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,
[53]
NEUTRAL SPHINGOMYELINASE FROM HUMAN URINE
541
Sphingomyelin---, ceramide + phosphocholine
(I)
or deficiency of the acid sphingomyelinase (a lysosomal enzyme) leading to the storage of sphingomyelin in Niemann-Pick disease is well established. Indirect evidence suggests that a sphingomyelinase (having a neutral pH optima) may be involved in catabolizing cell surface sphingomyelin: Ceramide released from such a reaction is speculated to be involved in the generation of sphingosine (via the action of ceramidase), and the sphingosine may be subsequently involved in the cascade of reactions leading to the regulation of protein kinase activity. 6'7 We have shown that decreased activity of neutral sphingomyelinase (N-SMase) and acid sphingomyelinase in oitro in cultured human proximal tubular cells incubated with gentamicin and in vivo in patients receiving gentamicin, leads to the accumulation of sphingomyelin.8"9
Assay Method The activity of neutral sphingomyelinase is measured, using [14C]sphingomyelin as the substrate, by a modification of the procedure of Brady et al. ~ The water-soluble product phosphocholine is separated from [~"C] sphingomyelin following trichloroacetic acid (TCA) precipitation of the mixture and extraction with ether. Reagents
[~4C]Sphingomyelin, purchased from Amersham Searle (Arlington Heights, IL), (specific activity 57 mCi/mmol) 0.2 M Tris-glycine buffer (pH 7.4) Human serum albumin (fatty acid-free) MgCI2 Cutscum (a detergent available through Fisher Scientific Company, Pittsburgh, PA) 10% Trichloroacetic acid (TCA) Bovine serum albumin (BSA) Ether (anhydrous) Aquasol II (New England Nuclear, Boston, MA) s C. W. Slife, E. Wang, R. Hunter, S. Wang, C. Burgess, D. C. Liotta, and A. H. Merrill, Jr., J. Biol. Chem. 264, 10371 (1989). 6 y. A. Hannun, C. R. Loomis, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 261, 12604 (1986). 7 y . A. Hannun and R. M. Bell, Science 2, 500 (1989). 8 S. Chatterjee, J. Biochem. Toxicol. 2, 181 (1987). 9 S. Chatterjee and S. Bose, J. Biochem. Toxicol. 3, 47 (1988).
542
PHOSPHOLIPASEC
[53]
Unless otherwise mentioned all other chemicals described here are available from Sigma Chemical Company (St. Louis, MO). Procedure. The assay mixture for the measurement of neutral sphingomyelinase activity consists of the following: 25 nmol of Tris-glycine buffer, pH 7.4, 2.5 nmol of Mg 2+, 50 nmol of [~4C]sphingomyelin [20,000 disintegrations per minute (dpm)], 0.5 mg of human serum albumin, 50/xg of Cutscum, and 10-50/zg of enzyme protein. The final volume of the assay mixture is adjusted to 200/zl with water, and incubation is carried out for 1 hr at 37°. The assay is terminated with 1 ml of ice-cold TCA. The contents of the tubes are mixed, allowed to settle for 5 min at room temperature, and then centrifuged for 5 min at 2000 rpm in a swinging bucket (IEC, bench top centrifuge) at 10% One milliliter of the supernatant is carefully withdrawn using disposable plastic Eppendorf tips and transferred to a separate set of glass test tubes (13 × 100 mm). One milliliter of ether is added to the samples mixed, allowed to settle for 5 min on ice, and centrifuged as above. The aqueous layer (-800/xl, maximum) is withdrawn and transferred into glass scintillation vials. Such samples are dried overnight, or the radioactivity is measured directly after mixing with 10 ml of Aquasol II in a scintillation spectrometer (Beckman LS-3801). Product Identification. Following enzyme assay, the products are analyzed by thin-layer chromatography. The aqueous phase containing [~4C]phosphocholine is mixed with unlabeled phosphocholine and separated by thin-layer chromatography using methanol, 0.5% NaCI, and NH4OH (100 : 100 : 2, v/v). The gel area corresponding to phosphocholine is scraped and the radioactivity measured in a scintillation spectrometer. Neutral Sphingomyelinase Purification We have developed the followign steps to purify N-SMase from urine concentrates, mTo isolate N-SMase from tissues, m step I below is replaced by subcellular fractionation and isolation of plasma membranes 2 that serve as the starting material.
Reagents Sephadex G-75 Biolyte (Bio-Rad, Richmond, CA) CH-Sepharose Sodium acetate buffer: 25 mM NaCl, I mM EDTA, 20 mM sodium acetate, pH 6.5 10 S. Chatterjee and N. Ghosh, J. Biol. Chem. 2,64, 12554 (1989).
[53]
NEUTRAL SPHINGOMYELINASE FROM HUMAN URINE
543
Phenylmethylsulfonyl fluoride Sodium azide Glycine Sphingosylphosphocholine Phenyl-Sepharose CL-4B Step I: Preparation of Urine Concentrate. We collect about 2-3 liters of normal urine from male subjects daily. Male volunteers are preferred over females because of lack of contamination by blood cells, excessive number of uroepithelial cells, and urogenital secretions. For best results, protease and other inhibitors should be added to the container and to all the purification buffers, namely 10/zM phenylmethylsulfonyl fluoride (PMSF) and 0.001% sodium azide. Without such inhibitors, the enzyme is susceptible to extensive proteolytic cleavage. The urine sample is immediately centrifuged at 16,270 g (30 min at 4°) in a Sorvall RC-2B refrigerated centrifuge. The cell pellet, a transparent layer at the bottom, is carefully removed, the supernatant is concentrated in a Millipore Minitan ultrafiltration device (Bedford, MA), using a filter (PTGC-OMT-05) with a nominal cutoff of Mr 10,000. Next, the urine concentrate is further concentrated to 5-10 ml in an Amicon (Danvers MA) YM10 membrane filter with a nominal cutoff of M r 10,000. The filters are washed with 1% NaOH and water and reused up to several weeks. Fresh filters are used daily for ultrafiltration purposes. The urine concentrate is dialyzed against 2 liters of sodium acetate buffer (25 mM NaC1, I mM EDTA, 20 mM sodium acetate, pH 6.5) for 24 hr. Urine concentrate from at least 10 liters of urine is used for the isolation of N-SMase. Step H: Chromatography on Sephadex G-75. The urine concentrate is applied to a Sephadex G-75 column (2.5 × 34 cm) preequilibrated with the acetate buffer above and eluted. First, absorbance at 280 nm is measured in all the fractions. The positive samples are used for protein and enzyme activity determinations. For example, fractions 8-10 in Fig. 1 containing high N-SMase activity are pooled and concentrated by ultrafiltration. Step III: Preparative Isoelectric Focusing. The concentrate (30 mg protein) obtained following Sephadex G-75 column chromatography is dialyzed against glycine and placed in a Biolyte gel bed containing carrier ampholytes of pH range 4-10 using a sample applicator provided by the manufacturer (LKB Instrument Co.). Electrofocusing is carried out at constant power of 8 W for 14-16 hr. To visualize the focused zones a print is made of the gel on a nitrocellulose paper and stained with Coomassie blue. The focused zones are cut out, suspended in water, and the pH measured. Since the pI value of N-SMase is approximately 6.5,1° gel areas corresponding to this pH range (fractions 12-15 in Fig. 2) are transferred to a Pasteur pipette column and eluted with 0.2 M Tris-glycine buffer, pH
544
PnosPFIOLIPASE C
[53]
,2I
E
I o
2
E
.$=
I0 -~ 8
>,
c
O
£I_
z 0
c
2
4
6
8
I0
12
14
16
18
20
Fraction N u m b e r
FIG. 1. Sephadex G-75 chromatography of neutral sphingomyelinase from human urine. Approximately 246 mg of urine concentrate was applied to a Sephadex G-75 column (2.5 x 34 cm) preequilibrated with a buffer containing 20 mM sodium acetate, 25 mM NaC1, 1 mM EDTA (pH 6.5). The column was washed with the same buffer at a flow rate of 25 ml/hr, and 5-ml fractions were collected in a Pharmacia (Piscataway, NJ) fraction collector (FRAC100). All operations were carded out at 4°. The content of protein and neutral sphingomyelinase activity were measured in the fractions. [Reproduced from S. Chatterjee and N. Ghosh, J. Biol. Chem. 264, 12554 (1989).]
7.4. The sample is concentrated and the a m p h o l y t e s r e m o v e d on columns loaded with Phenyl-Sepharose CL-4B (1 x 4 cm) by sequential elution with 0.2 M T r i s - g l y c i n e buffer and 0.1-1 M phosphate. Samples are withdrawn for the m e a s u r e m e n t of protein and e n z y m e activity. Alternatively, we h a v e d e v e l o p e d a relatively simple method to pursue preparative isoelectric focusing of N - S M a s e using the Rotofor (Bio-Rad, Richmond, CA) equipment. The concentrate ( - 5 0 ml) obtained following ultrafiltration (step I I above) is mixed with ampholytes (2%, w/v), p H range 4-10, and subjected to isoelectric focusing at a constant p o w e r of 12 W with initial voltage of 400 V. The total time required is approximately 4 - 5 hr. The latter p r o c e d u r e of preparative isoelectric focusing has several advantages o v e r the Biolyte gel isoelectric focusing method. (1) The samples are in a liquid state; accordingly, the step involving elution of protein from the Biolyte gel bed is not required. (2) p H m e a s u r e m e n t s and ampho-
[53]
545
NEUTRAL SPHINGOMYELINASE FROM HUMAN URINE
8
2.0
20
10-
.6
-
g 06-
>,
E
t'M t.)
-t- 4 -
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,
[53]
NEUTRAL SPHINGOMYELINASE FROM HUMAN URINE
541
Sphingomyelin---, ceramide + phosphocholine
(I)
or deficiency of the acid sphingomyelinase (a lysosomal enzyme) leading to the storage of sphingomyelin in Niemann-Pick disease is well established. Indirect evidence suggests that a sphingomyelinase (having a neutral pH optima) may be involved in catabolizing cell surface sphingomyelin: Ceramide released from such a reaction is speculated to be involved in the generation of sphingosine (via the action of ceramidase), and the sphingosine may be subsequently involved in the cascade of reactions leading to the regulation of protein kinase activity. 6'7 We have shown that decreased activity of neutral sphingomyelinase (N-SMase) and acid sphingomyelinase in oitro in cultured human proximal tubular cells incubated with gentamicin and in vivo in patients receiving gentamicin, leads to the accumulation of sphingomyelin.8"9
Assay Method The activity of neutral sphingomyelinase is measured, using [14C]sphingomyelin as the substrate, by a modification of the procedure of Brady et al. ~ The water-soluble product phosphocholine is separated from [~"C] sphingomyelin following trichloroacetic acid (TCA) precipitation of the mixture and extraction with ether. Reagents
[~4C]Sphingomyelin, purchased from Amersham Searle (Arlington Heights, IL), (specific activity 57 mCi/mmol) 0.2 M Tris-glycine buffer (pH 7.4) Human serum albumin (fatty acid-free) MgCI2 Cutscum (a detergent available through Fisher Scientific Company, Pittsburgh, PA) 10% Trichloroacetic acid (TCA) Bovine serum albumin (BSA) Ether (anhydrous) Aquasol II (New England Nuclear, Boston, MA) s C. W. Slife, E. Wang, R. Hunter, S. Wang, C. Burgess, D. C. Liotta, and A. H. Merrill, Jr., J. Biol. Chem. 264, 10371 (1989). 6 y. A. Hannun, C. R. Loomis, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 261, 12604 (1986). 7 y . A. Hannun and R. M. Bell, Science 2, 500 (1989). 8 S. Chatterjee, J. Biochem. Toxicol. 2, 181 (1987). 9 S. Chatterjee and S. Bose, J. Biochem. Toxicol. 3, 47 (1988).
542
PHOSPHOLIPASEC
[53]
Unless otherwise mentioned all other chemicals described here are available from Sigma Chemical Company (St. Louis, MO). Procedure. The assay mixture for the measurement of neutral sphingomyelinase activity consists of the following: 25 nmol of Tris-glycine buffer, pH 7.4, 2.5 nmol of Mg 2+, 50 nmol of [~4C]sphingomyelin [20,000 disintegrations per minute (dpm)], 0.5 mg of human serum albumin, 50/xg of Cutscum, and 10-50/zg of enzyme protein. The final volume of the assay mixture is adjusted to 200/zl with water, and incubation is carried out for 1 hr at 37°. The assay is terminated with 1 ml of ice-cold TCA. The contents of the tubes are mixed, allowed to settle for 5 min at room temperature, and then centrifuged for 5 min at 2000 rpm in a swinging bucket (IEC, bench top centrifuge) at 10% One milliliter of the supernatant is carefully withdrawn using disposable plastic Eppendorf tips and transferred to a separate set of glass test tubes (13 × 100 mm). One milliliter of ether is added to the samples mixed, allowed to settle for 5 min on ice, and centrifuged as above. The aqueous layer (-800/xl, maximum) is withdrawn and transferred into glass scintillation vials. Such samples are dried overnight, or the radioactivity is measured directly after mixing with 10 ml of Aquasol II in a scintillation spectrometer (Beckman LS-3801). Product Identification. Following enzyme assay, the products are analyzed by thin-layer chromatography. The aqueous phase containing [~4C]phosphocholine is mixed with unlabeled phosphocholine and separated by thin-layer chromatography using methanol, 0.5% NaCI, and NH4OH (100 : 100 : 2, v/v). The gel area corresponding to phosphocholine is scraped and the radioactivity measured in a scintillation spectrometer. Neutral Sphingomyelinase Purification We have developed the followign steps to purify N-SMase from urine concentrates, mTo isolate N-SMase from tissues, m step I below is replaced by subcellular fractionation and isolation of plasma membranes 2 that serve as the starting material.
Reagents Sephadex G-75 Biolyte (Bio-Rad, Richmond, CA) CH-Sepharose Sodium acetate buffer: 25 mM NaCl, I mM EDTA, 20 mM sodium acetate, pH 6.5 10 S. Chatterjee and N. Ghosh, J. Biol. Chem. 2,64, 12554 (1989).
[53]
NEUTRAL SPHINGOMYELINASE FROM HUMAN URINE
543
Phenylmethylsulfonyl fluoride Sodium azide Glycine Sphingosylphosphocholine Phenyl-Sepharose CL-4B Step I: Preparation of Urine Concentrate. We collect about 2-3 liters of normal urine from male subjects daily. Male volunteers are preferred over females because of lack of contamination by blood cells, excessive number of uroepithelial cells, and urogenital secretions. For best results, protease and other inhibitors should be added to the container and to all the purification buffers, namely 10/zM phenylmethylsulfonyl fluoride (PMSF) and 0.001% sodium azide. Without such inhibitors, the enzyme is susceptible to extensive proteolytic cleavage. The urine sample is immediately centrifuged at 16,270 g (30 min at 4°) in a Sorvall RC-2B refrigerated centrifuge. The cell pellet, a transparent layer at the bottom, is carefully removed, the supernatant is concentrated in a Millipore Minitan ultrafiltration device (Bedford, MA), using a filter (PTGC-OMT-05) with a nominal cutoff of Mr 10,000. Next, the urine concentrate is further concentrated to 5-10 ml in an Amicon (Danvers MA) YM10 membrane filter with a nominal cutoff of M r 10,000. The filters are washed with 1% NaOH and water and reused up to several weeks. Fresh filters are used daily for ultrafiltration purposes. The urine concentrate is dialyzed against 2 liters of sodium acetate buffer (25 mM NaC1, I mM EDTA, 20 mM sodium acetate, pH 6.5) for 24 hr. Urine concentrate from at least 10 liters of urine is used for the isolation of N-SMase. Step H: Chromatography on Sephadex G-75. The urine concentrate is applied to a Sephadex G-75 column (2.5 × 34 cm) preequilibrated with the acetate buffer above and eluted. First, absorbance at 280 nm is measured in all the fractions. The positive samples are used for protein and enzyme activity determinations. For example, fractions 8-10 in Fig. 1 containing high N-SMase activity are pooled and concentrated by ultrafiltration. Step III: Preparative Isoelectric Focusing. The concentrate (30 mg protein) obtained following Sephadex G-75 column chromatography is dialyzed against glycine and placed in a Biolyte gel bed containing carrier ampholytes of pH range 4-10 using a sample applicator provided by the manufacturer (LKB Instrument Co.). Electrofocusing is carried out at constant power of 8 W for 14-16 hr. To visualize the focused zones a print is made of the gel on a nitrocellulose paper and stained with Coomassie blue. The focused zones are cut out, suspended in water, and the pH measured. Since the pI value of N-SMase is approximately 6.5,1° gel areas corresponding to this pH range (fractions 12-15 in Fig. 2) are transferred to a Pasteur pipette column and eluted with 0.2 M Tris-glycine buffer, pH
544
PnosPFIOLIPASE C
[53]
,2I
E
I o
2
E
.$=
I0 -~ 8
>,
c
O
£I_
z 0
c
2
4
6
8
I0
12
14
16
18
20
Fraction N u m b e r
FIG. 1. Sephadex G-75 chromatography of neutral sphingomyelinase from human urine. Approximately 246 mg of urine concentrate was applied to a Sephadex G-75 column (2.5 x 34 cm) preequilibrated with a buffer containing 20 mM sodium acetate, 25 mM NaC1, 1 mM EDTA (pH 6.5). The column was washed with the same buffer at a flow rate of 25 ml/hr, and 5-ml fractions were collected in a Pharmacia (Piscataway, NJ) fraction collector (FRAC100). All operations were carded out at 4°. The content of protein and neutral sphingomyelinase activity were measured in the fractions. [Reproduced from S. Chatterjee and N. Ghosh, J. Biol. Chem. 264, 12554 (1989).]
7.4. The sample is concentrated and the a m p h o l y t e s r e m o v e d on columns loaded with Phenyl-Sepharose CL-4B (1 x 4 cm) by sequential elution with 0.2 M T r i s - g l y c i n e buffer and 0.1-1 M phosphate. Samples are withdrawn for the m e a s u r e m e n t of protein and e n z y m e activity. Alternatively, we h a v e d e v e l o p e d a relatively simple method to pursue preparative isoelectric focusing of N - S M a s e using the Rotofor (Bio-Rad, Richmond, CA) equipment. The concentrate ( - 5 0 ml) obtained following ultrafiltration (step I I above) is mixed with ampholytes (2%, w/v), p H range 4-10, and subjected to isoelectric focusing at a constant p o w e r of 12 W with initial voltage of 400 V. The total time required is approximately 4 - 5 hr. The latter p r o c e d u r e of preparative isoelectric focusing has several advantages o v e r the Biolyte gel isoelectric focusing method. (1) The samples are in a liquid state; accordingly, the step involving elution of protein from the Biolyte gel bed is not required. (2) p H m e a s u r e m e n t s and ampho-
[53]
545
NEUTRAL SPHINGOMYELINASE FROM HUMAN URINE
8
2.0
20
10-
.6
-
g 06-
>,
E
t'M t.)
-t- 4 -
