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[57] Ceramide trihexosidase from human placenta
[57]
CERAM|DE TRIHEXOSIDASE FROM HUMAN PLACEATA
533
[57] C e r a m i d e T r i h e x o s i d a s e f r o m H u m a n P l a c e n t a B y JOHN W. KUSIAK, JANE M. QUIRK, and Roscoe O. BRADY
A number of glycolytic enzymes are present in human tissues that are capable of hydrolyzing artificial substrates containing a-galactosidic linkages. One of these enzymes, in addition, has the ability to rapidly cleave the terminal a-galactosidic bond of the neutral glycolipid ceramide trihexoside (galactosylgalactosylglucosylceramide, CTH). This enzymic activity is lacking in patients with Fabry's disease, an X-linked recessive disorder in humans, and causes the deposition of CTH in kidney and peripheral blood vessels of afflicted individuals. Assay Methods Principle. Ceramidetrihexosidase activity can be quantified by measuring the release of [3H]galactose from ceramidetrihexoside specifically labeled at the C-6 portion of the terminal galactose by the galactose oxidase-sodium borohydride procedure, a-Galactosidase activity can be quantified by measuring the fluorescence of 4-methylumbelliferone released from its nonfluorescent a-D-galactopyranoside. Ceramidetrihexosidase. CTH was purified from Fabry kidney by the method of Esselman et al. ~ Specific tritium labeling of the CTH in the 6-position of the terminal galactose was carried out by the method of Radin et al. 2 The labeled CTH was diluted with unlabeled CTH to a specific activity of 1000 cpm/nmol for enzymic assays. Substrate (30 nmol) in chloroform : methanol, 2 : 1, was dried under a stream of nitrogen gas. To this was added 600 /~g of sodium taurocholate (Nutritional Biochemicals Corporation), 0.1 ml of 0.1 M acetate buffer, pH 4.1, an appropriately diluted aliquot of enzyme (1-10/xg of protein), and distilled water to a total volume of 0.2 ml. Incubations were carried out at 37° for 15-30 min. The reaction was terminated by the addition of 2.5 ml of chloroform : methanol (2 : 1), 0.1 ml of a solution of D-galactose (1 mg/ml), and 0.2 ml of H20. After mixing, the phases were separated by centrifugation. The aqueous upper phase was partitioned several times against chloroform; the aqueous solution was then transferred to a scintillation vial and evaporated to dryness. The residue was dissolved in a solution containing 7 g of PPO, 0.6 g of dimethyl-POPOP, 50 ml of Bio-Solv BBS-3 in 1 liter of toluene and counted in a Beckman LS 250 liquid scintillation
1w. J. Esselman,R. A. Laine, and C. C. Sweeley,this series Vol. 28 [8]. 2 N. S. Radin, L. Hof, R. M. Bradley,and R. O. Brady,Brain Res. 14, 497 (1969).
534
DEGRADAT|ON
[57]
system. Radioactivity in the incubations was 2-200 times the boiled and zero time enzyme blanks which were approximately 100 cpm. 4-Methylumbelliferyl-a-galactosidase. The fluorometric assay contained 0.05 ml of 5 mM 4-methylumbelliferyl-o~-o-galactopyranoside (4 MU-agal, Research Products International) in 0.15 M citrate-phosphate buffer, pH 4.4, and 0.01 ml of an appropriately diluted enzyme solution. Incubations were carried out for periods of up to 20 min. The reaction was terminated by the addition of l ml of 0.1 M glycine-sodium hydroxide buffer, pH 10.7. The fluorescence of the released 4-methylumbelliferone was quantitated on an Eppendorf fluorometer equipped with a primary filter transmitting at 366 nm and a secondary double-barrier filter transmitting at 430-470 nm. Protein values of pooled enzyme solutions were determined by the Lowry method, 3 and column fractions were measured at 280 nm. Purification The following purification scheme is based upon 10-15 kg of fresh human placenta obtained from local hospitals and kept on ice no longer than 24 hr before processing. All the subsequent procedures were carried out at 4 ° except where specifically noted. Placentas were washed free of adhering blood with distilled water, and fibrous tissue was dissected away. They were then minced in a meat grinder and portions (1 kg) were homogenized in a Waring Blendor (2× 1-min runs) in 2 liters of 25 mM phosphate buffer, pH 6.5. The homogenate was centrifuged at 16,000g for 30 min. The supernatant (20-30 liters) was then percolated over a column of concanavalin A-Sepharose (800 ml, Pharmacia) previously equilibrated with 25 mM phosphate buffer, pH 6.5. The loaded column was then washed with 3 liters of the same buffer and brought to room temperature (22°). The enzyme along with other glycoproteins was eluted from the column with 2 liters of buffer at 22 °, containing 1 M NaCl and 0.2 M a-methyl-D-mannopyranoside. The concanavalin A-Sepharose was washed with 4 liters of 0.1M acetate buffer, pH 6.0 containing 1 M NaC1, MnCI2, MgCI2, and CaC12, all 1 mM, and 0.01% Merthiolate and stored in this buffer until reuse. The enzyme solution (1.6 liters) was immediately cooled to 4 ° and concentrated on a Pellicon Ultrafiltration Cassette System (Millipore Corporation). The concentrate (400 ml) was dialyzed against four changes of 15 liters each of 25 mM phosphate buffer, pH 6.5. After centrifugation at 48,000 g for 30 min, the dialyzate was applied to an upward flowing column (7 liters, 10× 100 cm) of Cellex-D, (Bio-Rad) prea O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall,J. Biol. Chem. 193, 265 (1951).
[57]
CERAMIDE TRIHEXOSIDASE FROM HUMAN PLACEATA
535
viously equilibrated with 25 mM phosphate buffer, pH 6.5. Fractions containing 20 ml were collected at the rate of 150 ml/hr, and a linear salt-gradient was developed (0 to 1 M NaCl in phosphate buffer, total volume l0 liters) after the void volume (5 liters). Fractions containing ct-galactosidase activity were pooled, concentrated to 100 ml as above, and dialyzed against four changes of 15 liters each of l0 mM phosphate buffer, pH 6.5. The enzyme solution was titrated to pH 5.0 using dilute acetic acid, centrifuged to remove the precipitate, and applied to a column (1 liter, 5x60 cm) containing SP-Sephadex (Pharmacia) previously equilibrated with 35 mM acetate buffer pH 5.0. Eluent (15-ml fractions) was collected into tubes containing 1 ml of 0.4 M phosphate buffer, pH 6.5, and a linear gradient (0 to 1 M NaCl in acetate buffer, total volume 3 liters) was developed after the void volume. Two peaks of a-galactosidase activity were separately pooled. The a-galactosidase activity eluting in the void volume had low but detectable levels of ceramidetrihexosidase activity. The t~-galactosidase activity eluted in the salt gradient had high ceramidetrihexosidase activity and was further purified. Ceramidetrihexosidase-rich o~-galactosidase was applied to a column (1 liter, 5 x 60 cm) of Ultrogel AcA-34 (LKB) equilibrated with 5 mM EDTA, pH 6.5, containing 0.1 M NaC1. Fractions containing enzyme activity were pooled, concentrated on an Amicon ultrafiltration apparatus, dialyzed against 10 mM phosphate buffer at pH 6.5, and applied to a column (5 x 5 cm, 80 ml) of hydroxyapatite (Bio-Gel HT, Bio-Rad) equilibrated with the same buffer. A linear phosphate gradient (0.01 to 0.2 M phosphate, 250 ml total volume) was developed. Fractions containing a-galactosidase activity were pooled, concentrated, and dialyzed against 5 mM phosphate buffer, pH 6.5. The enzyme was finally applied to a column (10 ml, 1.3 x 14 cm) of Butyl Agarose (Miles Laboratories) equilibrated with the same buffer. A linear salt gradient was developed (0 to 0.2 M NaC1 in phosphate buffer, 200 ml total volume). The ct-galactosidase activity was pooled, concentrated, and stored at 4°. A summary of the purification is given in the table. Column fractions were routinely assayed using the artificial fluorogenic substrate once the identity of the ceramidetrihexosidase a-galactosidic activity had been determined using the natural lipid substrate. Both assay procedures are applicable to the relatively crude initial fractions and the final purified fractions although there is minor quenching of fluorescence by hemoglobin in the initial supernatant. Properties of the Purified Enzyme The ceramidetrihexosidase purified by this procedure is approximately 95% pure based upon disc gel electrophoretic patterns, and the
536
DEGRADATION
[57]
PURIFICATION SCHEME OF CERAMIDETRIHEXOSIDASE FROM HUMAN PLACENTA
Step Supernatant Concanavalin ASepharose Cellex D SP-Sephadexa AcA 34 Hydroxyapatite Butyl Agarose
Total protein (g) 900 10.8 1.2 0.23 0.04 0.012 0.003
4-Methylumbelliferyl-a-galactosidase activity (/xmol/min) 88.3 112.3 46.2 4.7 3.7 3.4 2.8
Specific activity (riM/ min/mg) 0.098 10.4 50.4 36.7 117.0 427.0 4020
Yield (%)
Pufffication (fold)
--
--
130 52 5 4 4 3
110 500 380 1200 4300 40,500
a At this step the major ct-galactosidase isozymes are separated. Enzyme eluting in the void volume not ceramidetrihexosidase, represented 83% of the recovered activity. yield is a p p r o x i m a t e l y 3%. There is slight contamination with a-glucosidase activity, which does not represent a minor activity associated with the a-galactosidase since the ratio of the activities changes during the purification. The e n z y m e has a molecular weight of 103,000 based upon gel filtration on S e p h a d e x G-200. The e n z y m e has a Km of 1.55 m M for the artificial substrate 4-methylumbelliferyl-a-Dgalactopyranoside but exhibits anomalous kinetic behavior with the natural substrate. Sodium taurocholate is n e c e s s a r y for activity with the natural substrate, and Triton X-100 is inhibitory. T h e ratio of artificial to natural activity o f the purified e n z y m e is 1 : 1. The other major i s o z y m e o f a-galactosidase has a ratio of 300: 1. The p H o p t i m u m for the artificial substrate is 4.4, and for the natural substrate 4.1. The isoelectric point of ceramidetrihexosidase is 4.7. The e n z y m e is a glycoprotein since it is stained positively with the periodic acid-Schiff p r o c e d u r e 4 and could be bound on concanavalin A - S e p h a r o s e columns. Its binding to anionic exchange columns is inhibited by neuraminidase treatment of the e n z y m e , suggesting that the c a r b o h y d r a t e chain contains sialic acid. The e n z y m e is heat labile, losing 90% o f its initial activity after 1 hr at 52 °. Comment Injection of purified h u m a n placental ceramidetrihexosidase has been shown to cause a decrease in the elevated quantity of ceramidetrihexoside 4 j. p. Segrest and R. L. Jackson, this series Vol. 28 [5].
[58]
ARYLSULFATASES A AND B
537
in the circulation of patients with Fabry's disease? Ceramidetrihexosidase prepared by the present procedure may provide for an evaluation of the clinical effects of enzyme replacement therapy in Fabry's disease. R. O. Brady, J. F. Tailman, W. G. Johnson, A. E. Gal, W. R. Leahy, J. M. Quirk, and A. S. Dekaban, N. Engl. J. Med. 289, 9 (1973).
[58] Arylsulfatases A and B f r o m H u m a n L i v e r By
A R V A N L . F L U H A R T Y a n d JOHN E D M O N D
Arylsulfatases A and B act physiologically as specific glycosulfatases. Arylsulfatase A acts on galactose 3-O-sulfate residues in cerebroside sulfate and certain other sulfolipids. 1 It also hydrolyzes ascorbic acid-2sulfate. 2 Arylsulfatase B acts on N-acetylgalactosamine 4-O-sulfate residues in chondroitin 4-sulfate, dermatan sulfate, and UDpoN-acetylgalactosamine 4-sulfate. 3 Assay Methods Synthetic Arylsulfates Synthetic substrate assays are convenient but may not reflectbiologically significantparameters. Oftcn thcy do not clearly differentiate between arylsulfatases A and B. Arylsulfatase A does show kinetic anomalies with nitrocatechol sulfate,the most commonly used synthetic substratc, and conditions havc been devised that allow arylsulfatascs A and B to be selectively assayed in samples of human origin? Methylumbcllifcrylsulfateprovidcs a more sensitive assay but does not diffcrcntiatc between sulfatascs.5
Differential Assays for Arylsulfatases A and B
Baum Type 4
Principle. Enzyme activity is measured by the hydrolysis of nitrocatechol sulfate to nitrocatechol, which is quantitated spect E. Mehl and H. Jatzkewitz, Biochim. Biophys. Acta 151,619 (1968). 2 A. B. Roy,Biochim. Biophys. Acta 377,356 (1975); A. L. Fluharty, R. L. Stevens, R. T. Miller, S. S. Shapiro, and H. Kihara, Biochim. Biophys. Acta 429, 508 (1976). 3 A. L. FLuharty, R. L. Stevens, D. Fung, S. Peak, and H. Kihara,Biochem. Biophys. Res. Commun. 64, 955 (1975). 4 H. Baum, K. S. Dodgson, and B. Spencer, Clin. Chim. Acta 4, 453 (1959). 5 B. C. Harinath and E. Robins, J. Neurochem. 18,237 (1971).
CERAM|DE TRIHEXOSIDASE FROM HUMAN PLACEATA
533
[57] C e r a m i d e T r i h e x o s i d a s e f r o m H u m a n P l a c e n t a B y JOHN W. KUSIAK, JANE M. QUIRK, and Roscoe O. BRADY
A number of glycolytic enzymes are present in human tissues that are capable of hydrolyzing artificial substrates containing a-galactosidic linkages. One of these enzymes, in addition, has the ability to rapidly cleave the terminal a-galactosidic bond of the neutral glycolipid ceramide trihexoside (galactosylgalactosylglucosylceramide, CTH). This enzymic activity is lacking in patients with Fabry's disease, an X-linked recessive disorder in humans, and causes the deposition of CTH in kidney and peripheral blood vessels of afflicted individuals. Assay Methods Principle. Ceramidetrihexosidase activity can be quantified by measuring the release of [3H]galactose from ceramidetrihexoside specifically labeled at the C-6 portion of the terminal galactose by the galactose oxidase-sodium borohydride procedure, a-Galactosidase activity can be quantified by measuring the fluorescence of 4-methylumbelliferone released from its nonfluorescent a-D-galactopyranoside. Ceramidetrihexosidase. CTH was purified from Fabry kidney by the method of Esselman et al. ~ Specific tritium labeling of the CTH in the 6-position of the terminal galactose was carried out by the method of Radin et al. 2 The labeled CTH was diluted with unlabeled CTH to a specific activity of 1000 cpm/nmol for enzymic assays. Substrate (30 nmol) in chloroform : methanol, 2 : 1, was dried under a stream of nitrogen gas. To this was added 600 /~g of sodium taurocholate (Nutritional Biochemicals Corporation), 0.1 ml of 0.1 M acetate buffer, pH 4.1, an appropriately diluted aliquot of enzyme (1-10/xg of protein), and distilled water to a total volume of 0.2 ml. Incubations were carried out at 37° for 15-30 min. The reaction was terminated by the addition of 2.5 ml of chloroform : methanol (2 : 1), 0.1 ml of a solution of D-galactose (1 mg/ml), and 0.2 ml of H20. After mixing, the phases were separated by centrifugation. The aqueous upper phase was partitioned several times against chloroform; the aqueous solution was then transferred to a scintillation vial and evaporated to dryness. The residue was dissolved in a solution containing 7 g of PPO, 0.6 g of dimethyl-POPOP, 50 ml of Bio-Solv BBS-3 in 1 liter of toluene and counted in a Beckman LS 250 liquid scintillation
1w. J. Esselman,R. A. Laine, and C. C. Sweeley,this series Vol. 28 [8]. 2 N. S. Radin, L. Hof, R. M. Bradley,and R. O. Brady,Brain Res. 14, 497 (1969).
534
DEGRADAT|ON
[57]
system. Radioactivity in the incubations was 2-200 times the boiled and zero time enzyme blanks which were approximately 100 cpm. 4-Methylumbelliferyl-a-galactosidase. The fluorometric assay contained 0.05 ml of 5 mM 4-methylumbelliferyl-o~-o-galactopyranoside (4 MU-agal, Research Products International) in 0.15 M citrate-phosphate buffer, pH 4.4, and 0.01 ml of an appropriately diluted enzyme solution. Incubations were carried out for periods of up to 20 min. The reaction was terminated by the addition of l ml of 0.1 M glycine-sodium hydroxide buffer, pH 10.7. The fluorescence of the released 4-methylumbelliferone was quantitated on an Eppendorf fluorometer equipped with a primary filter transmitting at 366 nm and a secondary double-barrier filter transmitting at 430-470 nm. Protein values of pooled enzyme solutions were determined by the Lowry method, 3 and column fractions were measured at 280 nm. Purification The following purification scheme is based upon 10-15 kg of fresh human placenta obtained from local hospitals and kept on ice no longer than 24 hr before processing. All the subsequent procedures were carried out at 4 ° except where specifically noted. Placentas were washed free of adhering blood with distilled water, and fibrous tissue was dissected away. They were then minced in a meat grinder and portions (1 kg) were homogenized in a Waring Blendor (2× 1-min runs) in 2 liters of 25 mM phosphate buffer, pH 6.5. The homogenate was centrifuged at 16,000g for 30 min. The supernatant (20-30 liters) was then percolated over a column of concanavalin A-Sepharose (800 ml, Pharmacia) previously equilibrated with 25 mM phosphate buffer, pH 6.5. The loaded column was then washed with 3 liters of the same buffer and brought to room temperature (22°). The enzyme along with other glycoproteins was eluted from the column with 2 liters of buffer at 22 °, containing 1 M NaCl and 0.2 M a-methyl-D-mannopyranoside. The concanavalin A-Sepharose was washed with 4 liters of 0.1M acetate buffer, pH 6.0 containing 1 M NaC1, MnCI2, MgCI2, and CaC12, all 1 mM, and 0.01% Merthiolate and stored in this buffer until reuse. The enzyme solution (1.6 liters) was immediately cooled to 4 ° and concentrated on a Pellicon Ultrafiltration Cassette System (Millipore Corporation). The concentrate (400 ml) was dialyzed against four changes of 15 liters each of 25 mM phosphate buffer, pH 6.5. After centrifugation at 48,000 g for 30 min, the dialyzate was applied to an upward flowing column (7 liters, 10× 100 cm) of Cellex-D, (Bio-Rad) prea O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall,J. Biol. Chem. 193, 265 (1951).
[57]
CERAMIDE TRIHEXOSIDASE FROM HUMAN PLACEATA
535
viously equilibrated with 25 mM phosphate buffer, pH 6.5. Fractions containing 20 ml were collected at the rate of 150 ml/hr, and a linear salt-gradient was developed (0 to 1 M NaCl in phosphate buffer, total volume l0 liters) after the void volume (5 liters). Fractions containing ct-galactosidase activity were pooled, concentrated to 100 ml as above, and dialyzed against four changes of 15 liters each of l0 mM phosphate buffer, pH 6.5. The enzyme solution was titrated to pH 5.0 using dilute acetic acid, centrifuged to remove the precipitate, and applied to a column (1 liter, 5x60 cm) containing SP-Sephadex (Pharmacia) previously equilibrated with 35 mM acetate buffer pH 5.0. Eluent (15-ml fractions) was collected into tubes containing 1 ml of 0.4 M phosphate buffer, pH 6.5, and a linear gradient (0 to 1 M NaCl in acetate buffer, total volume 3 liters) was developed after the void volume. Two peaks of a-galactosidase activity were separately pooled. The a-galactosidase activity eluting in the void volume had low but detectable levels of ceramidetrihexosidase activity. The t~-galactosidase activity eluted in the salt gradient had high ceramidetrihexosidase activity and was further purified. Ceramidetrihexosidase-rich o~-galactosidase was applied to a column (1 liter, 5 x 60 cm) of Ultrogel AcA-34 (LKB) equilibrated with 5 mM EDTA, pH 6.5, containing 0.1 M NaC1. Fractions containing enzyme activity were pooled, concentrated on an Amicon ultrafiltration apparatus, dialyzed against 10 mM phosphate buffer at pH 6.5, and applied to a column (5 x 5 cm, 80 ml) of hydroxyapatite (Bio-Gel HT, Bio-Rad) equilibrated with the same buffer. A linear phosphate gradient (0.01 to 0.2 M phosphate, 250 ml total volume) was developed. Fractions containing a-galactosidase activity were pooled, concentrated, and dialyzed against 5 mM phosphate buffer, pH 6.5. The enzyme was finally applied to a column (10 ml, 1.3 x 14 cm) of Butyl Agarose (Miles Laboratories) equilibrated with the same buffer. A linear salt gradient was developed (0 to 0.2 M NaC1 in phosphate buffer, 200 ml total volume). The ct-galactosidase activity was pooled, concentrated, and stored at 4°. A summary of the purification is given in the table. Column fractions were routinely assayed using the artificial fluorogenic substrate once the identity of the ceramidetrihexosidase a-galactosidic activity had been determined using the natural lipid substrate. Both assay procedures are applicable to the relatively crude initial fractions and the final purified fractions although there is minor quenching of fluorescence by hemoglobin in the initial supernatant. Properties of the Purified Enzyme The ceramidetrihexosidase purified by this procedure is approximately 95% pure based upon disc gel electrophoretic patterns, and the
536
DEGRADATION
[57]
PURIFICATION SCHEME OF CERAMIDETRIHEXOSIDASE FROM HUMAN PLACENTA
Step Supernatant Concanavalin ASepharose Cellex D SP-Sephadexa AcA 34 Hydroxyapatite Butyl Agarose
Total protein (g) 900 10.8 1.2 0.23 0.04 0.012 0.003
4-Methylumbelliferyl-a-galactosidase activity (/xmol/min) 88.3 112.3 46.2 4.7 3.7 3.4 2.8
Specific activity (riM/ min/mg) 0.098 10.4 50.4 36.7 117.0 427.0 4020
Yield (%)
Pufffication (fold)
--
--
130 52 5 4 4 3
110 500 380 1200 4300 40,500
a At this step the major ct-galactosidase isozymes are separated. Enzyme eluting in the void volume not ceramidetrihexosidase, represented 83% of the recovered activity. yield is a p p r o x i m a t e l y 3%. There is slight contamination with a-glucosidase activity, which does not represent a minor activity associated with the a-galactosidase since the ratio of the activities changes during the purification. The e n z y m e has a molecular weight of 103,000 based upon gel filtration on S e p h a d e x G-200. The e n z y m e has a Km of 1.55 m M for the artificial substrate 4-methylumbelliferyl-a-Dgalactopyranoside but exhibits anomalous kinetic behavior with the natural substrate. Sodium taurocholate is n e c e s s a r y for activity with the natural substrate, and Triton X-100 is inhibitory. T h e ratio of artificial to natural activity o f the purified e n z y m e is 1 : 1. The other major i s o z y m e o f a-galactosidase has a ratio of 300: 1. The p H o p t i m u m for the artificial substrate is 4.4, and for the natural substrate 4.1. The isoelectric point of ceramidetrihexosidase is 4.7. The e n z y m e is a glycoprotein since it is stained positively with the periodic acid-Schiff p r o c e d u r e 4 and could be bound on concanavalin A - S e p h a r o s e columns. Its binding to anionic exchange columns is inhibited by neuraminidase treatment of the e n z y m e , suggesting that the c a r b o h y d r a t e chain contains sialic acid. The e n z y m e is heat labile, losing 90% o f its initial activity after 1 hr at 52 °. Comment Injection of purified h u m a n placental ceramidetrihexosidase has been shown to cause a decrease in the elevated quantity of ceramidetrihexoside 4 j. p. Segrest and R. L. Jackson, this series Vol. 28 [5].
[58]
ARYLSULFATASES A AND B
537
in the circulation of patients with Fabry's disease? Ceramidetrihexosidase prepared by the present procedure may provide for an evaluation of the clinical effects of enzyme replacement therapy in Fabry's disease. R. O. Brady, J. F. Tailman, W. G. Johnson, A. E. Gal, W. R. Leahy, J. M. Quirk, and A. S. Dekaban, N. Engl. J. Med. 289, 9 (1973).
[58] Arylsulfatases A and B f r o m H u m a n L i v e r By
A R V A N L . F L U H A R T Y a n d JOHN E D M O N D
Arylsulfatases A and B act physiologically as specific glycosulfatases. Arylsulfatase A acts on galactose 3-O-sulfate residues in cerebroside sulfate and certain other sulfolipids. 1 It also hydrolyzes ascorbic acid-2sulfate. 2 Arylsulfatase B acts on N-acetylgalactosamine 4-O-sulfate residues in chondroitin 4-sulfate, dermatan sulfate, and UDpoN-acetylgalactosamine 4-sulfate. 3 Assay Methods Synthetic Arylsulfates Synthetic substrate assays are convenient but may not reflectbiologically significantparameters. Oftcn thcy do not clearly differentiate between arylsulfatases A and B. Arylsulfatase A does show kinetic anomalies with nitrocatechol sulfate,the most commonly used synthetic substratc, and conditions havc been devised that allow arylsulfatascs A and B to be selectively assayed in samples of human origin? Methylumbcllifcrylsulfateprovidcs a more sensitive assay but does not diffcrcntiatc between sulfatascs.5
Differential Assays for Arylsulfatases A and B
Baum Type 4
Principle. Enzyme activity is measured by the hydrolysis of nitrocatechol sulfate to nitrocatechol, which is quantitated spect E. Mehl and H. Jatzkewitz, Biochim. Biophys. Acta 151,619 (1968). 2 A. B. Roy,Biochim. Biophys. Acta 377,356 (1975); A. L. Fluharty, R. L. Stevens, R. T. Miller, S. S. Shapiro, and H. Kihara, Biochim. Biophys. Acta 429, 508 (1976). 3 A. L. FLuharty, R. L. Stevens, D. Fung, S. Peak, and H. Kihara,Biochem. Biophys. Res. Commun. 64, 955 (1975). 4 H. Baum, K. S. Dodgson, and B. Spencer, Clin. Chim. Acta 4, 453 (1959). 5 B. C. Harinath and E. Robins, J. Neurochem. 18,237 (1971).