Vol. 90, No. 4, 1979 October 29, 1979
BIOCHEMICAL
MUCOLIPIDOSIS Gideon
Bach,
Marcia
Department
Received
September
TYPE IV:
AND BIOPHYSICAL
GANGLIOSIDE Tamar
Zeigler,
RESEARCH COMMUNICATIONS Pages 1341-1347
SIALIDASE
Schaap
and Gertrude
of Human Genetics, Hadassah-Hebrew Medical Center, Jerusalem, Israel 11,
DEFICIENCY Kohn
University
1979 SUMMARY
A solubilized sialidase is partially deficient in cultured fibroblasts derived from skin biopsies of four mucolipidosis IV patients, Fibroblasts from two obligate heterozygotes also have sialidase activity lower than normal controls, Membrane-bound sialidase activity is not affected in this disease. Based on previous and present findings, we propose that this solubilized activity is probably lysosomal origin. Sialidase activity in mucolipidosis IV cells is normal when neuraminlactose is used as substrate,, Mixing cell homogenates from mucolipidosis IV patients and frcm controls results in the expected combined sialidase activity, indicating the absence of an internal inhibitor in the deficient cells. It is therefore suggested that the mutation in mucolipidosis IV specifically affects a lysosomal ganglioside sialidase, while the remaining non-lysosomal sialidases partially mask this deficiency. Mucolipidosis
type
as an autosomal
recessive
by progressive
As in other
of both
patients disialo-GD3)
for
we present possible
increased
and acid
responsible
this
evidence metabolic
and bilateral
congenital
levels
defect
in ML IV,
sialidase Mucolipidosis Mucolipidosis Mucolipidosis
was observed was assayed IV II I
skin
yet been
transmitted is
cornea1 accumul-
materials
has been
of ML IV patients obtained
(both
resolved.
from ML IV and
defect In this
sialidase
paper
as the
In contrast
to the ganglioside
between
the ML IV and control
neuraminlactose
(1,2,5,6).
monosialo-GM3
metabolic
of a ganglioside
with
characterized
intralysosomal
fibroblasts
The basic
has not
the deficiency
when
organs
of gangliosides
for
fibroblasts
increased
in various
of cultured
accumulation
no difference
4),
and lipid-like
mucopolysaccharides.
deficiency,
Abbreviations:
(i',g)
disease
the disorder
(3,
microscopy
storage
Clinically,
glycoconjugates
analysis
indicated
a lysosomal
(1,2).
mucolipidoses
by electron
chemical
is
retardation
water-soluble
demonstrated Direct
trait
psychomotor
opacities. ation
IV (ML IV)
sialidase
as substrate,
- ML IV - ML II -ML1 0006-291X/79/201341-07$01.00/0 1341
Copyright All rights
@ 1979
by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALSANDMETHODS Cell culture Cultured skin fibroblasts obtained from four ML IV patients, five normal controls and two obli ate heterozygotes (parents of an ML IV patient) were propagated in 75 cmi plastic tissue culture flasks (Falcon) to a density of 5-7 x lo6 cells per flask (1 mg protein) and maintained under Cells were harvested 14 days after conditions previously described (7,8), the last passage. The pH of the mediumaffected the ganglioside sialidase activity, which was barely detectable if the mediumpH was greater than pH 7.4. All comparisons between the ML IV and the control fibroblasts were therefore made in cells grown below pH 7.2. the cells were Sialidase determinations - Following harvest by trypsinization, washed twice with 0.15 M NaCl, resuspended in 0.5 ml of 0.1% Triton X-100 and homogenized by teflon-glass hand homogenizer. The suspension was then sonicated (Braun sonic, 330s sonicator, small prove, B. Braun, Germany) for a total of 30 sec. with 15 sec. bursts and 15 seco cooling period between sonications. The insoluble debris was precipitated by centrifugation at 40,000 g for 30 min. at 4°C and the supernatant was collected and kept at O'C prior to analysis. The precipitate, when used for further analysis, was suspended in 0.5 ml of H20. Ganglioside sialidase was determined using GM3 (Hematoside) as a substrate obtained and purified from a spleen of an adult Gaucher patient according to Svennerholm (9). The reaction mixture contained 0.1 mg of GM3dissolved in chloroform-methanol 2:l (v/v), dried under nitrogen stream, 0.1 M acetate buffer pH 4.6 (optimal in both extraction fractions), 0.007% sodium azide, 0.1% Triton X-100 and 0.10-0.15 mg protein in a finalvolume of 0.2 ml. Incubation was carried out at 37OCfor six hours followed by the determination of free sialic acid according to Warren (lo), using the equation to eliminate interfering substances. Controls containing enzyme or substrate alone, and zero time controls were determined in parallel. Neuraminlactose sialidase activity was determined with 0.04 mg neuraminlactose (Sigma Chemicals, type II) as substrate in an incubation mixture similar to that described above. The mixture was incubated for three hours followed by the determination of free sialic acid. RESULTS Table 1 represents the neuraminlactose and ganglioside sialidase in the two extraction
fractions
for a typical
lactose as substrate normal enzyme activities natant and precipitate
of ML IV.
in the supernatant fluid the control,
ganglioside sialidase activities
were observed in both the super-
of ML IV showed reduced activity difference
X-100 or when ultrasonication and approximately
When homogenization was omitted,
all
the
60%of the neuraminlactose sialidase
were found in the precipitate.
between the ML IV and controls
when compared to
could be detected in the
of the MI IV and control precipitates.
was done without Triton
With neuramin-
On the other hand, ganglioside sialidase
whereas no significant
enzyme activities
experiment.
activities
Under these conditions no difference
could be observed.
1342
Another important finding
BIOCHEMICAL
Vol. 90, No. 4, 1979
Table
Sialidase
1.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activity
in
supernatant
fibroblast
and
precipitate
extracts Sialidase
Cell
Neuraminlactose
Control
(A)
Ganglioside
Neuraminlactose
Ganglioside
1.38
1.67
1.17
3.85
(V)
1.51
0.67
1.25
4.05
ML II
(K)
0
0.33
0
2.63
*
Activities per
shown
are
expressed
as nmoles
sialic
acid
formed
per
hour
mg protein.
1.
Whereas
a total
was observed
in both
extraction
in
activity
Table
ganglioside natant
Precipitate
IV
Ml
is
activity*
Supernatant
origin
of
sialidase
activity
and precipitate
with
the
the
results
deficiency
of neuraminlactose
fractions
was only
in ML II
partially
supernatant
sialidase
fibroblasts,
deficient
in both
activity
somewhat
typical
experiments,
super-
lower
than
in ML IV. Table
2 summarizes
ganglioside
sialidase
patients, between
controls
(e.g.,
sensitivity
supposedly the specific
activity
between
cell
lines
ranking
of cell
are
lower indicated
when experiments 0.575.21 Furthermore,
represented
same harvest by S.D. between
ratio
considered
to small
out of four
Duplicate varied
values
is
also
considerable,
However,
of MI IV to control (Table
50% decrease
1343
the
3).
ML IV
variation due to in
determinations slightly.
as compared
experiments
for
variations
only
experiments.
separately
in which
2 and 3) is probably
conditions,
an approximately
in three
The considerable
activity
in ML IV patients by the
are
indicate
differ
was determined
experiment
enzyme
culture
in the
lines
the supernatant
between
tissue
six
heterozygotes.
of this
uniform
was consistently
in
and obligate
experiments
the extreme
results
activity
from
of
The variation and the
enzyme activity
to controls.
These
enzyme activities The mean ratio,
in ML IV enzyme activity. ratio
of enzyme activities
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1344
Vol. 90, No. 4, 1979
BIOCHEMICAL
Table
3.
supernatant
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Ratios of ganglioside sialidase activities in the fraction of fibroblasts of the three genotypes
Experiment
MLIV
Heterozygotes Control
*
Control
NO.
1
0.80
2
0.70
0.55
3
0.54
0.70
4
0.60
0.51
5
0.43
6
0.71
1.00
7
0.60
8
0.59
9
0.64
10
0.24
11
0.23
12
0.75
13
0.55
14
0.64
Mean
0.57
0.69
S.D.
0.21
0.22
*
Ratio
of specific
of heterozygotes indicating considerable
similar
in these
between
variability
enzyme activity
were
activity
differences
experiment
activities.
to controls
a reduced
*
to those cell
lines
ML IV patients
makes it
difficult
of ML IV to controls as well.
In spite
and controls,
to define
of the
the inter-
the normal
and ML IV
ranges.
The possibility
of an internal
the ML IV cells
was ruled
amounts
of cell
protein
combined
values
inhibitor
out by mixing
experiments.
from ML IV and control
of ganglioside
of ganglioside
sialidase
Mixtures
lines
activity
sialidase
resulted (data
not
in
of equal in the
expected
presented).
DISCUSSION The significantly in ML IV patients
reduced and the
activity
accumulation
of solubilized of sialogangliosides
1345
ganglioside (6,7)
sialidase suggest
BIOCHEMICAL
Vol. 90, No. 4, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
that the ML IV disease is associated with ganglioside sialidase In three out of four determinations, also lower than controls;
the heterozygote
deficiency.
enzyme activity
was
however, a larger number of heterozygotes
should
be studied before firm conclusions can be drawn. The fact that neuraminlactose sialidase is not surprising
in the disease
since neuraminlactose and ganglioside sialidases
been shown to be two distinct by the total
is not affected
deficiency
enzymes.(18,21).
This finding
of neuraminlactose sialidase
while ganglioside sialidase
is unaffected
have
is supported
in ML I (11,12,20,21),
(22).
Sialidases have not been sufficiently
characterized
in fibroblasts,
but the existence of more than one sialidase has been demonstrated in liver (14,15),
brain (16,18),
and heart muscle (19).
sides sialidases have been localized
Furthermore, various ganglio-
to different
cell organelles (plasma
membrane, microsomes and lysosomes) (19,23). In view of the fact that the plasma membraneand the microsomal-bound enzymes are practically are generally
more readily
ganglioside sialidase sialidase
insoluble
(16,23) and that lysosomal hydrolases
soluble (24), we suggest that the supernatant
activity
might be greatly
while the precipitate
enriched by the lysosomal
is mainly non-lysosomal.
tion is supported by the findings
in the ML II cell extract,
characterized
deficiency
hydrolases.
by an intracellular
of the neuraminlactose sialidase
indicating
its mainly lysosomal origin. activity
This may indicate
is affected
of most lysosomal
activity
in this cell line,
However, not all ganglioside
in ML II,
a non-lysosomal origin
single mutation affecting be partially
in fibroblasts
which is
In agreement with previous studies (11-13) there was a total
deficiency
sialidase
This interpreta-
particularly
in the precipitate.
for the remaining activity.
only a lysosomal isoenzyme may therefore
maskedby the unaffected
remaining isoenzymes.
A at least
Further
separation of the presumed isoenzymes is needed for precise determination of the lysosomal sialidase,
particularly
1346
for diagnostic purposes.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Acknowledgement: The skillful technical assistance of Mrs. B. Medan is greatly appreciated. The project was supported by grants from the National Foundation - March of Dimes (Noel-599) and the Stiftung Volkswagen (No. 34048). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22. 23.
24.
Berman, E.R., Livni, N., Shapira, E., Merin, S. and Levij, I.S. (1974) J. Pediat. 84, 519-526. S. (1975) Invest. Merin, S., Livni, N., Berman, E.R. and Yatsiv, Ophthalmol. z, 437-448. in Heritable disorders of connective tissue, McKusick, V.A. (1972) 4th ed. pp. 641-665, C.V. Mosby CO. Spranger, J.W. and Wiedemann, H.R. Humangenetik 2, 113-139. (1970) Amer. J. Ophthalmol. Newell, F.W., Matalon, R. and Meyer, S. (1975) so, 440-449. Tellez-Nagel, I., Rapin, I., Iwamoto, T., Johnson, A.B., Norton, W.T. and Nitowsky, H. (1976) Arch. Neurol. 33, 828-835. Bach, G., Cohen, M.M. and Kohn, G. (1975) Biochem. Biophys. Res. Commun. 66, 1483-1490. Bach, G., Zeigler, M., Kohn, G. and Cohen, M.M. (1977) Amer. J. Hum. Genet. 2, 610-618. Svennerholm, L. (1972) in Methods in carbohydrate chemistry. Vol. 6, pp. 464-474, Academic Press, N.Y. Warren, L. (1959) J. Biol. Chem. 234, 1971-1975. Cantz, M., Gehler, J. and Spranger, J.W. Biochem. Biophys. Res. (1977) Commun. 3, 732-738. Kelly, T.E. and Graetz, G. (1977) Amer. J. Med. Genet. 1, 31-46. Strecker, G., Michalski, J.C., Montreuill, J. and Farriaux, M. (1976) Biomed. Exp. 25, 238-240. Touster, O., Aronson, N.N., Dulaney, J.T. and Hendrickson, M. (1970) J. Cell Biol. 47, 604-618. Schengrund, C.L., Jensen, D.S. and Rosenberg, A. (1972) J. Biol. Chem. 247, 2742-2746. Leibowitz, Z. and Gatt, S. (1968) Biochim. Biophys. Acta 152, 136-143. Ohman, R., Rosenberg, A. and Svennerholm, L. (1970) Biochemistry 2, 3774-3783. Venerando, B., Tettamanti, G., Cestaro, B. and Zambotti, V. (1975) Biochim. Biophys. Acta 403, 461-472. Tallman, J.F. and Brady, R.O. (1973) Biochim. Biophys. Acta 3, 434-443. O'Brien, J.S. (1977). Biochem. Biophys. Res. Commun. 2, 1136-1141. Rapin, A., Goldfisher, S., Katzman, R., Engel, J. and O'Brien, J.S. (1978) Ann. Neurol. 2, 234-242. Cantz, M. and Messer, H. (1979) Europ. J. Biochem. 97, 113-118. Sandhoff, K. and Pallmann, B. (1978) Proc. Natl. Acad. Sci. (USA) 75, 122-126. Lucy, J.A. (1969) in Lysosomes, part 2, ed. Dingle, J.T. and Fell, H.B. pp. 321-327, North-Holland Publishing Co.
1347
BIOCHEMICAL
MUCOLIPIDOSIS Gideon
Bach,
Marcia
Department
Received
September
TYPE IV:
AND BIOPHYSICAL
GANGLIOSIDE Tamar
Zeigler,
RESEARCH COMMUNICATIONS Pages 1341-1347
SIALIDASE
Schaap
and Gertrude
of Human Genetics, Hadassah-Hebrew Medical Center, Jerusalem, Israel 11,
DEFICIENCY Kohn
University
1979 SUMMARY
A solubilized sialidase is partially deficient in cultured fibroblasts derived from skin biopsies of four mucolipidosis IV patients, Fibroblasts from two obligate heterozygotes also have sialidase activity lower than normal controls, Membrane-bound sialidase activity is not affected in this disease. Based on previous and present findings, we propose that this solubilized activity is probably lysosomal origin. Sialidase activity in mucolipidosis IV cells is normal when neuraminlactose is used as substrate,, Mixing cell homogenates from mucolipidosis IV patients and frcm controls results in the expected combined sialidase activity, indicating the absence of an internal inhibitor in the deficient cells. It is therefore suggested that the mutation in mucolipidosis IV specifically affects a lysosomal ganglioside sialidase, while the remaining non-lysosomal sialidases partially mask this deficiency. Mucolipidosis
type
as an autosomal
recessive
by progressive
As in other
of both
patients disialo-GD3)
for
we present possible
increased
and acid
responsible
this
evidence metabolic
and bilateral
congenital
levels
defect
in ML IV,
sialidase Mucolipidosis Mucolipidosis Mucolipidosis
was observed was assayed IV II I
skin
yet been
transmitted is
cornea1 accumul-
materials
has been
of ML IV patients obtained
(both
resolved.
from ML IV and
defect In this
sialidase
paper
as the
In contrast
to the ganglioside
between
the ML IV and control
neuraminlactose
(1,2,5,6).
monosialo-GM3
metabolic
of a ganglioside
with
characterized
intralysosomal
fibroblasts
The basic
has not
the deficiency
when
organs
of gangliosides
for
fibroblasts
increased
in various
of cultured
accumulation
no difference
4),
and lipid-like
mucopolysaccharides.
deficiency,
Abbreviations:
(i',g)
disease
the disorder
(3,
microscopy
storage
Clinically,
glycoconjugates
analysis
indicated
a lysosomal
(1,2).
mucolipidoses
by electron
chemical
is
retardation
water-soluble
demonstrated Direct
trait
psychomotor
opacities. ation
IV (ML IV)
sialidase
as substrate,
- ML IV - ML II -ML1 0006-291X/79/201341-07$01.00/0 1341
Copyright All rights
@ 1979
by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALSANDMETHODS Cell culture Cultured skin fibroblasts obtained from four ML IV patients, five normal controls and two obli ate heterozygotes (parents of an ML IV patient) were propagated in 75 cmi plastic tissue culture flasks (Falcon) to a density of 5-7 x lo6 cells per flask (1 mg protein) and maintained under Cells were harvested 14 days after conditions previously described (7,8), the last passage. The pH of the mediumaffected the ganglioside sialidase activity, which was barely detectable if the mediumpH was greater than pH 7.4. All comparisons between the ML IV and the control fibroblasts were therefore made in cells grown below pH 7.2. the cells were Sialidase determinations - Following harvest by trypsinization, washed twice with 0.15 M NaCl, resuspended in 0.5 ml of 0.1% Triton X-100 and homogenized by teflon-glass hand homogenizer. The suspension was then sonicated (Braun sonic, 330s sonicator, small prove, B. Braun, Germany) for a total of 30 sec. with 15 sec. bursts and 15 seco cooling period between sonications. The insoluble debris was precipitated by centrifugation at 40,000 g for 30 min. at 4°C and the supernatant was collected and kept at O'C prior to analysis. The precipitate, when used for further analysis, was suspended in 0.5 ml of H20. Ganglioside sialidase was determined using GM3 (Hematoside) as a substrate obtained and purified from a spleen of an adult Gaucher patient according to Svennerholm (9). The reaction mixture contained 0.1 mg of GM3dissolved in chloroform-methanol 2:l (v/v), dried under nitrogen stream, 0.1 M acetate buffer pH 4.6 (optimal in both extraction fractions), 0.007% sodium azide, 0.1% Triton X-100 and 0.10-0.15 mg protein in a finalvolume of 0.2 ml. Incubation was carried out at 37OCfor six hours followed by the determination of free sialic acid according to Warren (lo), using the equation to eliminate interfering substances. Controls containing enzyme or substrate alone, and zero time controls were determined in parallel. Neuraminlactose sialidase activity was determined with 0.04 mg neuraminlactose (Sigma Chemicals, type II) as substrate in an incubation mixture similar to that described above. The mixture was incubated for three hours followed by the determination of free sialic acid. RESULTS Table 1 represents the neuraminlactose and ganglioside sialidase in the two extraction
fractions
for a typical
lactose as substrate normal enzyme activities natant and precipitate
of ML IV.
in the supernatant fluid the control,
ganglioside sialidase activities
were observed in both the super-
of ML IV showed reduced activity difference
X-100 or when ultrasonication and approximately
When homogenization was omitted,
all
the
60%of the neuraminlactose sialidase
were found in the precipitate.
between the ML IV and controls
when compared to
could be detected in the
of the MI IV and control precipitates.
was done without Triton
With neuramin-
On the other hand, ganglioside sialidase
whereas no significant
enzyme activities
experiment.
activities
Under these conditions no difference
could be observed.
1342
Another important finding
BIOCHEMICAL
Vol. 90, No. 4, 1979
Table
Sialidase
1.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activity
in
supernatant
fibroblast
and
precipitate
extracts Sialidase
Cell
Neuraminlactose
Control
(A)
Ganglioside
Neuraminlactose
Ganglioside
1.38
1.67
1.17
3.85
(V)
1.51
0.67
1.25
4.05
ML II
(K)
0
0.33
0
2.63
*
Activities per
shown
are
expressed
as nmoles
sialic
acid
formed
per
hour
mg protein.
1.
Whereas
a total
was observed
in both
extraction
in
activity
Table
ganglioside natant
Precipitate
IV
Ml
is
activity*
Supernatant
origin
of
sialidase
activity
and precipitate
with
the
the
results
deficiency
of neuraminlactose
fractions
was only
in ML II
partially
supernatant
sialidase
fibroblasts,
deficient
in both
activity
somewhat
typical
experiments,
super-
lower
than
in ML IV. Table
2 summarizes
ganglioside
sialidase
patients, between
controls
(e.g.,
sensitivity
supposedly the specific
activity
between
cell
lines
ranking
of cell
are
lower indicated
when experiments 0.575.21 Furthermore,
represented
same harvest by S.D. between
ratio
considered
to small
out of four
Duplicate varied
values
is
also
considerable,
However,
of MI IV to control (Table
50% decrease
1343
the
3).
ML IV
variation due to in
determinations slightly.
as compared
experiments
for
variations
only
experiments.
separately
in which
2 and 3) is probably
conditions,
an approximately
in three
The considerable
activity
in ML IV patients by the
are
indicate
differ
was determined
experiment
enzyme
culture
in the
lines
the supernatant
between
tissue
six
heterozygotes.
of this
uniform
was consistently
in
and obligate
experiments
the extreme
results
activity
from
of
The variation and the
enzyme activity
to controls.
These
enzyme activities The mean ratio,
in ML IV enzyme activity. ratio
of enzyme activities
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1344
Vol. 90, No. 4, 1979
BIOCHEMICAL
Table
3.
supernatant
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Ratios of ganglioside sialidase activities in the fraction of fibroblasts of the three genotypes
Experiment
MLIV
Heterozygotes Control
*
Control
NO.
1
0.80
2
0.70
0.55
3
0.54
0.70
4
0.60
0.51
5
0.43
6
0.71
1.00
7
0.60
8
0.59
9
0.64
10
0.24
11
0.23
12
0.75
13
0.55
14
0.64
Mean
0.57
0.69
S.D.
0.21
0.22
*
Ratio
of specific
of heterozygotes indicating considerable
similar
in these
between
variability
enzyme activity
were
activity
differences
experiment
activities.
to controls
a reduced
*
to those cell
lines
ML IV patients
makes it
difficult
of ML IV to controls as well.
In spite
and controls,
to define
of the
the inter-
the normal
and ML IV
ranges.
The possibility
of an internal
the ML IV cells
was ruled
amounts
of cell
protein
combined
values
inhibitor
out by mixing
experiments.
from ML IV and control
of ganglioside
of ganglioside
sialidase
Mixtures
lines
activity
sialidase
resulted (data
not
in
of equal in the
expected
presented).
DISCUSSION The significantly in ML IV patients
reduced and the
activity
accumulation
of solubilized of sialogangliosides
1345
ganglioside (6,7)
sialidase suggest
BIOCHEMICAL
Vol. 90, No. 4, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
that the ML IV disease is associated with ganglioside sialidase In three out of four determinations, also lower than controls;
the heterozygote
deficiency.
enzyme activity
was
however, a larger number of heterozygotes
should
be studied before firm conclusions can be drawn. The fact that neuraminlactose sialidase is not surprising
in the disease
since neuraminlactose and ganglioside sialidases
been shown to be two distinct by the total
is not affected
deficiency
enzymes.(18,21).
This finding
of neuraminlactose sialidase
while ganglioside sialidase
is unaffected
have
is supported
in ML I (11,12,20,21),
(22).
Sialidases have not been sufficiently
characterized
in fibroblasts,
but the existence of more than one sialidase has been demonstrated in liver (14,15),
brain (16,18),
and heart muscle (19).
sides sialidases have been localized
Furthermore, various ganglio-
to different
cell organelles (plasma
membrane, microsomes and lysosomes) (19,23). In view of the fact that the plasma membraneand the microsomal-bound enzymes are practically are generally
more readily
ganglioside sialidase sialidase
insoluble
(16,23) and that lysosomal hydrolases
soluble (24), we suggest that the supernatant
activity
might be greatly
while the precipitate
enriched by the lysosomal
is mainly non-lysosomal.
tion is supported by the findings
in the ML II cell extract,
characterized
deficiency
hydrolases.
by an intracellular
of the neuraminlactose sialidase
indicating
its mainly lysosomal origin. activity
This may indicate
is affected
of most lysosomal
activity
in this cell line,
However, not all ganglioside
in ML II,
a non-lysosomal origin
single mutation affecting be partially
in fibroblasts
which is
In agreement with previous studies (11-13) there was a total
deficiency
sialidase
This interpreta-
particularly
in the precipitate.
for the remaining activity.
only a lysosomal isoenzyme may therefore
maskedby the unaffected
remaining isoenzymes.
A at least
Further
separation of the presumed isoenzymes is needed for precise determination of the lysosomal sialidase,
particularly
1346
for diagnostic purposes.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Acknowledgement: The skillful technical assistance of Mrs. B. Medan is greatly appreciated. The project was supported by grants from the National Foundation - March of Dimes (Noel-599) and the Stiftung Volkswagen (No. 34048). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22. 23.
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