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Purification of β-glucosidase activities from bovine spleen by affinity chromatography
ANALYTICAL
60, %)0-%5 (1974)
BIOCHEMISTRY
Purification
of ,@-Glucosidase Spleen
JULIAN
by Affinity
N. KANFER, RICHARD RAGHAVAN, AND
Activities
from
Bovine
Chromatography A. MUMFORD, JAMES BYRD
Eunice Kennedy Shiver Center, Walter E. 02164 and Neurology Research, Massachusetts
Fernald General
SRINIVASA
State School, Waltham, Hospital, Boston, MA
S. MA OZll4
Received December 10, 1973; accepted January 28, 1974 Bovine spleen p-n-glucosidase, glucosylceramide : ,6-n-glucosidase and glucosylsphingosine: p-n-giueosidase were purified by chromatography on a “gluconate” Sepharose column. Ten other lysosomal acid hydrolases, present in the preparation applied to the column, were absent from the glucosidase fraction.
Enzymes have been purified from bovine brain (1) as well as from both human (2) and beef spleen (3) as well as human placental tissue (4) which catalyze the hydrolysis of the &glucosidic linkages present in glucosylceramide and 4MU-P-glucoside. These preparations have usually been obtained as a result of a series of classical steps including salt fractionation, ion-exchange, and gel exclusion chromatography. The technique of affinity chromatography is useful as a mild and semispecific procedure for enzyme purification, however, there have been only a few reports employing this method in the purification of glycosidases. Bacterial P-galactosidase has been obtained from thiophenyl-/3-n-galactoside Sepharose (5), neuraminidase from oxamic acid Sepharose (6), bovine P-glucuronidase from saccaronalactone Sepharose (7)) plasma a-galactosidases on aminophenylmellibiose Sepharose (8)) plant a- and /3-galactosidase as well
as hexosaminidase
on
“lactone”
Sepharoses
(9),
mammalian
/3-
hexosaminidase on a galactosamine-containing glycopeptide Sepharose (lo), ceramide-galactosyl-3-sulfate: sulfatase from psychosine sulfateSepharose (11). This communication describes the purification of a fraction containing the following three activities of glucosylceramide: ,@-D-glucosidase, glucosylsphingosine: p-n-glucosidase, and a P-n-glucosidase active toward a 4-methylumbelliferyl derivative on a “gluconate-Sepharose” affinity chromatographic column. 206 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
p-GLUCOSIDASE
201
mrmIcATI0~ METHODS
The gluconate column was prepared according to a published procedure (9)) except that 6-n-gluconolactone (Pfanstiehl Co., Waukegan, Illinois) was employed instead of n-galactono-&lactone. Bovine spleen was obtained at the local abattoir and stored at -20°C prior to use at which time 20 g of tissue was homogenized with 100 ml of 000-
700.
600
60
70
80
90
FRACTION FIG. 1. Purification of glucosylceramide : P-n-glucosidase, glucosylsphingosine P-Dglucosidase and /3-n-glucosidase by affinity chromatography on “gluconate’‘-Sepharose. Fractions l-46 were obtained with 0.1 M citrate pH 4.5 while 46-90 were obtained with 0.1 M citrate pH 4.5 containing 1% Triton X-100. Open triangles = N-acetyl-fln-glucosaminidase ; closed squares = .a-n-galactosidase; closed circles = cu-n-mannosidase; crosses = acid phosphatase; open circles = a-n-glucosidase; Xs = p-n-glucosidase ; open squares = glucosylsphingosine /3-o-glucosidase ; closed triangles = glucosylsphingosine : /3-D-glucosidase.
202
KANFER
ET
AL.
a solution of .Ol M monobasic sodium phosphate, 0.2% cutscum, and 1% sodium deoxycholate (4). The homogenate was centrifuged at 3000g for 30 min and a 5-ml aliquot of the supernatant containing 130 mg protein was routinely applied to a “gluconate-Sepharose” column (1 cm X 20 cm) which had been previously equilibrated with a 0.1 M citrate buffer pH 4.75 solution at 3°C. The column was initially eluted with 300 ml of 0.1 M citrate buffer pH 4.75 in 6-ml fractions, and was continued with a buffer containing 0.1 M citrate-l% Triton X-100 pH 4.75. A flow rate of l-2 ml/min was achieved employing a modest positive pressure. Aliquots of each fraction were assayed for several acid hydrolases, these included glucosylceramide : /3-D-glucoside (4)) glucosylsphingosine : p-n-glucoside (12) as previously described and various artificial substrates according to published methods (13). Protein was determined according to Itzhaki et al. with serum albumin as the standard (14). RESULTS
A typical elution profile obtained demonstrating the separation of the three p-n-glucosidase activities from several other lysosomal acid hydrolases is shown in Fig. 1. It is apparent that /3-D-N-acetylglucosaminidase, ac-n-galactosidase, 8cY-D-mannosidase, acid phosphatase, and a-Dglucosidase were not retained by this column and were recovered in the first eluant as was the bulk of the protein applied to the column. The secSeparation
TABLE 1 of Glucosidase Activities from wit,h a “Gluconate’‘-Senharose
Enzyme @-o-N-acetylgluoosaminidase (A-A a-n-galactosidase (em) B-n-galactosidase a-n-mannosidsae (o-0 ) Acid phosphatase (+-+) p-n-glucuronidaae &Lfucosidase a-n-fucosidase a-n-xylosidase a-n-glucosidase (O0 ) j3-o-glucosidase ( X-X ) Glucosylceramide: ,9-n-glucosidase Glucosylsphingosine: @-n-glucosidase
Other Acid Column
Applied )
(w-0) (A-A)
6978 524 777 1893 924 1329 33 1338 262 517 1750 2420 78
to columnD f + f + + + f * If: f * + zk
740 6 67 30 42 67 9 74 18 29 80 95 50
Hydrolases
Recovered in the second eluateb
39
3586 3427 110
tr 0 0 f 0 0 0 0 0 0 k + *
4
200 210 40
Valuesa expressed as total activity in nanomoles substrate hydrolyzed applied to the affinity chromatographic column. Value@ expressed as total activity recovered in the Triton X-100 eluent. All values represent the mean of two experiments Z!I SD; tr = trace.
203
P-GLUCOSIDASE PURIFICATION TABLE 2 Specific Activity of p-Glucosidase, Glucosylceramide: p-Glucosidase and Glucosylsphingosine: p-Glucosidase Applied and Recovered from the “Gluconate” Columna
fi-o-glucosidase+ Glucosylceramide: &glucosidase++ Glucosylsphingosine: j3-D-glucosidase+++
Applied
Recovered
13.4 1X.53 0.29
7172 6854 220
Values* expressed as nmoles hydrolyzed/mg protein/hr with + 4MU-#3-n-glucoaide as substrate or + + glucosyl-I-*% ceramide as subst.rate or + + + glucosyl-PC sphingosine as substrate.
ond eluant, containing the Triton X-100, effectively removes the glucosylceramide: /3-D-glucosidase, glucosylsphingosine: P-n-glucosidase and p-D-glucosidase activities. These results are further documented in Table 1. It is apparent that, except for a trace of a-mannosidase activity, ten acid hydrolases which were present in the original extract applied to the column were absent from the p-glucosidase containing fractions. The recovery of the three glucosidases in excess of 100% has been previously observed (8) and may reflect the removal of inhibiting substances. The reported specific activit,ies of the glucosidases as presented in Table 2 can only be regarded as minimal and as approximate values since the amount of protein recovered was below the limit of detection (15-25 pg) in this particular assay procedure. Therefore, the calculated values are based upon the lower limits of the specific analytic method. The pH optimum for all three glucosidase activities was 4.5, as shown in Fig. 2. DISCUSSION
The ability of lactones to inhibit the corresponding acid hydrolase was the principal employed for the enzyme purification by affinity chromatography as reported in these studies. This usefulness was previously demonstrated for galactosidase purification on Sepharose-4B-“galactonate” geIs (9). The general applicability is further supported by the 550-fold purification of /3-glucosidase, the 377-fold purification of glucosylceramide: P-Dglucosidase and the 758-fold purification of glucosylsphingosine: P-Dglucosidase on the Sepharose-4B-“gluconate” gel. At least ten other hydrolytic enzyme activities originally applied to the column were absent from these glucosidases. The galactonate columns previously described were unable to separate Ficin-a-galactosidase and *Jack bean meal pgalactoside (9). In the current, studies, the n-glucosidase a&vi@ present in the original spleen extract did not appear in the Triton X-100 eluatc, resulting in complete separation of (Y- and /?-n-glucosidases. This labo-
204
KANFER
3.5
4.0
4.5
ET
5.0
AL.
5.5
6.0
6.5
3
PH FIG. 2. Effect of varying pH upon the hydrolysia of ,8-n-glucosidase (Xs), glucosylceramide : /3-n-glucoside (open squares) and glucosylsphingosine ,8-n- glucoside (closed triangles).
ratory has recently demonstrated decreased glucosylsphingosine hydrolysis in Gaucher’s tissue and skin fibroblasts (12). This suggested that the genetic modification which results in the enzymatic defect characteristic of this disease reduces detectable glucosylceramide: ,8-n-glucosidase (15), glucosylsphingosine: ,8-D-glucosidase (12) and p-n-glucosidase (16). The ability to purify and prepare these ,B-glucosidase activities from mammalian tissues by affinity chromatography should facilitate the understanding of the basic enzymatic defect. ACKNOWLEDGMENTS Supported by Grants from the USPHS HD 05515 and NS 10330 and Maternal Child Health Project 906.
and
P-GLUCOSIDASE
~ummmox
205
REFERENCES 1. GATT, S. (1969) in Methods in Enzymology (Colowick, S., and Kaplan, N. 0.. eds.), Vol. 14, pp. 152, Academic Press, New York and London. 2. BRADY, R. O., AND KANFER, J. N. (1969) in Methods in Enzymology (Colowick, S., and Kaplan, N. O., eds.), Vol. 14, pp. 591, Academic Press, New York and London. 3. WEINREB, N. J., AND BRADY, R. 0. (1972) in Methods in Enzymology (Colowick, S., and Kaplan, N. O., eds.), Vol. 28, pp. 830, Academic Press, New York and London. 4. PENTCHEV, R. G., BRADY, R. O., HIBBERT, S. R., GAL, A. E., SHAPIRO, D., MOOR, G., AND CAMBIER, H. (1973) J. Biol. Ckem. 248,5256. 5. STEERS, E., CUATRECASAS, P., AND POLLARD, H. (1971) J. Biol. Chem. 246, 196. 6. CUATRECASAS, P., AND ILLIANO, G. (1971) Biochem. Biophys. Res. Commun. 44, 178. 7. HARRIS, R. B., ROWE, J. J. M., STEWART, P. S., AND WILLIAMS, D. C. (1973) FEBB Lett. 29, 189. 8. MAPES. C. A.. AND SWEELEY, C. C. (1973) J. Biol. Chem. 248, 2461. 9. KANFER, J. N., PETROVICH, G., AND MUMFORD, R. A. (1973) Anal. Biochem. 55, 301. 10. DAWSON, G., PROPPER, R. L., AND DORFMAN. A. (1973) Biochem. Biophys. Res. Commun. 54, 1102. 11. BRESMV, J. L., AND SLOANE, H. R. (1972) Biochem. Biophys. Res. Commun. 46, 919. 12. RAGHAVAN, S., MUMFORD, R. A., AND KANFER, J. 1”IJ. (1973) Biochem. Biophys. Res. Commun. 54, 256. 13. RACHAVAN, S.. RHOADS, D. B., AND KANFER. J. N. (1972) Biochim. Biophys. Acta 268, 755. 14. ITZHAHI, R. F., AND GILL, D. M. (1964) Anal. Biochem. 9,401. 15. BRADY, R. O., KANFER, J. N., AND SHAPIRO? D. (1965) Biochem. Biophys, Res. Commun. 18, 221. 16. PATRICK, A. D. (1965) Biochem. J. 97, 17~.
60, %)0-%5 (1974)
BIOCHEMISTRY
Purification
of ,@-Glucosidase Spleen
JULIAN
by Affinity
N. KANFER, RICHARD RAGHAVAN, AND
Activities
from
Bovine
Chromatography A. MUMFORD, JAMES BYRD
Eunice Kennedy Shiver Center, Walter E. 02164 and Neurology Research, Massachusetts
Fernald General
SRINIVASA
State School, Waltham, Hospital, Boston, MA
S. MA OZll4
Received December 10, 1973; accepted January 28, 1974 Bovine spleen p-n-glucosidase, glucosylceramide : ,6-n-glucosidase and glucosylsphingosine: p-n-giueosidase were purified by chromatography on a “gluconate” Sepharose column. Ten other lysosomal acid hydrolases, present in the preparation applied to the column, were absent from the glucosidase fraction.
Enzymes have been purified from bovine brain (1) as well as from both human (2) and beef spleen (3) as well as human placental tissue (4) which catalyze the hydrolysis of the &glucosidic linkages present in glucosylceramide and 4MU-P-glucoside. These preparations have usually been obtained as a result of a series of classical steps including salt fractionation, ion-exchange, and gel exclusion chromatography. The technique of affinity chromatography is useful as a mild and semispecific procedure for enzyme purification, however, there have been only a few reports employing this method in the purification of glycosidases. Bacterial P-galactosidase has been obtained from thiophenyl-/3-n-galactoside Sepharose (5), neuraminidase from oxamic acid Sepharose (6), bovine P-glucuronidase from saccaronalactone Sepharose (7)) plasma a-galactosidases on aminophenylmellibiose Sepharose (8)) plant a- and /3-galactosidase as well
as hexosaminidase
on
“lactone”
Sepharoses
(9),
mammalian
/3-
hexosaminidase on a galactosamine-containing glycopeptide Sepharose (lo), ceramide-galactosyl-3-sulfate: sulfatase from psychosine sulfateSepharose (11). This communication describes the purification of a fraction containing the following three activities of glucosylceramide: ,@-D-glucosidase, glucosylsphingosine: p-n-glucosidase, and a P-n-glucosidase active toward a 4-methylumbelliferyl derivative on a “gluconate-Sepharose” affinity chromatographic column. 206 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
p-GLUCOSIDASE
201
mrmIcATI0~ METHODS
The gluconate column was prepared according to a published procedure (9)) except that 6-n-gluconolactone (Pfanstiehl Co., Waukegan, Illinois) was employed instead of n-galactono-&lactone. Bovine spleen was obtained at the local abattoir and stored at -20°C prior to use at which time 20 g of tissue was homogenized with 100 ml of 000-
700.
600
60
70
80
90
FRACTION FIG. 1. Purification of glucosylceramide : P-n-glucosidase, glucosylsphingosine P-Dglucosidase and /3-n-glucosidase by affinity chromatography on “gluconate’‘-Sepharose. Fractions l-46 were obtained with 0.1 M citrate pH 4.5 while 46-90 were obtained with 0.1 M citrate pH 4.5 containing 1% Triton X-100. Open triangles = N-acetyl-fln-glucosaminidase ; closed squares = .a-n-galactosidase; closed circles = cu-n-mannosidase; crosses = acid phosphatase; open circles = a-n-glucosidase; Xs = p-n-glucosidase ; open squares = glucosylsphingosine /3-o-glucosidase ; closed triangles = glucosylsphingosine : /3-D-glucosidase.
202
KANFER
ET
AL.
a solution of .Ol M monobasic sodium phosphate, 0.2% cutscum, and 1% sodium deoxycholate (4). The homogenate was centrifuged at 3000g for 30 min and a 5-ml aliquot of the supernatant containing 130 mg protein was routinely applied to a “gluconate-Sepharose” column (1 cm X 20 cm) which had been previously equilibrated with a 0.1 M citrate buffer pH 4.75 solution at 3°C. The column was initially eluted with 300 ml of 0.1 M citrate buffer pH 4.75 in 6-ml fractions, and was continued with a buffer containing 0.1 M citrate-l% Triton X-100 pH 4.75. A flow rate of l-2 ml/min was achieved employing a modest positive pressure. Aliquots of each fraction were assayed for several acid hydrolases, these included glucosylceramide : /3-D-glucoside (4)) glucosylsphingosine : p-n-glucoside (12) as previously described and various artificial substrates according to published methods (13). Protein was determined according to Itzhaki et al. with serum albumin as the standard (14). RESULTS
A typical elution profile obtained demonstrating the separation of the three p-n-glucosidase activities from several other lysosomal acid hydrolases is shown in Fig. 1. It is apparent that /3-D-N-acetylglucosaminidase, ac-n-galactosidase, 8cY-D-mannosidase, acid phosphatase, and a-Dglucosidase were not retained by this column and were recovered in the first eluant as was the bulk of the protein applied to the column. The secSeparation
TABLE 1 of Glucosidase Activities from wit,h a “Gluconate’‘-Senharose
Enzyme @-o-N-acetylgluoosaminidase (A-A a-n-galactosidase (em) B-n-galactosidase a-n-mannosidsae (o-0 ) Acid phosphatase (+-+) p-n-glucuronidaae &Lfucosidase a-n-fucosidase a-n-xylosidase a-n-glucosidase (O0 ) j3-o-glucosidase ( X-X ) Glucosylceramide: ,9-n-glucosidase Glucosylsphingosine: @-n-glucosidase
Other Acid Column
Applied )
(w-0) (A-A)
6978 524 777 1893 924 1329 33 1338 262 517 1750 2420 78
to columnD f + f + + + f * If: f * + zk
740 6 67 30 42 67 9 74 18 29 80 95 50
Hydrolases
Recovered in the second eluateb
39
3586 3427 110
tr 0 0 f 0 0 0 0 0 0 k + *
4
200 210 40
Valuesa expressed as total activity in nanomoles substrate hydrolyzed applied to the affinity chromatographic column. Value@ expressed as total activity recovered in the Triton X-100 eluent. All values represent the mean of two experiments Z!I SD; tr = trace.
203
P-GLUCOSIDASE PURIFICATION TABLE 2 Specific Activity of p-Glucosidase, Glucosylceramide: p-Glucosidase and Glucosylsphingosine: p-Glucosidase Applied and Recovered from the “Gluconate” Columna
fi-o-glucosidase+ Glucosylceramide: &glucosidase++ Glucosylsphingosine: j3-D-glucosidase+++
Applied
Recovered
13.4 1X.53 0.29
7172 6854 220
Values* expressed as nmoles hydrolyzed/mg protein/hr with + 4MU-#3-n-glucoaide as substrate or + + glucosyl-I-*% ceramide as subst.rate or + + + glucosyl-PC sphingosine as substrate.
ond eluant, containing the Triton X-100, effectively removes the glucosylceramide: /3-D-glucosidase, glucosylsphingosine: P-n-glucosidase and p-D-glucosidase activities. These results are further documented in Table 1. It is apparent that, except for a trace of a-mannosidase activity, ten acid hydrolases which were present in the original extract applied to the column were absent from the p-glucosidase containing fractions. The recovery of the three glucosidases in excess of 100% has been previously observed (8) and may reflect the removal of inhibiting substances. The reported specific activit,ies of the glucosidases as presented in Table 2 can only be regarded as minimal and as approximate values since the amount of protein recovered was below the limit of detection (15-25 pg) in this particular assay procedure. Therefore, the calculated values are based upon the lower limits of the specific analytic method. The pH optimum for all three glucosidase activities was 4.5, as shown in Fig. 2. DISCUSSION
The ability of lactones to inhibit the corresponding acid hydrolase was the principal employed for the enzyme purification by affinity chromatography as reported in these studies. This usefulness was previously demonstrated for galactosidase purification on Sepharose-4B-“galactonate” geIs (9). The general applicability is further supported by the 550-fold purification of /3-glucosidase, the 377-fold purification of glucosylceramide: P-Dglucosidase and the 758-fold purification of glucosylsphingosine: P-Dglucosidase on the Sepharose-4B-“gluconate” gel. At least ten other hydrolytic enzyme activities originally applied to the column were absent from these glucosidases. The galactonate columns previously described were unable to separate Ficin-a-galactosidase and *Jack bean meal pgalactoside (9). In the current, studies, the n-glucosidase a&vi@ present in the original spleen extract did not appear in the Triton X-100 eluatc, resulting in complete separation of (Y- and /?-n-glucosidases. This labo-
204
KANFER
3.5
4.0
4.5
ET
5.0
AL.
5.5
6.0
6.5
3
PH FIG. 2. Effect of varying pH upon the hydrolysia of ,8-n-glucosidase (Xs), glucosylceramide : /3-n-glucoside (open squares) and glucosylsphingosine ,8-n- glucoside (closed triangles).
ratory has recently demonstrated decreased glucosylsphingosine hydrolysis in Gaucher’s tissue and skin fibroblasts (12). This suggested that the genetic modification which results in the enzymatic defect characteristic of this disease reduces detectable glucosylceramide: ,8-n-glucosidase (15), glucosylsphingosine: ,8-D-glucosidase (12) and p-n-glucosidase (16). The ability to purify and prepare these ,B-glucosidase activities from mammalian tissues by affinity chromatography should facilitate the understanding of the basic enzymatic defect. ACKNOWLEDGMENTS Supported by Grants from the USPHS HD 05515 and NS 10330 and Maternal Child Health Project 906.
and
P-GLUCOSIDASE
~ummmox
205
REFERENCES 1. GATT, S. (1969) in Methods in Enzymology (Colowick, S., and Kaplan, N. 0.. eds.), Vol. 14, pp. 152, Academic Press, New York and London. 2. BRADY, R. O., AND KANFER, J. N. (1969) in Methods in Enzymology (Colowick, S., and Kaplan, N. O., eds.), Vol. 14, pp. 591, Academic Press, New York and London. 3. WEINREB, N. J., AND BRADY, R. 0. (1972) in Methods in Enzymology (Colowick, S., and Kaplan, N. O., eds.), Vol. 28, pp. 830, Academic Press, New York and London. 4. PENTCHEV, R. G., BRADY, R. O., HIBBERT, S. R., GAL, A. E., SHAPIRO, D., MOOR, G., AND CAMBIER, H. (1973) J. Biol. Ckem. 248,5256. 5. STEERS, E., CUATRECASAS, P., AND POLLARD, H. (1971) J. Biol. Chem. 246, 196. 6. CUATRECASAS, P., AND ILLIANO, G. (1971) Biochem. Biophys. Res. Commun. 44, 178. 7. HARRIS, R. B., ROWE, J. J. M., STEWART, P. S., AND WILLIAMS, D. C. (1973) FEBB Lett. 29, 189. 8. MAPES. C. A.. AND SWEELEY, C. C. (1973) J. Biol. Chem. 248, 2461. 9. KANFER, J. N., PETROVICH, G., AND MUMFORD, R. A. (1973) Anal. Biochem. 55, 301. 10. DAWSON, G., PROPPER, R. L., AND DORFMAN. A. (1973) Biochem. Biophys. Res. Commun. 54, 1102. 11. BRESMV, J. L., AND SLOANE, H. R. (1972) Biochem. Biophys. Res. Commun. 46, 919. 12. RAGHAVAN, S., MUMFORD, R. A., AND KANFER, J. 1”IJ. (1973) Biochem. Biophys. Res. Commun. 54, 256. 13. RACHAVAN, S.. RHOADS, D. B., AND KANFER. J. N. (1972) Biochim. Biophys. Acta 268, 755. 14. ITZHAHI, R. F., AND GILL, D. M. (1964) Anal. Biochem. 9,401. 15. BRADY, R. O., KANFER, J. N., AND SHAPIRO? D. (1965) Biochem. Biophys, Res. Commun. 18, 221. 16. PATRICK, A. D. (1965) Biochem. J. 97, 17~.