536
PHOSPHOLIPASEC
[52]
[52] H u m a n A c i d S p h i n g o m y e l i n a s e f r o m H u m a n U r i n e By L. E. QUINTERN and K. SANDHOFF Introduction
Acid sphingomyelinase (EC 3.1.4.12, sphingomyelin phosphodiesterase) is a lysosomal enzyme catalyzing the hydrolysis of sphingomyelin to ceramide and phosphorylcholine.~ In addition to the lysosomal enzyme, a neutral sphingomyelinase and other sphingomyelinases have also been described. 2-4 Deficiency of acid sphingomyelinase activity results in the accumulation of sphingomyelin as seen in Niemann-Pick disease, types A and B, which are severe lysosomal lipid storage disorders. 5,6 In order to characterize the enzyme, several purification procedures have been developed using different human tissues as starting material. The presence of acid sphingomyelinase in human urine offers the possibility of purifying the enzyme from a source which is easily obtained. The purified enzyme has a low hydrolytic activity if the assay contains pure sphingomyelin liposomes; the addition of detergents stimulates activity. The natural stimulating agent may be a small heat-stable glycoprotein as discussed by Morimoto et al. 7 Assay Methods
Reagents. The incubation mixture contains (in a volumc of 200 ~l) 100 I~M [choline-methyl-3H]sphingomyelin from bovine brain (37 x 101° Bq/mol); 200 m M sodium/acetate buffer (pH 4.5); 0.05% (w/v) Nonidet P-40 (NP-40) as detergent; and an aliquot of the enzyme preparation [diluted to an extent giving not more than 10% substrate utilizationduring the time of incubation (see below)] in I0 m M Tris-HCl buffer (pH 7.2), 0.1% (w/w) Nonidct P-40 or crude enzyme solution up to a volume of 20 pA. i j . N . K a n f e r , O . Y o u n g , D . S h a p i r o , a n d R . O . B r a d y , J. Biol. Chem. 241, 1081 (1966).
2 S. Chatterjee and N. Gosh, J. Biol. Chem. 264, 12554 (1989). 3 M. W. Spence, D. M. Byers, F. B. St.C. Palmer, and H. Cook, J. Biol. Chem. 264, 5358 (1989). 4 T. Vanha-Perttula, FEBS Lett. 233, 263 (1988). 5 R. O. Brady, J. N. Kanfer, M. B. Mock, and D. S. Fredrickson, Proc. Natl. Acad. Sci. U.S.A. 55, 366 (1966). 6 p. B. Schneider and E. P. Kennedy, J. Lipid Res. 8, 202 (1967). 7 S. Morimoto, B. M. Martin, Y. Kishimoto, and J. S. O'Brien, Biochim. Biophys. Res. Commun. 156, 403 (1988).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
[52]
ACID SPHINGOMYELINASE FROM HUMAN URINE
537
Procedure. The assay should be carried out as follows. [3H]Sphingomyelin dissolved in 20/.d toluene/ethanol (2 : 1, v/v), or in another organic solvent, should be added to each test tube and the solvent evaporated in a stream of nitrogen at room temperature. After adding the buffer and detergent, the solution should be mixed (e.g., for 5 sec with a Vortex Genie Mixer) and then cooled to 0° on ice; the enzyme can now be added. The reaction (e.g., 60 min at 37° in a shaking water bath) can be stopped by the addition of 800/zl chloroform/methanol (2 : 1, v/v) and 100/zl of 0.1% (w/w) unlabeled phosphorylcholine in water. After mixing the two phases can be separated by centrifugation (i.e., 5 min in an uncooled tabletop centrifuge). The upper water phase contains [3H]phosphorylcholine released by the enzyme. In order to measure enzyme activity 100 ~1 of the upper water layer is removed and mixed with scintillation fluid, and the radioactivity measured in a scintillation counter. Appropriate blanks should be carded out with denatured enzyme (15 min, 95°). Purification Procedure The purification steps are carried out at 4° unless otherwise stated. The urine of certain male or female hospital patients contains much higher amounts of acid sphingomyelinase than normal controls. Apart from an increase in the excretion of the enzyme into the urine of patients with peritonitis and some other diseases, 8 there is often a greatly enhanced excretion of other proteins as well. For successful enzyme purification only urine with slightly elevated protein content but a highly elevated level of sphingomyelinase activity should be used. Urine should be collected daily. After the addition of sodium acetate buffer (20 raM; pH 5.0) and sodium azide (0.01%), the urine can be stored for several days; pH 5.0 is chosen because the enzyme in the undialyzed urine is stable at this pH. Table I summarizes the data for a purification of the enzyme from the urine of patients with peritonitis. The purification is done as follows: the clear supernatant from stored urine is concentrated by ultrafiltration [i.e., 50 liters is reduced to 1 liter with a Millipore (Bedford, MA) ultrafiltration system using a cassette filter Type PTGC MG 10,000]. After dialysis (3 x 24 hr, 3 x I0 liters buffer) against 20 mM sodium acetate (pH 5.0)/50 mM NaCI/0.01% NaN3 and dialysis for 24 hr against 10 liters I0 mM Tris-HC1 buffer (pH 7.2), 100 mM NaC1/0.01% NaN3 (buffer A), the solution is clarified by centrifugation (1 hr, 10,000 g). Step 1: Octyl-Sepharose Chromatography. The supernatant is then loaded onto an octyl-Sepharose column previously equilibrated with buffer 8 L. E. Quintern, T. S. Zenk, and K. Sandhoff. Biochim. Biophys. Acta 1003, 121 (1989).
538
PHOSPHOLIPASE C
[52]
TABLE I PURIFICATION OF ACID SPHINGOMYELINASEFROM URINE OF PATIENTS WITH PERITONITISa
Step Urine concentrate (from 49 liters) Octyl-Sepharose Concanavaiin A-Sepharose Blue-Sepharose DEAE-cellulose a
Volume (ml)
Protein (mg)
Activity (/zmol/hr)
1150
19,750
61,000
400 460
760 37
63 68
3 2
Specific activity (p,mol/hr/mg)
Yield (%)
Enrichment (-fold)
2
100
--
24,900 28,600
33 777
41 47
15 353
16,000 13,500
5550 6900
26 22
2523 3136
Acid sphingomyelinase activity was measured in a test volume of 200/zl which contained 200 mM sodium acetate buffer, pH 4.5, 50/~M [3H]sphingomyelin, and 0.05% Nonidet P-40. Data from L. E. Quintern, T. S. Zenk, and K. Sandhoff, Biochim. Biophys. Acta 1003, 121 (1989).
A. Unbound proteins are removed with at least 800 ml of buffer A. The bound sphingomyelinase elutes in high yield (-40%) with 300 ml of 1% (w/v) Nonidet P-40 in a buffer containing 10 mM Tris-HC1 (pH 7.2)/100 mM NaCI/1 mM CaC12/1 mM MgC12/1 mM MnC12/0.01% NAN3. The flow rate should not be higher than 30 ml/hr during loading, washing, and elution; pH 7.2 is chosen because in the presence of Nonidet P-40 the enzyme is most stable at this pH. During loading, washing, and elution the column should always be run in such a way that solutions of higher density enter the column from the bottom and solutions of lower density from the top; these precautions should be followed during every chromatographic step. Step 2: Concanavalin A-Sepharose Chromatography. The octyl-Sepharose eluate containing the enzyme is loaded onto a concanavalin ASepharose column (1.6 × 24 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.2), I00 mM NaCI, 1 mM CaC12, 1 mM MgC12, 1 mM MnC12, 0.01%, NAN3, and 0.1% Nonidet P-40 (buffer B); the column should be washed with at least 200 ml of buffer B. We have had good results by performing a further washing step with 200 ml of buffer B containing 0.2% (w/w) amethyl-D-glucopyranoside, followed by at least another 200 ml of buffer B. The enzyme is eluted in good yield at room temperature with 15% (w/w) a-methyl-o-glucopyranoside in buffer B using a fraction collector; the flow rate should not exceed 10, 30, and 2 ml/hr during loading, washing, and elution, respectively. It is important to maintain the flow direction in the column. In this way the first fractions of the eluate contain the bulk
[52]
ACID SPHINGOMYELINASE FROM HUMAN URINE
539
of contaminating proteins and the acid sphingomyelinase elutes later. Fractions with the highest specific activity are pooled and dialyzed against 6 changes (6 × 12 hr) of 4 liters each of 30 mM Tris-HC1 buffer (pH 7.2), 0.01% NaN3, 0.1% Nonidet P-40 (buffer C). Step 3: Blue-Sepharose Chromatography. The dialyzed fractions are loaded onto a freshly prepared Blue-Sepharose column (1.6 × 8 cm) equilibrated with buffer C. After washing with 200 ml buffer C the bound enzyme is eluted with a linear sodium chloride gradient in buffer C (0-1 M NaC1; total volume 50 + 50 ml); the flow rate during loading, washing, and elution should be 3, 30, and 2 ml/hr, respectively. Most of the enzyme elutes in the presence of around 500 mM NaC1. Fractions with the highest specific activities are pooled and then dialyzed against 6 changes of 2 liters of 40 mM Tris-HC1 (pH 7.6), 0. I% NP-40 (buffer D). Step 4: DEAE-Cellulose Chromatography. The dialyzed fractions of the Blue-Sepharose eluate are applied to a DEAE-cellulose column (1 × 2 cm) equilibrated with buffer D. Under these conditions most of the enzyme does not bind to the column, but most of the contaminating proteins do.
Additional Remarks In order to obtain pure acid sphingomyelinase protein, the enzyme (post-DEAE-cellulose) should be concentrated and separated from the detergent Nonidet P-40 by ethanol precipitation, alkylated with iodoacetamide, 9 and purified on a hydrophobic HPLC column (e.g., HIBAR Lichrospher, 500 CH-2/10/xm) with a linear gradient of acetonitrile (0-70%, v/v) in 0.05% trifluoroacetic acid. This procedure gives denatured but pure enzyme protein (e.g., for immunochemical experiments). Properties The purified glycosylated enzyme has an apparent molecular mass of 72 kDa determined with SDS-gel electrophoresis, which was reduced to 61 kDa after deglycosylation. It has an isoelectric point (p/) of 7.5, and the optimum for enzyme activity occurs in the range pH 4.5-5.0. Enzyme activity can be stimulated with Nonidet P-40, various cholates, or by the addition of certain 1/pids to a detergent-free assay system. 10Under some conditions the freshly prepared enzyme hydrolyzes phosphatidylcholine and phosphatidylglycerol with phospholipase C-like activity) ° The en9 j. Hempel, H. v. Bahr-Lindstr0m, and H. J6rnvall, Eur. J. Biochem. 141, 21 0984). l0 L. E. Quintern, G. Weffz, H. Nehrkorn, J. M. Tager, A. W. Schram, and K. Sandhoff, Biochim. Biophys. Acta 922, 323 (1987).
540
PHOSPHOLIPASEC
[53]
zyme has an apparent K m value of around 60/zM toward the three lipids sphingomyelin, phosphatidylcholine, and phosphatidylglycerol in the presence of Nonidet P-40, sodium taurodeoxycholate, and Nonidet P-40, respectively. Reducing agents (e.g., dithiothreitol) inactivate the enzyme. Divalent cations (Ca 2÷ , Co 2÷ , Mg 2÷ , Mn 2÷) or the cation-chelating agent EDTA up to a concentration of 20 mM have no effect on enzyme activity. AMP (Ki 45-74/,M), 9-/3-D-arabinofuranosyladenine 5'-monophosphate (Ki - 4 / z M ) and phosphatidylinositol 4',5'-bisphosphate (Ki - 1 / z M ) are potent inhibitors of acid sphingomyelinase. In the presence of 0.1% Nonidet P-40 and greater than 100 mM NaCI the enzyme can be stored for several weeks with only a small loss in total hydrolytic activity. Additional Remarks
The cDNA of the enzyme has been cloned. 11The occurrence of alternatively processed transcripts indicates the presence of isoenzymes of this enzyme. Acknowledgments We are grateful to Roger Klein for proofreading the manuscript and the Deutsche Forschungsgemeinschaft for financial support (Grant Sa 257/12-1). tl L. E. Quintern, E. H. Schuchmann, O. Levran, M. Suchi, K. Ferlinz, H. Reinke, K. Sandhoff, and R. J. Desnick, EMBO J. 8, 2469 (1989).
[53] P u r i f i c a t i o n o f N e u t r a l S p h i n g o m y e l i n a s e f r o m Human Urine B y SUBROTO CHATTERJEE a n d N U P U R GHOSH
Introduction Sphingomyelinase (EC 3.1.4.12) catalyzes the hydrolytic cleavage of sphingomyelin via reaction (1)) It has been shown that sphingomyelin may be cleaved at both acid pH optima and neutral pH optima. 2-4 The lack t R. O. Brady, J. N. Kanfer, M. B. Mock, and D. S. Fredrickson, Proc. Natl. Acad. Sci. U.S.A. 55, 366 (1966). 2 p. Ghosh and S. Chatterjee, J. Biol. Chem. 262, 12550 (1987). 3 S. Gatt, Biochem. Biophys. Res. Commun. 68, 235 (1976). 4 B. G. Rao and M. W. Spence, J. Lipid Res. 17, 506 (1976).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc, All fightsof reproductionin any form reserved,
PHOSPHOLIPASEC
[52]
[52] H u m a n A c i d S p h i n g o m y e l i n a s e f r o m H u m a n U r i n e By L. E. QUINTERN and K. SANDHOFF Introduction
Acid sphingomyelinase (EC 3.1.4.12, sphingomyelin phosphodiesterase) is a lysosomal enzyme catalyzing the hydrolysis of sphingomyelin to ceramide and phosphorylcholine.~ In addition to the lysosomal enzyme, a neutral sphingomyelinase and other sphingomyelinases have also been described. 2-4 Deficiency of acid sphingomyelinase activity results in the accumulation of sphingomyelin as seen in Niemann-Pick disease, types A and B, which are severe lysosomal lipid storage disorders. 5,6 In order to characterize the enzyme, several purification procedures have been developed using different human tissues as starting material. The presence of acid sphingomyelinase in human urine offers the possibility of purifying the enzyme from a source which is easily obtained. The purified enzyme has a low hydrolytic activity if the assay contains pure sphingomyelin liposomes; the addition of detergents stimulates activity. The natural stimulating agent may be a small heat-stable glycoprotein as discussed by Morimoto et al. 7 Assay Methods
Reagents. The incubation mixture contains (in a volumc of 200 ~l) 100 I~M [choline-methyl-3H]sphingomyelin from bovine brain (37 x 101° Bq/mol); 200 m M sodium/acetate buffer (pH 4.5); 0.05% (w/v) Nonidet P-40 (NP-40) as detergent; and an aliquot of the enzyme preparation [diluted to an extent giving not more than 10% substrate utilizationduring the time of incubation (see below)] in I0 m M Tris-HCl buffer (pH 7.2), 0.1% (w/w) Nonidct P-40 or crude enzyme solution up to a volume of 20 pA. i j . N . K a n f e r , O . Y o u n g , D . S h a p i r o , a n d R . O . B r a d y , J. Biol. Chem. 241, 1081 (1966).
2 S. Chatterjee and N. Gosh, J. Biol. Chem. 264, 12554 (1989). 3 M. W. Spence, D. M. Byers, F. B. St.C. Palmer, and H. Cook, J. Biol. Chem. 264, 5358 (1989). 4 T. Vanha-Perttula, FEBS Lett. 233, 263 (1988). 5 R. O. Brady, J. N. Kanfer, M. B. Mock, and D. S. Fredrickson, Proc. Natl. Acad. Sci. U.S.A. 55, 366 (1966). 6 p. B. Schneider and E. P. Kennedy, J. Lipid Res. 8, 202 (1967). 7 S. Morimoto, B. M. Martin, Y. Kishimoto, and J. S. O'Brien, Biochim. Biophys. Res. Commun. 156, 403 (1988).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
[52]
ACID SPHINGOMYELINASE FROM HUMAN URINE
537
Procedure. The assay should be carried out as follows. [3H]Sphingomyelin dissolved in 20/.d toluene/ethanol (2 : 1, v/v), or in another organic solvent, should be added to each test tube and the solvent evaporated in a stream of nitrogen at room temperature. After adding the buffer and detergent, the solution should be mixed (e.g., for 5 sec with a Vortex Genie Mixer) and then cooled to 0° on ice; the enzyme can now be added. The reaction (e.g., 60 min at 37° in a shaking water bath) can be stopped by the addition of 800/zl chloroform/methanol (2 : 1, v/v) and 100/zl of 0.1% (w/w) unlabeled phosphorylcholine in water. After mixing the two phases can be separated by centrifugation (i.e., 5 min in an uncooled tabletop centrifuge). The upper water phase contains [3H]phosphorylcholine released by the enzyme. In order to measure enzyme activity 100 ~1 of the upper water layer is removed and mixed with scintillation fluid, and the radioactivity measured in a scintillation counter. Appropriate blanks should be carded out with denatured enzyme (15 min, 95°). Purification Procedure The purification steps are carried out at 4° unless otherwise stated. The urine of certain male or female hospital patients contains much higher amounts of acid sphingomyelinase than normal controls. Apart from an increase in the excretion of the enzyme into the urine of patients with peritonitis and some other diseases, 8 there is often a greatly enhanced excretion of other proteins as well. For successful enzyme purification only urine with slightly elevated protein content but a highly elevated level of sphingomyelinase activity should be used. Urine should be collected daily. After the addition of sodium acetate buffer (20 raM; pH 5.0) and sodium azide (0.01%), the urine can be stored for several days; pH 5.0 is chosen because the enzyme in the undialyzed urine is stable at this pH. Table I summarizes the data for a purification of the enzyme from the urine of patients with peritonitis. The purification is done as follows: the clear supernatant from stored urine is concentrated by ultrafiltration [i.e., 50 liters is reduced to 1 liter with a Millipore (Bedford, MA) ultrafiltration system using a cassette filter Type PTGC MG 10,000]. After dialysis (3 x 24 hr, 3 x I0 liters buffer) against 20 mM sodium acetate (pH 5.0)/50 mM NaCI/0.01% NaN3 and dialysis for 24 hr against 10 liters I0 mM Tris-HC1 buffer (pH 7.2), 100 mM NaC1/0.01% NaN3 (buffer A), the solution is clarified by centrifugation (1 hr, 10,000 g). Step 1: Octyl-Sepharose Chromatography. The supernatant is then loaded onto an octyl-Sepharose column previously equilibrated with buffer 8 L. E. Quintern, T. S. Zenk, and K. Sandhoff. Biochim. Biophys. Acta 1003, 121 (1989).
538
PHOSPHOLIPASE C
[52]
TABLE I PURIFICATION OF ACID SPHINGOMYELINASEFROM URINE OF PATIENTS WITH PERITONITISa
Step Urine concentrate (from 49 liters) Octyl-Sepharose Concanavaiin A-Sepharose Blue-Sepharose DEAE-cellulose a
Volume (ml)
Protein (mg)
Activity (/zmol/hr)
1150
19,750
61,000
400 460
760 37
63 68
3 2
Specific activity (p,mol/hr/mg)
Yield (%)
Enrichment (-fold)
2
100
--
24,900 28,600
33 777
41 47
15 353
16,000 13,500
5550 6900
26 22
2523 3136
Acid sphingomyelinase activity was measured in a test volume of 200/zl which contained 200 mM sodium acetate buffer, pH 4.5, 50/~M [3H]sphingomyelin, and 0.05% Nonidet P-40. Data from L. E. Quintern, T. S. Zenk, and K. Sandhoff, Biochim. Biophys. Acta 1003, 121 (1989).
A. Unbound proteins are removed with at least 800 ml of buffer A. The bound sphingomyelinase elutes in high yield (-40%) with 300 ml of 1% (w/v) Nonidet P-40 in a buffer containing 10 mM Tris-HC1 (pH 7.2)/100 mM NaCI/1 mM CaC12/1 mM MgC12/1 mM MnC12/0.01% NAN3. The flow rate should not be higher than 30 ml/hr during loading, washing, and elution; pH 7.2 is chosen because in the presence of Nonidet P-40 the enzyme is most stable at this pH. During loading, washing, and elution the column should always be run in such a way that solutions of higher density enter the column from the bottom and solutions of lower density from the top; these precautions should be followed during every chromatographic step. Step 2: Concanavalin A-Sepharose Chromatography. The octyl-Sepharose eluate containing the enzyme is loaded onto a concanavalin ASepharose column (1.6 × 24 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.2), I00 mM NaCI, 1 mM CaC12, 1 mM MgC12, 1 mM MnC12, 0.01%, NAN3, and 0.1% Nonidet P-40 (buffer B); the column should be washed with at least 200 ml of buffer B. We have had good results by performing a further washing step with 200 ml of buffer B containing 0.2% (w/w) amethyl-D-glucopyranoside, followed by at least another 200 ml of buffer B. The enzyme is eluted in good yield at room temperature with 15% (w/w) a-methyl-o-glucopyranoside in buffer B using a fraction collector; the flow rate should not exceed 10, 30, and 2 ml/hr during loading, washing, and elution, respectively. It is important to maintain the flow direction in the column. In this way the first fractions of the eluate contain the bulk
[52]
ACID SPHINGOMYELINASE FROM HUMAN URINE
539
of contaminating proteins and the acid sphingomyelinase elutes later. Fractions with the highest specific activity are pooled and dialyzed against 6 changes (6 × 12 hr) of 4 liters each of 30 mM Tris-HC1 buffer (pH 7.2), 0.01% NaN3, 0.1% Nonidet P-40 (buffer C). Step 3: Blue-Sepharose Chromatography. The dialyzed fractions are loaded onto a freshly prepared Blue-Sepharose column (1.6 × 8 cm) equilibrated with buffer C. After washing with 200 ml buffer C the bound enzyme is eluted with a linear sodium chloride gradient in buffer C (0-1 M NaC1; total volume 50 + 50 ml); the flow rate during loading, washing, and elution should be 3, 30, and 2 ml/hr, respectively. Most of the enzyme elutes in the presence of around 500 mM NaC1. Fractions with the highest specific activities are pooled and then dialyzed against 6 changes of 2 liters of 40 mM Tris-HC1 (pH 7.6), 0. I% NP-40 (buffer D). Step 4: DEAE-Cellulose Chromatography. The dialyzed fractions of the Blue-Sepharose eluate are applied to a DEAE-cellulose column (1 × 2 cm) equilibrated with buffer D. Under these conditions most of the enzyme does not bind to the column, but most of the contaminating proteins do.
Additional Remarks In order to obtain pure acid sphingomyelinase protein, the enzyme (post-DEAE-cellulose) should be concentrated and separated from the detergent Nonidet P-40 by ethanol precipitation, alkylated with iodoacetamide, 9 and purified on a hydrophobic HPLC column (e.g., HIBAR Lichrospher, 500 CH-2/10/xm) with a linear gradient of acetonitrile (0-70%, v/v) in 0.05% trifluoroacetic acid. This procedure gives denatured but pure enzyme protein (e.g., for immunochemical experiments). Properties The purified glycosylated enzyme has an apparent molecular mass of 72 kDa determined with SDS-gel electrophoresis, which was reduced to 61 kDa after deglycosylation. It has an isoelectric point (p/) of 7.5, and the optimum for enzyme activity occurs in the range pH 4.5-5.0. Enzyme activity can be stimulated with Nonidet P-40, various cholates, or by the addition of certain 1/pids to a detergent-free assay system. 10Under some conditions the freshly prepared enzyme hydrolyzes phosphatidylcholine and phosphatidylglycerol with phospholipase C-like activity) ° The en9 j. Hempel, H. v. Bahr-Lindstr0m, and H. J6rnvall, Eur. J. Biochem. 141, 21 0984). l0 L. E. Quintern, G. Weffz, H. Nehrkorn, J. M. Tager, A. W. Schram, and K. Sandhoff, Biochim. Biophys. Acta 922, 323 (1987).
540
PHOSPHOLIPASEC
[53]
zyme has an apparent K m value of around 60/zM toward the three lipids sphingomyelin, phosphatidylcholine, and phosphatidylglycerol in the presence of Nonidet P-40, sodium taurodeoxycholate, and Nonidet P-40, respectively. Reducing agents (e.g., dithiothreitol) inactivate the enzyme. Divalent cations (Ca 2÷ , Co 2÷ , Mg 2÷ , Mn 2÷) or the cation-chelating agent EDTA up to a concentration of 20 mM have no effect on enzyme activity. AMP (Ki 45-74/,M), 9-/3-D-arabinofuranosyladenine 5'-monophosphate (Ki - 4 / z M ) and phosphatidylinositol 4',5'-bisphosphate (Ki - 1 / z M ) are potent inhibitors of acid sphingomyelinase. In the presence of 0.1% Nonidet P-40 and greater than 100 mM NaCI the enzyme can be stored for several weeks with only a small loss in total hydrolytic activity. Additional Remarks
The cDNA of the enzyme has been cloned. 11The occurrence of alternatively processed transcripts indicates the presence of isoenzymes of this enzyme. Acknowledgments We are grateful to Roger Klein for proofreading the manuscript and the Deutsche Forschungsgemeinschaft for financial support (Grant Sa 257/12-1). tl L. E. Quintern, E. H. Schuchmann, O. Levran, M. Suchi, K. Ferlinz, H. Reinke, K. Sandhoff, and R. J. Desnick, EMBO J. 8, 2469 (1989).
[53] P u r i f i c a t i o n o f N e u t r a l S p h i n g o m y e l i n a s e f r o m Human Urine B y SUBROTO CHATTERJEE a n d N U P U R GHOSH
Introduction Sphingomyelinase (EC 3.1.4.12) catalyzes the hydrolytic cleavage of sphingomyelin via reaction (1)) It has been shown that sphingomyelin may be cleaved at both acid pH optima and neutral pH optima. 2-4 The lack t R. O. Brady, J. N. Kanfer, M. B. Mock, and D. S. Fredrickson, Proc. Natl. Acad. Sci. U.S.A. 55, 366 (1966). 2 p. Ghosh and S. Chatterjee, J. Biol. Chem. 262, 12550 (1987). 3 S. Gatt, Biochem. Biophys. Res. Commun. 68, 235 (1976). 4 B. G. Rao and M. W. Spence, J. Lipid Res. 17, 506 (1976).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc, All fightsof reproductionin any form reserved,
