clinical Science (1983)65, 325-331
325
Detection of Morquio A syndrome using radiolabelled
substrates derived from keratan sulphate for the estimation
of galactose 6-sulphate sulphatase
J O H N J . HOPWOOD A N D H E L E N E L L I O T T
Department of Clinical Pathology, The Adelaide Children's Hospital Inc., North Adelaide, South Australia, Australia
(Received 20 December 198213 March 1983; accepted 31 March 1983)
3. Galactose 6-sulphate sulphatase activity in
fibroblast homogenates assayed with Gal6S1. The following radiolabelled disaccharide and anM6S as substrate clearly distinguished Morquio
tetrasaccharide were prepared from keratan A patients from normal controls, and Morquio B
sulphate and evaluated as substrates for gaiac- and Sanfdippo D patients.
tose 6-sulphate sulphatase present in cultured
4. We recommend the use of both the radiohuman skin fibroblasts: O-(p-~-6-sulphogalactosyl)- labelled disaccharide derived from keratan sulphate
(1-+ 4)-2,5 -anhydro -D-[ 1-3H] mannitol 6-sulphate
and the trisaccharide derived from chondroitin
(Gal6S-anM6S) and the tetrasaccharide equivalent
6-sulphate to assess sulphatase activity in fibroGal6S-GlcNAc6S-Gal6S-anM6S.
Radiolabelled triblasts of atypical Morquio patients.
saccharide substrates, N-acetylgalactosamine 6sulphate (p, 1+4)-glucuronic acid@, 1+3)-N- Key words: galactose 6-sulphate sulphatase deficiacetyl[l~H]galactosminitol6-sulphate(GalNAc6Sency, keratan sulphate, Morquio A syndrome.
GlcAGalitolNAc6S), and N - acetylgalactosamine
6-sulphate @, 1+4)-glucose-@, 1+3)-N-acetylD -sulphOAbbreviations: Gal6S -anM6S, 0-@[1-3H]galactosaminitol 6-sulphate (GalNAc6S-Glcgalactosy1)-(1+ 4)-2,5-anhydro-D- [ l-'H]mannitol
Galitol-NAc6S), were prepared from chondroitin
6-sulphate; GalNAc6SGlcA-GalitolNAc6S, O(-&D6-sulphate and used to assay N-acetylgalactosamine
6-sulpho-2-acetamido-2-deoxygalactosyl)-(1+ 4)-06-sulphate sulphatase.
/3 - D -glucuronosyl -(1+ 3)-0-/3-~
-6-sulpho-2-aceta2. Sulphatase activity obtained with Gal6Smido-2-deoxy-[ 1-3H]galactitol; GalNAc6S-GlcanM6S was approximately the same, 100 and 30-50
- D -6-sulpho -2-acetamid0 -2 GalitolNAc6S, 0-/3
times less than values obtained with Gal6SdeoxygalactosyL(1 + 4) -O-fh-glucosyl-(l+
3)-OQG ~ c N A c ~ S G ~ ~ ~ SGalNAc6S-GlcAGalitol-~~M~S,
D -6 -sulpho -2-acetamido-2-deoxy-[ 1-3H]galactitol;
NAc6S and GalNAc6SGlc-GalitolNAc respectively.
MPS, mucopolysaccharidosis; C6S, chondroitin
Less than 5% of normal activity resulted when
6-sulphate.
these substrates were incubated with fibroblasts
from Morquio A patients. These results demonstrate that the trisaccharide substrates derived
Lntroduction
from chondroitin 6-sulphate although de-0sulphated at a considerably higher rate than was The Morquio A syndrome (mucopolysaccharidosis
type IVA; MPS IVA) is caused by a profound
observed for the de-0-sulphation of the disacchardeficiency of the lysosomal enzyme N-acetylide and tetrasaccharide derived from keratan
galactosamine 6-sulphate sulphatase [1,2]. This
sulphate, are acted upon by the same sulphatase.
enzyme has been shown to be involved in the stepCorrespondence: Dr John J.'Hopwood, Depart- wise degradation of the glycosaminoglycan chonment of Chemical Pathology, The Adelaide Chil- droitin 6-sulphate (C6S). However, a deficiency of
the enzyme activity leads t o an accumulation of
dren's Hospital Inc., North Adelaide, South
partially degraded C6S and another glycosaminoAustralia, 5006, Australia.
Summary
326
J. J. Hopwood and H. Elliott
glycan, keratan sulphate, in lysosomes of cells and
tissues, which presumably causes the typical
skeletal deformities of the Morquio syndrome. The
syndrome can be enzymically diagnosed by assay
of N-acetylgalactosamine 6-sulphate sulphatase
activity in fibroblasts by using a trisaccharide substrate isolated from chondroitin 6-sulphate [3,4].
Genetic evidence from patients with MPS IVA
disease strongly suggests that the accumulation of
keratan sulphate in this disease results from a lack
of galactose 6-sulphate sulphatase activity, which
resides in the same enzyme protein as the N-acetylgalactosamine 6-sulphate sulphatase activity. However, Gloss1 et al. [S] reported that purified
N-acetylgalactosamine 6-sulphate sulphatase would
not hydrolyse galactitol 6-sulphate, a substrate
used by Ginsberg et al. [6] to demonstrate the
deficiency of galactose 6-sulphate sulphatase in
MPS IVA fibroblasts. Fujimoto & Horwitz [7] have
recently described two related patients with clinical phenotypes consistent with a diagnosis of a
very mild form of MPS IV. There was no evidence
of keratan sulphaturia, the total glycosaminoglycan
content was approximately twice as high as normal
and the majority of glycosaminoglycan was identified as C6S. The absence of keratan sulphaturia
and the reported deficiency of N-acetylgalactosamine 6-sulphate sulphatase in leucocytes and
fibroblasts from these two patients suggests that
the mutation affects the catabolism of C6S but
not keratan sulphate.
Experience with the determination of the activities of mutant enzymes produced in genetic disorders has underlined the need to use substrates
the structures of which closely match the structure
of the natural substrate [8-121. This is particularly
important with N-acetylgalactosamine 6-sulphate
sulphatase since this enzyme is also likely to
degrade keratan sulphate, a structurally different
substrate .
We have prepared radiolabelled disaccharide
and tetrasaccharide substrates, from keratan
sulphate, with a non-reducing end galactose
6-sulphate residue and shown that cultured skin
fibroblasts from MPS IVA patients are deficient in
the enzyme activity involved in both the desulphation of these keratan sulphate substrates and a
radiolabelled trisaccharide substrate with a nonreducing end N-acetylgalactosamine 6-sulphate
residue derived from C6S. After this work was
complete Yutaka et al. [13] reported similar
observations with the degradation of a keratan
sulphate-derived trisaccharide (Gal6S-GlcNAc6Sgalactitol) by fibroblasts from normal and MPS
IVA patients. We conclude, as have [13], that
galactose 6-sulphate and N-acetylgalactosamine
6-sulphate residues are desulphated by the same
CH.OSO
CH,OSO
"'~O,(&
Gal6S - arlM6S
H.OH
OH
HNCOCH,
OH
HNCOCH
GalNAc6S
HNCOCH ,
,
- GlcA - GalilolNAc6S
HNCOCH.
GalNAc6S
. Glc . Gal810lNAc6S
FIG. 1. Proposed structures for the radiolabelled
oligosaccharides derived from keratan sulphate
(Gal6S-anM6S and Gal6S-GlcNAc6SGal6S-anM6S)
and C6S (GalNAc6S-GlcA-GalitolNAc6S and
GalNA16S-Glc-GalitolNAc6S).
enzyme and describe substrates, derived from
keratan sulphate, for assay of galactose 6-sulphate
sulphatase activity.
The proposed structures of the oligosaccharides
used in the study that follows are shown in Fig. 1.
Materials and methods
Preparation of radiolabelled substrates
The radiolabelled disccharide O-(p-~d-sulphogalactosyl) -(1 +4)-2,5-anhydro -D-[ 1-3H]mannitol
6-sulphate (Gal6S-anM6S) and the tetrasaccharide
equivalent galactose 6-sulphate (p, 1+4)-N-acetylglucosamine 6-sulphate @, 1+3)-galactose 6-sulmannitol 6 phate - (p, 1+ 4) - anhydro - D -[1
sulphate (Gal6S -GlcNAc6S-Gal6S-anM6S) were
prepared by deamination of de-N-acetylated bovine
intervertebral disc keratan sulphate by published
methods [ 141. The specific radioactivity of disaccharide and tetrasaccharide substrates was 30
Ci/mol and 500 Ci/mol respectively.
For the preparation of GalNAc6S-GlcAGalitolNAc6S, 0.4 g of C6S was dissolved in 10 ml of
Diagnosis of Morquio A syndrome
NaCl(O.15 mol/l)/sodium acetate (0.1 mol/l) buffer,
pH 5.0, and digested with 7000 units of ovine
testicular hyaluronidase (Sigma Chemical Co.) for
16 h at 37°C. The reaction mixture was applied to
a column (1 cm X 200 cm) of Sephadex G-25 and
eluted with NaCl (0.2 mol/l)/sodium acetate (0.05
mol/l) buffer (PH 5.2). The uronic acid positive
material eluting at a position corresponding to a
tetrasaccharide was pooled, desalted on a column
(1 cm x 100 cm) of Sephadex G-10, rotary evaporated to dryness, dissolved in 1 ml of water and
0.5 ml of sodium acetate (0.2 mol/l) buffer, pH
4.9, and digested with 25000 units of p-Dglucuronidase for 16 h at 37°C. The digest was
then applied and eluted from the column of
Sephadex G-25, used above, to separate trisaccharide and monosaccharide uronic acid positive peaks.
The trisaccharide fractions were pooled and desalted on a column of Sephadex (3-10 and reductively labelled with sodium boro [jH] hydride in
sodium borate buffer (pH 7.8) and desalted as
previously described [15,16]. The desalted radiolabelled components were applied to a column'
(5 cm x 2 cm) of Dowex I (Cl- form) and eluted
with a linear gradient of LiCl from 0 to 3.0 mol/l.
More than 90% of the total radioactivity was
eluted in a single peak between 1.1 and 1.4 mol of
LiCl/l, pooled, desalted on a column of Sephadex
G-10, rotary evaporated to 100 p1 and subjected to
preparative paper chromatography on Whatman
3MM paper for 5 days in isobutyric acid (2.0 mol/l)/
aq. NH3 solution (150 : 90,vlv); the trailing 50% of
radioactivity free of 4-sulphate esters was eluted
and subjected to preparative high-voltage electrophoresis at pH 1.7. The specific radioactivity of
GalNAc6S-GlcAGalitolNAc6S was 90 Cilmol.
GalNAc6S-Glc-GalitolNAc6S was prepared by
carboxyl group reduction of the radiolabelled
GalNAc6S-GlcA-GalitolNAc6S. GalNAc6S-GlcAGalitolNAc6S (60 nmol) was mixed with 0.5 mg of
1-ethyl- 3 4 3 - dimethylaminopropyl) carbodi - imide
hydrochloride in l m l of water at 25°C. After
90 min 76 mg of NaBH,, was added, the mixture
incubated at 50°C for 2 h , acidified with acetic
acid, applied to a column containing 5 g of Dowex
50 (H+ form) and washed with 2 0 m l of water.
The eluate was rotary-evaporated to dryness in
the presence of methanol, dissolved in water (1 ml),
desalted on Sephadex (2-10 as described above,
rotary-evaporated to dryness, dissolved in water
(1OOpl) and subjected to preparative high-voltage
electrophoresis at pH 5.0. Two peaks of radioactive
material were identified. The faster moving and
minor component had the same mobility as
GalNAc6S-GlcA-GalitolNAc6Swhereas the slower
moving major component had the mobility expected for a trisaccharide with two negative charges.
327
We have tentatively identified this component as
GalNAc6SGlc-GalitolNAc6S.
D -[1-'4C] Galactose 6-sulphate and 2-acetamido2-deoxy-~-[l-'~C]galactose
6-sulphate were prepared by 0-sulphation of the radiolabelled monosaccharides by methods previously described [16].
Enzyme preparation
Fibroblast cultures were established from skin
biopsies taken in or provided to this hospital [ 171.
Mucopolysaccharidosis type IVB (MPS IVB) fibroblasts (P-D-galactosidase deficiency GM 325) were
obtained from the Human Genetic Cell Repository
(Institute for Medical Research, Camden, NJ,
U.S.A.). All patients classified as MPS IVA excreted
excessive amounts of both C6S and keratan sulphate in their urine [18], had normal levels of
0-D-galactosidase [19] and were deficient in Nacetylgalactosamine 6-sulphate sulphatase activity
when cultured fibroblasts were assayed with
GalNAc6SGlcA-GalitolNAc6S [3,4].
Peripheral blood leucocytes were prepared as
previously described [17]. All cell types were
suspended in aqueous Triton X-100 (lg/l) to a
concentration of 5-10 g/l and were disrupted by
rapid freezing and thawing six times in a solid
Codethanol mixture.
Enzyme assays
Incubation mixtures contained 15-30 pg of
protein from cultured fibroblast homogenates or
30 pg of protein from leucocyte homogenates in
sodium formate buffer (50 mmol/l), pH 4.0,
containing NaN3 (4 mmol/l) and approximately
500 pmol of substrate in a final volume of 20 pl in
a sealed polypropylene tube. Incubation was at
37°C for 16 h for fibroblast homogenate hydrolysis
of Gal6S-anM6S, Gal6S-GlcNAc6S-Gal6S-anM6S
and 2 h for hydrolysis of GalNAc6S-GlcA-GalitolNAc6S and GalNAc6S-Glc-GalitolNAc6S. At the
end of the incubation period, products and residual
substrate were separated by either high-voltage
electrophoresis at pH 1.7 or descending chromatography as described below. Incubation mixtures
not immediately subjected to electrophoresis were
stored at -20°C. Sulphatase activity was expressed
as pmol of product produced m i d ' mg-' of
protein.
General methods
High-voltage electrophoresis was performed on
Whatman 3MM chromatography paper in formic
acid (1.74 mol/l), pH 1.7, at 4 2 V/cm for 60 min
or in sodium acetate (50 mmol/l) buffer, pH 5.0,
J. J. Hopwood and H. ElIiott
328
at 4 5 V/cm for 50 min using a Shandon Southern
model L-24 System (Shandon Southern Products
Ltd, Runcorn, Cheshire, U.K.). Descending chromatograms of the tetrasaccharide substrate and
product were run in the solvent ethyl acetate/
acetic acid/water (4: 3 : 3, by vol.) for 49 h at
25OC.
Protein was determined by the Fohn method of
Lowry et al. [20] with crystalline bovine serum
albumin (Sigma Chemical Co.) as standard. Radioactivity was measured as previously described at
approximately 30% efficiency [2 1 1.
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Materials
Results
Fibroblast sulphatase activity was measured at pH
4.0 with a variety of substrates, containing galactose
6-sulphase or N-acetylgalactosamine 6-sulphate
residues. Hydrolysis o f Gal6S-anM6S by galactose
6-sulpliate sulphatase yields unlabelled sulphate
and the corresponding radiolabelled monosulphated
disaccharide as shown in Fig. 2. Sulphatase activity
toward Gal6S-anM6S was 2.1 pmol niin-' mg-' of
OH
I
galactose 6-sulphate sulphatase
OH
Proposed degradation sequence of Gal6SanM6S by galactose 6sulphate sulphatase.
FIG. 2 .
2
0.
Sodium boro [3H] hydride, D-[ 1-I4C]galactose
and 2-acetamido-2-deoxy-~-[
1-14C]galactose were
purchased from The Radiochemical Centre (Amersham, Bucks, U.K.).
Chondroitin 6-sulphate (Sigma Chemical Co.)
contained approximately 8% of 4-sulphated isomer
when the disaccharides produced by chondroitinase
ABC digestion were separated by paper chromatography and assayed [22].
Disaccharide and tetrasaccharide reference compounds were prepared b y nitrous acid degradation
of heparin as previously described [ 151.
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protein and approximately the same (3.0 pmol
min-' mg-') as that observed for the tetrasulphated
tetrasaccharide derived from keratan sulphate and
100 times less (192 pmol min-I mg-' of protein) than
that for the trisaccharide GalNAc6S-GlcA-GalitolNAc6S derived from C6S. The monosulphated
disaccharide produced from Gal6S-anM6S by the
action o f the galactose 6-sulphate sulphatase and
shown t o be a substrate for 0-D-galactosidase [14]
was not degraded under the assay conditions used
above. Hydrolysis by fibroblast homogenates of
the C6 sulphated monosaccharide equivalents,
Gal6S and GalNAc6S, was not detected. Reduction o f the glucuronic acid carboxyl in the trisaccharide substrate t o glucose reduced the sulphatase
activity by approximately one half t o 9 2 pmol
min-' mg-' of protein. No detectable enzyme
activity toward any of these substrates was shown
by fibroblast cultures from one MPS IVA patient.
The effect of incubation p H o n fibroblast
enzyme activity is recorded in Fig. 3. Gal6S-anM6S
and GalNAc6S-GlcA-GalitoINAc6S have similar
profiles with maximum activity at pH 4.04.5,
with more than 50% of the maximum activity still
remaining at pH 3.6 and 5.0. This pH optimum is
similar t o that recently described by Gloss1 et al.
[23] for the trisaccharide substrate. However,
unlike the 40-fold increase in activity reported by
these authors reduction of acetate buffer concentration from 80 mmol/l t o 2 0 mniol/l resulted in
a twofold increase in enzyme activity toward the
trisaccharide without a shift in pH optimum.
Dingnosis of Morquio A syndrome
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FIG. 4. High-voltage separation of incubation mixtures containing normal fibroblast homogenate
and GalNAc6SGlcA-GalitolNAc6S in ( a ) sodium
formate (0.05 mol/l) buffer, pH 4.0 ( 0 - - - 0 ) ,
and sodium acetate (0.02mol/l) buffer, pH 4.0
(0-o), and ( b ) sodium acetate (0.08 molll)
buffer, pH 4.0.
Hydrolysis of trisaccharide substrate in sodium
acetate (20 mmol/l) buffer and the standard sodium
formate (50 mmol/l) buffer by fibroblast homogenates produced two products. The first, GalNAcGlcA-GalitolNAc6S, is a product of the sulphatase
and the second product, probably GlcA-GalitolNAc6S, results from the action of 0-N-acetylhexosaminidase on the first product (Fig. 4a). However,
on incubation of the trisaccharide substrate with
fibroblast homogenates in sodium acetate (80
mmol/l) buffer (Fig. 4b) only the product,
GalNAc-GlcA-GalitolNAc-6S, of the sulphatase,
could be detected. Thesc results suggest that
80 mmol/l acetate inhibits 0-N-acetylhexosaminidase activity toward the sulphatase product
(GalNAc-GlcA-GalitolNAc6S) and that, particularly in low acetate or sodium formate buffers,
the level of sulphatase will be an underestimate
unless account is made of the contribution of the
0-N-acetylhexosaminidase product (GlcA-GalitolNAc6S) t o the total hydrolysis by the action of
the sulphatase. Addition of bovine serum albumin
329
(0.03%,w/v) did not shift the pH profile or alter
sulphatase activity. The effect of protein concentration and incubation time on enzyme activity
was linear u p to 10%desulphation of both keratan
sulphate and chondroitin sulphate derived substrates.
The apparent K,,,for Gal6S-anM6S with fibroblast galactose 6-sulphate sulphatase was approximately 80 pmol/l and the Vma. value was 6 pmol
min-' mg-', compared with K, 35 pmolll and
V,,.
870 pmol m i d ' mg-' for GalNAc6SGlcAGalitolNAc6S. Leucocyte activity toward Gal6SanM6S was not detected. The enzyme activity
toward Gal6S-anM6S in fibroblast homogenates
was inhibited 50% and 75% by NaCl at 100mmol/l
and Na2S04 at 2 mmol/l respectively.
Fibroblasts from MPS IVA patients had very
low or no detected activity toward the disaccharide
and trisaccharide substrates derived from keratan
sulphate and C6S respectively (Table 1). Homogenates of fibroblasts from MPS IVB and IIID
patients had enzyme activities within the normal
range.
Discussion
The isolation of a radiolabelled disaccharide with
galactose 6-sulphate at the non-reducing terminus
provided a substrate for the estimation of galactose 6-sulphate sulphatase. The properties of
galactose 6-sulphate sulphatase resemble those
of the sulphatase activity toward the trisaccharide
with a N-acetylgalactosamine 6-sulphate at the
non-reducing terminus. In the past the lack of
hydrolysis of the monosaccharide galactose 6sulphate has prevented direct comparison of mam-
TABLE 1. Galactose 6-sulphate and N-acetylgalactosamine 6-sulphate sulphatase activities (toward
Gal6S-anMBS and GalNAc6S-GlcA-GalitolNAc6S
respectively) o f cultured skin fibroblast homogenates from MPS IVA, MPS IVB and MPS IIID
patients and unrelated normal persons
n.d., None detected; n , number of individuals.
Sulphatase activity
(pmol min-' mg-'of protein)
MPS IVA
MPS IVB
Normal
MPS IIID
Gal6SanM6S
GalNAcLSGlcAGalitolNAc6S
n.d.
(n = 5)
n.d.-l.7
(r; = 6 )
264-378
(n = 3)
58-268
(n = 13)
153
2.8-5.2
(n = 3)
0.7-3.5
(n = 12)
2.1
330
J. J. Hopwood and H. Elliott
malian sulphatase activity toward galactose
6-sulphate and N-acetylgalactosamine 6-sulphate
residues. Nakazawa & Kagabe [24] have described
a bacterial sulphatase activity toward these two
substrates. The latter substrate was hydrolysed at
a rate 2.4 times that observed for galactose 6sulphate. N-Acetylgalactosamine 6-sulphate sulphatase, partially purified from rat skin [25] and
human liver [26], was reported to release sulphate
from 35S-labelled keratan sulphate. Recently
Yutaka et al. [13] reported desulphation of an
oligosaccharide derived from keratan sulphate that
was not observed when fibroblasts from M P S IVA
patients were used. Likewise in this paper we
report that activity toward both Gal6S-anM6S
and the C6S trisaccharide was not detected in
fibroblasts from MPS IVA patients. We conclude
from these results (Table 1) that the sulphatase
responsible for the MPS IVA lesion is specific for
sulphate esters linked C-6 to hexose or N-acetylhexosamine with the galactose configuration. The
100-fold higher activity toward the C6S trisaccharide than that observed for the keratan sulphate
disaccharide can perhaps be partly explained by
the combined higher GalNAc 6-sulphatase activity
and the influence (twofold) of the C-6 carboxyl
group on the adjacent residue.
We conclude that the radiolabelled disulphated
disaccharide and tetrasulphated tetrasaccharide
substrates isolated from keratan sulphate are
effective substrates for galactose-6-sulphate sulphatase and may be used to diagnose MPS IVA.
Although the trisaccharide substrate derived from
C6S is a much more sensitive substrate t o use,
enzyme activity toward a keratan sulphate substrate
such as Gal6S-anM6S should be determined in
certain diagnostic circumstances. For example, in
patients presenting with a clinical phenotype suggestive of a Morquio diagnosis but excreting either
excess keratan sulphate or C6S, but not both
keratan sulphate and C6S (as found with typical
MPS IVA), it is important to assess activity toward
substrates derived from both keratan sulphate and
C6S. Such a patient, excreting excess C6S but not
keratan sulphate, with deficient fibroblast and
leucocyte sulphatase activity toward a C6S derived
substrate, has been described by Fujimoto & Horwitz [7]. These phenotypes may result from mutations that affect the relative enzyme activity
toward C6S and keratsn sulphate.
Acknowledgments
This work was supported by grants from the
Research Trust of The Adelaide Children’s Hospital
Inc., and the National Health and Medical Research
Council of Australia.
References
1 . Matalon, R., Arbogast, B., Justice, P., Brandt, I.K. &
Dorfman, A. (1974) Morquio’s syndrome: deficiency
of a chondroitin sulphate N-acetylhexosamine sulfate
sulfatase. Biochemical and Biophysical Research Communications, 61, 759-764.
2. Singh, J., Di Ferrante, N., Niebes, P. & Tavella, D.
(1976) N-Acetylgalactosamined-sulfate sulfatase in
man: absence of the enzyme in Morquio disease.
Journal of Clinical Investigation, 57,1036-1040.
3. Glossl, J. & Kresse, H. (1978) A sensitive procedure
for the diagnosis of N-acetylgalactosamined-sulfate
sulfatase deficiency in classical Morquio’s disease.
Clinica Chimica Acta, 88,111-1 19.
4. Horwitz, A. & Dorfman, A. (1978) The enzyme
defect in Morquio’s disease: the specificity of N acetylhexosamine sulfatases. Biochemical and Biophysical Research Communications, 80, 819-825.
5 . Glossl, J., Truppe, W. & Kresse, H. (1979) Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta. Biochemical
Journal, 181, 37-46.
6. Ginsberg, L.C., Donnelly, P.V., Di Ferrante, D.T., Di
Ferrante, N. & Coskey, C.T. (1978) N-Acetylgluco:
samined-sulfate sulfatase in man: deficiency of the
enzyme in a new mucopolysaccharidosis. Pediatric
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7. Fujimoto, A. & Horwitz, A. (1981) Biochemical
defect of non-keratan-sulfate-excreting Morquio syndrome (Abstract). Pediatric Research, 15, 561.
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(1974) Ganglioside catabolism in hexosaminidase Adeficient adults. Nature (London), 252, 254-255.
9. Owada, M., Sakiyama, T. & Kitagawa, T. (1977)
Neuropathic Gaucher’s disease with normal 4-methylumbelliferyl-p-glucosidase activity in the liver. Pediatric
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10. O’Brien, J.G., Norden, A.G.W., Miller, A.L., Frost,
R.G. & Kelly, T.E. (1977) Ganglioside GM2 N-acetylp-D-galactosaminidase and asialo GM2 (CA2) N-acetylp-Dgalactosaminidase; studies in human skin fibroblasts. Clinical Genetics, 11, 171-183.
11. Bcn-Yoseph, Y. & Nadler, H.L. (1978) Pitfalls in the
use of artificial substrates for the diagnosis of Gaucher’s
diseasc. Journal of Clinical Pathology, 31, 1091-1093.
12. Hopwood, J.J. & Muller, V. (1979) Biochemical discrimination of Hurler and Scheie syndrome. Clinical
Science, 57,265-212.
13. Yutaka, T., Okada, S., Kato, T., Inui, K. & Yabuuhi,
H. (1982) Galactose 6-sulfate sulfatase activity in
Morquio syndrome. Clinical Chimica Acta, 122, 169180.
14. Hopwood, J.J. & Elliott, H. (1983) Selective depolymerisation of keratan sulfate: production of radiolabclled substrates for 6-sulfogalactosc sulfatase and
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