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A micromethod for the detection of arylsulfatases A and B in cultured fibroblasts and amniocytes
ANALYTICAL
BIOCHEMISTRY
119, 360-364 (1982)
A Micromethod for the Detection Cultured Fibroblasts R. A. GRAVEL,*
A. LEUNG,*
of Arylsulfatases and Amniocytes
A and B in
AND E. H. KOLODNYt
F. TSUI,*
*The Hospital For Sick Children, Toronto, Ontario MSG 1X8. Canada: and TEunice Kennedy Shriver Center for Menial Retardation. Inc.. at the Walter E. Farnald State School, Waltham. Massachusetts 021.54 Received July 27, 1981 A micromethod for the determination of arylsulfatase activity in cultured fibroblasts or amniocytes is described. The method depends on the hydrolysis of methylumbelliferyl sulfate in an assay volume of 0.3 ~1 using extracts from as few as lo-20 cells. Arylsulfatase A and B activities are distinguished using the silver ion inhibition method of H. Christomanou and K. Sandhoff (1977) C/in. Chim. Acla 79, 527-531. The method is illustrated for the detection of metachromatic leukodystrophy resulting from a deficiency of arylsulfatase A activity.
Several disorders of arylsulfatase deficiency have been described in the human population. They include metachromatic leukodystrophy resulting from a deficiency of arylsulfatase A activity (1) MarotauxLamy syndrome with arylsulfatase B deficiency (2,3), and multiple sulfatase deficiency in which activities of both of these isoenzymes as well as other sulfatases are deficient (4). Routine methods for the assay of the arylsulfatases depend on the hydrolysis of a sulfate ester attached to a chromogenic or fluorogenic aromatic substituent. The most sensitive substrate currently available is methylumbelliferyl sulfate (MUS),’ the hydrolysis of which produces the intensely fluorescent methylumbelliferone (5,6). Recently Christomanou and Sandhoff (7) reported a method for distinguishing between arylsulfatase A and B activities on the basis of the differential inhibition of arylsulfatase B in the presence of silver ions. Hijsli (8) and Galjaard (9,lO) have established micromethods for the detection of sev’ Abbreviation fate.
used: MUS, methylumbelliferyl
0003-2697/82/020360-05502.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved
sul-
360
era1 lysosomal hydrolases using methylumbelliferyl substrates. These methods have made possible the detection of enzyme activity in extracts of as few as 5- 10 fibroblasts or amniocytes. These procedures have made possible prenatal diagnosis on limited numbers of cells ( 1 l- 14) and complementation experiments in isolated fibroblast heterokaryons (11,15,16). In this study we reported a method for the detection of arylsulfatases A and B by miniaturization of the methylumbelliferyl sulfate method with silver ion inhibition of arylsulfatase A (7) following microprocedures as developed by H&Ii (8). We have used this method for the assay of fibroblast and amniocyte cultures and have demonstrated its efficacy in the detection of arylsulfatase A deficiency in metachromatic leukodystrophy using both cell types. MATERIALS
AND METHODS
Fibroblast and amniocyte cultures were obtained from the cell repository at The Hospital for Sick Children, Toronto, and from P. Chang, McMaster University, Hamilton. Three fibroblast strains were used: strain 1025 (normal), and strains Bi and
MICROMETHOD
FOR
ARYLSULFATASE
2035 from patients diagnosed with metachromatic leukodystrophy and with demonstrated arylsulfatase A deficiency. One normal and one amniocyte culture from a confirmed diagnosis of metachromatic leukodystrophy at this hospital (Lowden, personal communication) were also included. Growth conditions and culture medium have been described previously ( 17). Chemicals were of reagent grade and were chosen to have the lowest concentration of chloride impurity available. Sodium acetate and human serum albumin were purchased from Sigma; aminomethylpropanediol and lead acetate were from Fisher; and silver nitrate was from J. T. Baker. Methylumbelliferyl sulfate (MUS), recrystallized from methanol four times ( 16), was from KochLight. A detailed description of the microtechnique methodology has been given by Hiisli (8). The nature of the plastic film dish, a modified petri dish with a polypropylene film as the growing surface (Tecnomara AG, Zurich), is illustrated in the same article. The preparation of the Parafilm microwells is also described. These are small cuvettes designed to hold 0.34 assay volumes. They are constructed by placing an indentation in Parafilm with a blunt needle using a Teflon template and are sealed airtight with a covering layer of Parafilm (8). Cells were prepared by plating approximately 2 X lo5 fibroblasts or 5 X IO5 amniocytes per plastic film dish followed by incubation overnight in medium at 37°C. The next day the plates were washed with phosphate-buffered saline and thoroughly drained by suction. (A final wash with unbuffered saline can be used to ensure complete removal of phosphate which interferes with the enzyme assay.) The last bit of liquid was removed with the dishes standing on edge of facilitate draining. The plates were frozen in liquid nitrogen and lyophilized overnight. It was essential to start the lyophilization with the dish filled with liquid nitrogen to prevent warming up of the plates before suf-
ACTIVITY
DETERMINATION
361
ficient vacuum was achieved. Following lyophilization the plates were removed and placed in an environmental chamber at 18°C and 30% relative humidity for isolating cells for assay. We hve found that it is best to have the lyophilizer inside the environmental chamber connected to a vacuum pump on the outside. Otherwise the plates should be placed in a desiccant to prevent “wetting” of the dried cells during transport to the chamber. Cells were recovered for assay by cutting small leaflets of 5-20 cells each from the plate using a scalpel under low-power microscopy (8). Such leaflets are typically 0.5-1.0 mm* in size. The leaflets were collected by placing them in a plastic petri dish or other suitable container with the cell side facing up. To detect total arylsulfatase activity the assay mix contained 35 mM sodium acetate buffer, pH 5.0, 10 mM MUS, 3 mM lead acetate, and 0.1% human serum albumin. Arylsulfatase B alone was detected by including 0.3 mM silver nitrate to inhibit arylsulfatase A activity. A 30 mM stock of lead acetate was maintained in 0.1 M acetate buffer to prevent precipitation on standing. A 20 mM stock of MUS was prepared in 0.05 M acetate buffer to prevent spontaneous hydrolysis of the substrate in more acidic solutions. Stock solutions of the remaining reagents were prepared in water. All reagents were prepared fresh just before use with chloride- and carbonate-free water purified by filtration through Super Q (Millipore). These precautions were essential to prevent precipitation of reagents during the assays. Assays were accomplished by pipetting 0.34 aliquots of assay mix into Parafilm microwells using a 0.54 syringe (Scientific Glass Engineering, Melbourne). A sharp needle was used to pick up individual plastic film leaflets and insert them into the assay mix. The complete procedure was done under low-power microscopy. Sets of 10 cuvettes were prepared at a time on a single strip of Parafilm. Each strip was covered
362
GRAVEL
with a second layer of Parafilm and sealed as described (8). The Parafilm strips were incubated for 30 min by immersing them in a 37°C water bath. At the end of incubation the covering strip was peeled off and the assay mix was removed by washing with 100 ~1 of diluent consisting of 0.1 M aminomethylpropanediol adjusted to pH 10.4 with NaOH and containing 10e3 M sodium ethylenediaminetetraacetate. Results were read on a Perkin-Elmer 650-40 fluorometer at 365 nm excitation and 448 nm emission using low-scatter quartz microcuvettes (No. 1O-0497).
ET AL.
0
10 Number
20 cS cells
FIG. 2. Dependence of arylsulfatase activity in normal fibroblasts on cell number. Enzyme activity was determined after 30 min incubation and is given as the mean + SD of seven replicate assays. Symbols are identical to those in Fig. 1.
RESULTS AND DISCUSSION
The dependence of MUS hydrolysis by fibroblasts on time and cell number was examined by micromethods. Using extracts of 20 cells per determination, the assay was approximately linear for 30 min and gave about equal activities of arylsulfatase A and B (Fig. 1). This compares favorably with the standard assay procedures which also are linear for a similar length of time. In addition the assay was approximately linear for
/ r
15
/ ______-
T
f-------
igv’/’ I/“\; 1
e -cl 2 IO-
2 =
i
5-
i
1
r
0
r
15
30 he
I
45
extracts of 10 or 20 cells (Fig. 2). These results give a specific activity of l-2 X 1O-‘3 mol MUS hydrolyzed/h/cell for total arylsulfatase activity, a result in keeping with the level of activity of several, low-activity lysosomal hydrolases previously analyzed by this approach (8). Figure 3 shows the results of the determination of arylsulfatase activity in cells from two patients with metachromatic leukodystrophy. In this case the total arylsulfatase activity and that due to arylsulfatase B alone gave identical results. Hence these results demonstrate a virtually complete absence of arylsulfatase A activity in this disorder in agreement with results produced by macromethods. An important application of the microas-
!
60
(Illmuter)
FIG. 1. Time course of arylsulfatase activity in normal fibroblasts. Enzyme activity was determined in extracts of 20 cells as described under Materials and Methods. Each determination represents the mean + SD of seven replicate assays. (0) Total arylsulfatase (A + B); (0) arylsulfatase B; (- - -) arylsulfatase A obtained by subtracting B from total activity.
j&?ezc! 0
10
20 0 Number of cells
10
20
FIG. 3. Arylsulfatase activity in fibroblasts of patients with metachromatic leukodystrophy. Method and symbols as given in Figs. 1 and 2. Graph at left for strain Bi and at right for strain 2035.
MICROMETHOD
FOR ARYLSULFATASE
20
10
0 Number
of cells
FIG. 4. Dependence of arylsulfatase activity in normal amniocytes on cell number. Enzyme activity was determined after 30 min incubation and is given as the mean + SD of 10 replicate assays. Symbols are identical to those in Fig. 1.
say techniques is in the area of prenatal diagnosis. Therefore we examined the enzyme activity in amniocytes of a culture previously frozen in liquid nitrogen after the diagnosis of metachromatic leukodystrophy was made. The time dependence of the assay in normal cells was determined by macromethods and also was linear for 30 min (data not shown). For micromethods cells showing fibroblastic morphology was selected for assay. Figure 4 shows the assay to be linear for extracts of 10 or 20 amniocytes. The specific activity of total arylsulfatase was similar to that of fibroblasts at l-2 X lo-l3 mol MUS hydrolyzed/h/cell with roughly equal amounts of arylsulfatase A and B activity. Table 1 shows the results of arylsulfatase determination in normal and mutant amniocytes. Raw data TABLE
1
ACTIVITY
ACTIVITY
OF ARYLSULFATASE
IN CULTURED
Relative
ACKNOWLEDGMENTS We are grateful to P. Chang for suggestions concerning the arylsulfatase assay procedure. These studies were suported by a Basil O’Connor Starter Grant to R. A. Gravel from the National Foundation March of Dimes. R. A. Gravel is a Scholar of the Medical Research Council of Canada.
REFERENCES 1.
Austin, J., McAlfie, D., and Shearer, L. (1965) Arch.
Neural.
CeIlS
Iv
A+0
B only
12, 441-455.
Beratis, N. Cl., Turner, B. M., Weiss, R., and Hirschhorn, K. (1975) Pediatr. Res. 9, 475-480. 3. Fluharty, A. L., Stevens, R. L., Fung, D., Peak, S., and Kihara, H. (1975) Biochem. Biophys. Rex 2.
4.
AMNIOCYTES
units
64,955-961.
Horwitz, A. L. (1979) Proc. Nat. Acad. Sci. USA 76,6496-6499.
Sherman, W. R., and Stanfield, E. F. (1967)
X10” mol/20 cells/30 min
AC
Biochem.
J 102,905-909.
Kolodny, E. H., and Mumford, R. A. (1976) Advan. Exp.
El
Med.
Bioi.
68, 239-251.
Christomanou, H., and Sandhoff, K. (1977) Normal
10
PatiCnlS
10
83 k 29 32f 8
36 * 9 29 + 5
363
(relative units) as well as the calculated specific activity are given in the table to illustrate the sensitivity of the assay. About 80% of the blank is a constant resulting from interference by a Raman band of water. The results show a near-complete deficiency of arylsulfatase A activity in the patient cells in agreement with the diagnosis previously made. The experiments extend the use of microtechniques to the determination of arylsulfatase. As with most other lysosomal hydrolases we have used a methylumbelliferyl substrate to achieve a sensitivity sufficient to detect enzyme activity in lo-20 cells. The determination of a complete defect of arylsulfatase A activity in fibroblasts and amniocytes of cases of metachromatic leukodystrophy confirmed that the silver ion inhibition procedure can effectively distinguish between arylsulfatases A and B.
Commun. MICRODETERMINATION
DETERMINATION
4.4 0.1
’ N, Number cd nplicatos of 20 cells each per away. ‘A all-free blank of 24 units wa8 subtracted from the raw data. ‘Obtained by subtracting B activity from total (A + B) activity.
3.4 2.8
Chim. 8. 9.
79, 527-531. (1977) Clin. Chem.
Clin.
Acla
H&Ii, P. 23, 1476-1484. Galjaard, H., Hoogeveen, A., Keijzer, W., de WitVerbeek, E., and Vlek-Noot, C. (1974) Histothem.
J. 6, 491-509.
364
GRAVEL
10. Galjaard, H., Van Hoogstraten, J. J., de Josselin de Jong, J. E., and Mulder, M. P. (1974) Histochem. J. 6, 409-429. 11. H&Ii, P. ( 1976) Advun. Exp. Med. Biol. 68, 1- 13. 12. Galjaard, H., Niermeijer, M. F., Hahnemann, N., Mohr, J., and Sorensen, S. A. ( 1974) Clin. Genet. 5, 368-377.
13. Niermeijer, M. F., Koster, J. F., Jahodova, M., Fernandes, J., Heukels-Dully, M. J., and Galjaard, H. (1975) Pediatr. Rex 9, 498-503.
ET AL. 14. Kleijer, W. J., Van der Veer, E., and Niermeijer, M. J. (1976) Hum. Genet. 33, 299-305. 15. Galjaard, H., Hoogevean, A., de Wit-Verbeek, H. A., Reuser, A. J. J., Ho, M. W., and Robinson, D. (1975) Nature (London) 257, 60-62. 16. Gravel, R. A., Leung, A., Saunders, M., and Hbsli, P. (1979) Proc. Nat. Acad. Sri. USA 76, 65206524.
17. Gravel, R. A., Lam, K. F., Scully, K. J., and Hsia, Y. E. (1977) Amer. J. Hum. Genet. 29,378-388.
BIOCHEMISTRY
119, 360-364 (1982)
A Micromethod for the Detection Cultured Fibroblasts R. A. GRAVEL,*
A. LEUNG,*
of Arylsulfatases and Amniocytes
A and B in
AND E. H. KOLODNYt
F. TSUI,*
*The Hospital For Sick Children, Toronto, Ontario MSG 1X8. Canada: and TEunice Kennedy Shriver Center for Menial Retardation. Inc.. at the Walter E. Farnald State School, Waltham. Massachusetts 021.54 Received July 27, 1981 A micromethod for the determination of arylsulfatase activity in cultured fibroblasts or amniocytes is described. The method depends on the hydrolysis of methylumbelliferyl sulfate in an assay volume of 0.3 ~1 using extracts from as few as lo-20 cells. Arylsulfatase A and B activities are distinguished using the silver ion inhibition method of H. Christomanou and K. Sandhoff (1977) C/in. Chim. Acla 79, 527-531. The method is illustrated for the detection of metachromatic leukodystrophy resulting from a deficiency of arylsulfatase A activity.
Several disorders of arylsulfatase deficiency have been described in the human population. They include metachromatic leukodystrophy resulting from a deficiency of arylsulfatase A activity (1) MarotauxLamy syndrome with arylsulfatase B deficiency (2,3), and multiple sulfatase deficiency in which activities of both of these isoenzymes as well as other sulfatases are deficient (4). Routine methods for the assay of the arylsulfatases depend on the hydrolysis of a sulfate ester attached to a chromogenic or fluorogenic aromatic substituent. The most sensitive substrate currently available is methylumbelliferyl sulfate (MUS),’ the hydrolysis of which produces the intensely fluorescent methylumbelliferone (5,6). Recently Christomanou and Sandhoff (7) reported a method for distinguishing between arylsulfatase A and B activities on the basis of the differential inhibition of arylsulfatase B in the presence of silver ions. Hijsli (8) and Galjaard (9,lO) have established micromethods for the detection of sev’ Abbreviation fate.
used: MUS, methylumbelliferyl
0003-2697/82/020360-05502.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved
sul-
360
era1 lysosomal hydrolases using methylumbelliferyl substrates. These methods have made possible the detection of enzyme activity in extracts of as few as 5- 10 fibroblasts or amniocytes. These procedures have made possible prenatal diagnosis on limited numbers of cells ( 1 l- 14) and complementation experiments in isolated fibroblast heterokaryons (11,15,16). In this study we reported a method for the detection of arylsulfatases A and B by miniaturization of the methylumbelliferyl sulfate method with silver ion inhibition of arylsulfatase A (7) following microprocedures as developed by H&Ii (8). We have used this method for the assay of fibroblast and amniocyte cultures and have demonstrated its efficacy in the detection of arylsulfatase A deficiency in metachromatic leukodystrophy using both cell types. MATERIALS
AND METHODS
Fibroblast and amniocyte cultures were obtained from the cell repository at The Hospital for Sick Children, Toronto, and from P. Chang, McMaster University, Hamilton. Three fibroblast strains were used: strain 1025 (normal), and strains Bi and
MICROMETHOD
FOR
ARYLSULFATASE
2035 from patients diagnosed with metachromatic leukodystrophy and with demonstrated arylsulfatase A deficiency. One normal and one amniocyte culture from a confirmed diagnosis of metachromatic leukodystrophy at this hospital (Lowden, personal communication) were also included. Growth conditions and culture medium have been described previously ( 17). Chemicals were of reagent grade and were chosen to have the lowest concentration of chloride impurity available. Sodium acetate and human serum albumin were purchased from Sigma; aminomethylpropanediol and lead acetate were from Fisher; and silver nitrate was from J. T. Baker. Methylumbelliferyl sulfate (MUS), recrystallized from methanol four times ( 16), was from KochLight. A detailed description of the microtechnique methodology has been given by Hiisli (8). The nature of the plastic film dish, a modified petri dish with a polypropylene film as the growing surface (Tecnomara AG, Zurich), is illustrated in the same article. The preparation of the Parafilm microwells is also described. These are small cuvettes designed to hold 0.34 assay volumes. They are constructed by placing an indentation in Parafilm with a blunt needle using a Teflon template and are sealed airtight with a covering layer of Parafilm (8). Cells were prepared by plating approximately 2 X lo5 fibroblasts or 5 X IO5 amniocytes per plastic film dish followed by incubation overnight in medium at 37°C. The next day the plates were washed with phosphate-buffered saline and thoroughly drained by suction. (A final wash with unbuffered saline can be used to ensure complete removal of phosphate which interferes with the enzyme assay.) The last bit of liquid was removed with the dishes standing on edge of facilitate draining. The plates were frozen in liquid nitrogen and lyophilized overnight. It was essential to start the lyophilization with the dish filled with liquid nitrogen to prevent warming up of the plates before suf-
ACTIVITY
DETERMINATION
361
ficient vacuum was achieved. Following lyophilization the plates were removed and placed in an environmental chamber at 18°C and 30% relative humidity for isolating cells for assay. We hve found that it is best to have the lyophilizer inside the environmental chamber connected to a vacuum pump on the outside. Otherwise the plates should be placed in a desiccant to prevent “wetting” of the dried cells during transport to the chamber. Cells were recovered for assay by cutting small leaflets of 5-20 cells each from the plate using a scalpel under low-power microscopy (8). Such leaflets are typically 0.5-1.0 mm* in size. The leaflets were collected by placing them in a plastic petri dish or other suitable container with the cell side facing up. To detect total arylsulfatase activity the assay mix contained 35 mM sodium acetate buffer, pH 5.0, 10 mM MUS, 3 mM lead acetate, and 0.1% human serum albumin. Arylsulfatase B alone was detected by including 0.3 mM silver nitrate to inhibit arylsulfatase A activity. A 30 mM stock of lead acetate was maintained in 0.1 M acetate buffer to prevent precipitation on standing. A 20 mM stock of MUS was prepared in 0.05 M acetate buffer to prevent spontaneous hydrolysis of the substrate in more acidic solutions. Stock solutions of the remaining reagents were prepared in water. All reagents were prepared fresh just before use with chloride- and carbonate-free water purified by filtration through Super Q (Millipore). These precautions were essential to prevent precipitation of reagents during the assays. Assays were accomplished by pipetting 0.34 aliquots of assay mix into Parafilm microwells using a 0.54 syringe (Scientific Glass Engineering, Melbourne). A sharp needle was used to pick up individual plastic film leaflets and insert them into the assay mix. The complete procedure was done under low-power microscopy. Sets of 10 cuvettes were prepared at a time on a single strip of Parafilm. Each strip was covered
362
GRAVEL
with a second layer of Parafilm and sealed as described (8). The Parafilm strips were incubated for 30 min by immersing them in a 37°C water bath. At the end of incubation the covering strip was peeled off and the assay mix was removed by washing with 100 ~1 of diluent consisting of 0.1 M aminomethylpropanediol adjusted to pH 10.4 with NaOH and containing 10e3 M sodium ethylenediaminetetraacetate. Results were read on a Perkin-Elmer 650-40 fluorometer at 365 nm excitation and 448 nm emission using low-scatter quartz microcuvettes (No. 1O-0497).
ET AL.
0
10 Number
20 cS cells
FIG. 2. Dependence of arylsulfatase activity in normal fibroblasts on cell number. Enzyme activity was determined after 30 min incubation and is given as the mean + SD of seven replicate assays. Symbols are identical to those in Fig. 1.
RESULTS AND DISCUSSION
The dependence of MUS hydrolysis by fibroblasts on time and cell number was examined by micromethods. Using extracts of 20 cells per determination, the assay was approximately linear for 30 min and gave about equal activities of arylsulfatase A and B (Fig. 1). This compares favorably with the standard assay procedures which also are linear for a similar length of time. In addition the assay was approximately linear for
/ r
15
/ ______-
T
f-------
igv’/’ I/“\; 1
e -cl 2 IO-
2 =
i
5-
i
1
r
0
r
15
30 he
I
45
extracts of 10 or 20 cells (Fig. 2). These results give a specific activity of l-2 X 1O-‘3 mol MUS hydrolyzed/h/cell for total arylsulfatase activity, a result in keeping with the level of activity of several, low-activity lysosomal hydrolases previously analyzed by this approach (8). Figure 3 shows the results of the determination of arylsulfatase activity in cells from two patients with metachromatic leukodystrophy. In this case the total arylsulfatase activity and that due to arylsulfatase B alone gave identical results. Hence these results demonstrate a virtually complete absence of arylsulfatase A activity in this disorder in agreement with results produced by macromethods. An important application of the microas-
!
60
(Illmuter)
FIG. 1. Time course of arylsulfatase activity in normal fibroblasts. Enzyme activity was determined in extracts of 20 cells as described under Materials and Methods. Each determination represents the mean + SD of seven replicate assays. (0) Total arylsulfatase (A + B); (0) arylsulfatase B; (- - -) arylsulfatase A obtained by subtracting B from total activity.
j&?ezc! 0
10
20 0 Number of cells
10
20
FIG. 3. Arylsulfatase activity in fibroblasts of patients with metachromatic leukodystrophy. Method and symbols as given in Figs. 1 and 2. Graph at left for strain Bi and at right for strain 2035.
MICROMETHOD
FOR ARYLSULFATASE
20
10
0 Number
of cells
FIG. 4. Dependence of arylsulfatase activity in normal amniocytes on cell number. Enzyme activity was determined after 30 min incubation and is given as the mean + SD of 10 replicate assays. Symbols are identical to those in Fig. 1.
say techniques is in the area of prenatal diagnosis. Therefore we examined the enzyme activity in amniocytes of a culture previously frozen in liquid nitrogen after the diagnosis of metachromatic leukodystrophy was made. The time dependence of the assay in normal cells was determined by macromethods and also was linear for 30 min (data not shown). For micromethods cells showing fibroblastic morphology was selected for assay. Figure 4 shows the assay to be linear for extracts of 10 or 20 amniocytes. The specific activity of total arylsulfatase was similar to that of fibroblasts at l-2 X lo-l3 mol MUS hydrolyzed/h/cell with roughly equal amounts of arylsulfatase A and B activity. Table 1 shows the results of arylsulfatase determination in normal and mutant amniocytes. Raw data TABLE
1
ACTIVITY
ACTIVITY
OF ARYLSULFATASE
IN CULTURED
Relative
ACKNOWLEDGMENTS We are grateful to P. Chang for suggestions concerning the arylsulfatase assay procedure. These studies were suported by a Basil O’Connor Starter Grant to R. A. Gravel from the National Foundation March of Dimes. R. A. Gravel is a Scholar of the Medical Research Council of Canada.
REFERENCES 1.
Austin, J., McAlfie, D., and Shearer, L. (1965) Arch.
Neural.
CeIlS
Iv
A+0
B only
12, 441-455.
Beratis, N. Cl., Turner, B. M., Weiss, R., and Hirschhorn, K. (1975) Pediatr. Res. 9, 475-480. 3. Fluharty, A. L., Stevens, R. L., Fung, D., Peak, S., and Kihara, H. (1975) Biochem. Biophys. Rex 2.
4.
AMNIOCYTES
units
64,955-961.
Horwitz, A. L. (1979) Proc. Nat. Acad. Sci. USA 76,6496-6499.
Sherman, W. R., and Stanfield, E. F. (1967)
X10” mol/20 cells/30 min
AC
Biochem.
J 102,905-909.
Kolodny, E. H., and Mumford, R. A. (1976) Advan. Exp.
El
Med.
Bioi.
68, 239-251.
Christomanou, H., and Sandhoff, K. (1977) Normal
10
PatiCnlS
10
83 k 29 32f 8
36 * 9 29 + 5
363
(relative units) as well as the calculated specific activity are given in the table to illustrate the sensitivity of the assay. About 80% of the blank is a constant resulting from interference by a Raman band of water. The results show a near-complete deficiency of arylsulfatase A activity in the patient cells in agreement with the diagnosis previously made. The experiments extend the use of microtechniques to the determination of arylsulfatase. As with most other lysosomal hydrolases we have used a methylumbelliferyl substrate to achieve a sensitivity sufficient to detect enzyme activity in lo-20 cells. The determination of a complete defect of arylsulfatase A activity in fibroblasts and amniocytes of cases of metachromatic leukodystrophy confirmed that the silver ion inhibition procedure can effectively distinguish between arylsulfatases A and B.
Commun. MICRODETERMINATION
DETERMINATION
4.4 0.1
’ N, Number cd nplicatos of 20 cells each per away. ‘A all-free blank of 24 units wa8 subtracted from the raw data. ‘Obtained by subtracting B activity from total (A + B) activity.
3.4 2.8
Chim. 8. 9.
79, 527-531. (1977) Clin. Chem.
Clin.
Acla
H&Ii, P. 23, 1476-1484. Galjaard, H., Hoogeveen, A., Keijzer, W., de WitVerbeek, E., and Vlek-Noot, C. (1974) Histothem.
J. 6, 491-509.
364
GRAVEL
10. Galjaard, H., Van Hoogstraten, J. J., de Josselin de Jong, J. E., and Mulder, M. P. (1974) Histochem. J. 6, 409-429. 11. H&Ii, P. ( 1976) Advun. Exp. Med. Biol. 68, 1- 13. 12. Galjaard, H., Niermeijer, M. F., Hahnemann, N., Mohr, J., and Sorensen, S. A. ( 1974) Clin. Genet. 5, 368-377.
13. Niermeijer, M. F., Koster, J. F., Jahodova, M., Fernandes, J., Heukels-Dully, M. J., and Galjaard, H. (1975) Pediatr. Rex 9, 498-503.
ET AL. 14. Kleijer, W. J., Van der Veer, E., and Niermeijer, M. J. (1976) Hum. Genet. 33, 299-305. 15. Galjaard, H., Hoogevean, A., de Wit-Verbeek, H. A., Reuser, A. J. J., Ho, M. W., and Robinson, D. (1975) Nature (London) 257, 60-62. 16. Gravel, R. A., Leung, A., Saunders, M., and Hbsli, P. (1979) Proc. Nat. Acad. Sri. USA 76, 65206524.
17. Gravel, R. A., Lam, K. F., Scully, K. J., and Hsia, Y. E. (1977) Amer. J. Hum. Genet. 29,378-388.