁺ did not stimulate the activity of the enzyme (Fig. 9A). However, Ni²⁺, and especially Cu²⁺ potently inhibited the activity of Mg²⁺ (1 mM)-stimulated-N-SMase activity (Fig. 9B).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Effects of cations on N-SMase activity. A, activity was determined in a standard assay system containing the indicated amounts of the indicated individual cations. B, the effects of cations on the Mg2+-stimulated N-SMase activity were determined by inclusion of the indicated amounts of the cations shown in an assay system containing 1 mM Mg2+.

Effects of pH and Isoelectric Point (pI)-- The enzyme had a pH optimum of 7.5. Some activity was detected at pH up to 9.0 using Tris-HCl buffer. No activity was detected in the acidic pH (4-6) or alkaline pH range (10-11). While similar activities were obtained with Tris-HCl, HEPES, or PIPES buffer in the neutral pH range (7-8), HEPES gave slightly higher activity at pH 7.5 than Tris-HCl or PIPES. Using the Sephacryl S300 column purified N-SMase as starting material, a pI of 5.3-5.4 was obtained (Fig. 10). When glycerol gradient purified N-SMase was used, a pI of 5.2 was obtained. However, the recovery of activity after this step was extremely low.

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Determination of pI for purified N-SMase. The pI of N-SMase was determined by isoelectric focusing as described under "Methods."

Effects of Glutathione-- Previously, we reported that the DEAE-purified N-SMase from cultured human Molt-4 leukemic cells was inhibited in vitro by glutathione (21). When the highly purified N-SMase (Superose 12 column) was examined for effects by GSH, a nearly complete inhibition was observed with 3 mM GSH (Fig. 11A). However, DTT significantly stimulated the enzyme activity. In the presence of 3 mM DTT, the enzyme activity was enhanced 2-fold (Fig. 11A). To examine the cooperativity of GSH inhibition of N-SMase, enzyme from the Mono Q column was incubated with GSH at a series of concentrations in small increments (0.1 or 0.2 mM each) up to 4 mM. The inhibition of N-SMase by GSH was observed with concentrations of GSH between 2 and 3 mM and a nearly complete inhibition was achieved with 3 mM GSH, similar to the profiles observed with N-SMase purified from Molt-4 cells (21). A cooperativity number of 8 was obtained when a Hill plot was constructed from GSH inhibition data obtained with the Mono Q purified N-SMase (Fig. 11B).

View larger version (12K): [in this window] [in a new window]   Fig. 11.   Effects of reducing agents on purified N-SMase and estimation of cooperativity for GSH inhibition of N-SMase. A, N-SMase from Superose 12 column was preincubated with the indicated concentrations of glutathione or DTT for 5 min at 37 °C before assayed for N-SMase activity in the presence of SM (2.5 mol %) and PS (5 mol %) as described under "Methods." B, Hill plot for GSH inhibition.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

In this study, we significantly purified the membrane-associated neutral magnesium-dependent sphingomyelinase from rat brain. The purified enzyme has an optimal pH of 7.4 and a pI of 5.2. The rat brain N-SMase does not hydrolyze phosphatidylcholine. Its activity is totally dependent upon either magnesium or manganese, but the magnesium stimulated activity is potently inhibited by copper or nickel. The enzyme displays highly hydrophobic properties, and its activity is significantly stimulated by anionic lipids, especially phosphatidylserine. On a sizing column, the enzyme migrates as a complex with detergent micelles with a size of approximately 150 kDa. Given the size of a Triton X-100 micelle at around 90 kDa, the actual molecular size of the rat brain membrane N-SMase is estimated at around 60 kDa, presuming that the enzyme associates with the Triton X-100 micelles as a monomer. The highly purified enzyme is significantly stimulated by dithiothreitol and potently inhibited by glutathione with a Hill number of approximately 8.

The properties of N-SMase have not been well determined at a biochemical level because the enzyme has not been purified to homogeneity from any source. Over the last three decades, many attempts have been made to purify this enzyme from rat brain (25, 26), rat liver (27), rat ascites hepatoma (28), human brain (18, 29), and human urine (30). The purification efforts so far have not been very successful, probably due to multiple factors. This study defines several of these factors including the nature of the enzyme as a highly hydrophobic integral membrane protein, its dependence on detergent during most if not all of the purification steps, its existence, at least in crude preparations, as a heavily lipidated enzyme, its extreme dependence on PS for activity, and its requirement for reducing agent such as DTT for stability.

Very recently, a group reported the cloning of a cDNA for a candidate neutral SMase for human and mouse based on distal sequence homology with bacterial SMases (31). Biochemical characterization of homogenate from cells overexpressing the candidate murine neutral SMase revealed differences with the enzyme purified from rat brain N-SMase. The candidate murine neutral SMase did not respond to DTT stimulation and was not examined for stimulation by PS. More importantly, the candidate murine neutral SMase had a significant activity toward phosphatidylcholine, in stark contrast to the enzyme described here which has a strict substrate specificity toward SM. In preliminary studies, we have expressed the putative cloned sphingomyelinase in HEK 293 cells. These studies show a number of differences between this protein and the enzyme purified in this study, including response to GSH and effects on sphingomyelin synthesis. Further studies are required to establish the relationship between these two enzymes.

N-SMase has been implicated in the regulation of a variety of cellular functions for a wide range of cell types. These include: cell growth, differentiation and apoptosis in response to TNF (6, 32-34), Fas (9), IgM cross-linking (35), CD40 activation (36), the chemotherapeutic agents daunorubicin (37), dexamethasone (38), cytosine arabinocide (39, 40), and pharmacologic agents such as protein kinase C inhibitors (41). Activation of N-SMase has been reported for neural cell differentiation induced by either neural growth factors (42) or retinoic acid (43). Cell cycle arrest induced by serum withdrawal (44) or cellular senescence human fibroblasts SMase (45) also involve the activity of N-SMase. In addition, N-SMase is believed to play a role in regulation of cholesterol metabolism in response to TNF (46, 47) or insulin (48). Finally, N-SMase has recently been proposed to act in hepatic regeneration (49), lung development (50), and phagocytosis (51). How these diverse signals, stimuli, stresses converge on N-SMase has become somewhat of a puzzle. Indeed, enormous efforts have been devoted to attempt to delineate the signaling pathways leading to activation of N-SMase. For example, it has been proposed that a putative protein (FAN) is linked to N-SMase in TNF signaling (52). The finding that the cellular reducing agent GSH may act to inhibit N-SMase in cells sheds light on one regulatory mechanism for this enzyme. According to this hypothesis, oxidative stress and/or agents that induce a depletion of cellular GSH (such as TNF and Fas) cause activation of N-SMase by relieving inhibition of the enzyme by GSH. The high cooperativity of inhibition of N-SMase by GSH then serves as a biochemical switch to turn the enzyme from an off to an on position. Once relieved of inhibition, the enzyme acts on SM and causes the accumulation of ceramide and other subsequent metabolites which together serve to signal specific functions.

In conclusion, these studies define several biochemical properties and mechanisms of regulation of membrane N-SMase, including regulation by magnesium, phosphatidylserine, and glutathione.

	ACKNOWLEDGEMENTS

We thank Alicja Bielawska for expert assistance with synthesis of ceramides and sphingomyelin, Mary Moyer, Will Burkhart, Kevin Blackburn, and Marcos Milla of GlaxoWellcome for helpful discussions.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants GM43825 (to Y. A. H.) and GM-17426 (to B. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ A Terry Seelinger Fellow in Cancer. Present address: Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709.

^** To whom correspondence should be addressed. Current address: Dept. of Biochemistry, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322.

The abbreviations used are: SMase, sphingomyelinase; DTT, dithiothreitol; GSH, glutathione; SM, sphingomyelin; N-SMase, neutral magnesium-dependent sphingomyelinase; PS, L--phosphatidylserine; TNF, tumor necrosis factor-; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PC, phosphatidylcholine; PNS, postnuclear supernatant.

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