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Human mannosidosis — The enzymic defect
BIOCHEMICAL
Vol. 49, No. 2,1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HUMAN MANNOSIDOSIS -Carroll,
M.
N. Dance,
THE ENZYMIC DEFECT
P. K. Masson,
D. Robinson
and B. G. Winchester
Department of Biochemistry Queen Elizabeth College Campden Hill London Wg 7AH Received
August
31,1972
Summary Normal human liver a-mannosidase exists in at least 3 forms, separable by DEAE cellulose chromatography. The A and B forms are most active at pH 4.4 In two cases of mannosidosis, while activity of form C is maximal at pH 6.0. examined by ion exchange chromatography and isoelectric focusing, both A and B forms were absent and the residual a-mannosidase activity was due to the presence of the C form in normal amounts. The presence tissues rare
of a-mannosidase
including but
human (1,2).
Genetic
a case has been described
oligosaccharides activity
in the tissues Hultberg
(3).
and found optimum
it
to have
than
(4)
the normal
separated human liver of which
are absent
Materials
accumulation
the residual lower
molecular
has now
in liver
With
at approximately
but
they
normal
into
two patients
levels
in
were
stored
patient
and more neutral
of the existence could
the use of ion
from
a-mannosidase
in this
weight
been separated
samples
enzyme are
of mannose-rich
activity
The possibility
focusing.
this
by a decreased
humans was raised
in
a-mannosidase
Form C remained
examined
in many mammalian
involving
was accompanied
enzyme.
by isoelectric
disorders
in which
an apparently
one form of a-mannosidase
of more than
not
exchange
be clearly chromatography
at least with
pli
3 forms,
two
mannosidosis.
the affected
individuals.
and Methods
Post-mortem
samples
to Dr.
P. A. &kerman
Tissue
homogenates
pH 6.0
at 4O with
at 36,000
of normal
for
liver
the frozen
(10% w/v) a ground
g supernatants
(Whatman DE23, syringes
has been demonstrated
were glass
were
W. R. Balston,
and pre-equilibrated
Copynkht 0 1972 by Academic Press, Inc. AN rights of reproduction in any form reserved.
We are grateful
liver
samples
from mannosidosis
prepared
in 0.01
M sodium
homogeniser.
applied Maidstone, with
at -12O.
0.01
to ion
After exchange
Kent, M sodium 579
U.K.)
phosphate
centrifugation columns packed
phosphate
patients.
in
buffer, for
5 min
of DEAB cellulose 2 ml disposable
buffer,
pH 6.0.
Vol. 49, No. 2, 1972
Samples
were
chloride
BIOCHEMICAL
first
eluted
gradient
at a flow
(O-O.2
the same buffer
M) in that
buffer.
and then
and assayed
with
mannopyranoside
substrate
(Koch Light
Laboratories,
The final
(1 ml)
assay 0.05
pH 6.0.
M sodium
After
citrate
incubation
fluorescence
measured
Isoelectric was carried
out
(1.5
ml enzyme sample
for
of distilled
110 ml capacity
(6).
and assayed
M sodium
collected
water
homogenates,
40 h the
in
buffer, and
(5).
centrifuged
as above,
in an LKB column
column
that
U.K.).
phosphate
assays
a pH 3-8 gradient
After
Bucks.,
was stopped
glycosidase
as above except
at pH 4.0 and 6.0 were
ml) were
and 1.0 mM substrate
other
producing
sodium
Colnbrook,
pH 4.4 or 0.05
as described
a linear
4-methylumbelliferyl-o-D-
2-4 h at 30° the reaction
in ampholines
2.2 ml fractions
0.2
buffer, for
focusing
approximately
buffers
contained
with
Fractions
of 60 ml/h
either
rate
with
AND BIOPHYSICAL RESEARCH COMMUNJCATIONS
was drained
of
off
McIlvaine
phosphate-citrate
and shift
of pH optimum
into
used.
Results The large towards
decrease
in u-mannosidase
a more neutral
value
in mannosidosis
has been confirmed.
At pH 4.4 activities
nmoles/min/g
tissue,
within
mannosidosis
liver
samples
The loss
of activity
lo-73
the normal
in
On DEAE cellulose resolved
into
to 8.1
components
A and the two peaks
concentration
gradient
active
curves
at pH 4.4
optimum
pH of 6.0
In contrast eluted
with than
were
range
(7),
from
while
the
marked,
the a-mannosidase
70 to 80
tissue.
falling
of normal
The unabsorbed
1).
of activity
at pH 4.4
at pH 6.0.
varied
(4)
from
in mannosidosis.
named B and C.
maxima
liver
of 1.2 and 3.3 nmoles/min/g
and 31.4
(Fig.
to normal
liver
at pH 6.0 was much less
a-mannosidase
pH-activity
normal
had activities
chromatography
three
as compared of normal
the accepted
measured liver
activity
eluted
liver
activity
was called
by the chloride
Forms A and B had very
and were
about
Form C was about
was
twice
three
times
as active
similar more at its
as at pH 4.4. almost
all
as form C, the amount
the activity of this
present form being
580
in mannosidosis closely
comparable
liver
was
to the
BIOCHEMICAL
Vol. 49, No. 2, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IO
5 ‘; .C 5
0
t !
IO
L
f i P. .% 24
5
0 IO
20 Fraction
30
40
50
Number
Fig. 1. DEAE-cellulose column chromatography of supernatant from homogenates of (a) control human liver, (b) mannosidosis a-mannosidase activity at pH 6.0; o, u-mannosidase activity NaCl gradient.
fractions (2 ml) liver. l , at pH 4.4; -,
8-
6.
12 IO 3 6 4
2 PH ‘*.
b
Fraction Number
Fig. 2. Isoelectric focusing over the range pH 3-8 of supematant fractions (2.4 ml) from homogenates of (a) control human liver, (b) mannosidosis liver. a, a-mannosidase activity at pH 6.0; o, a-mannosidase activity at pH 4.0; ---, pH gradient.
581
Vol. 49, No. 2,1972
amount
BIOCHEMICAL
of a-mannosidase In order
forms
to differentiate
of the enzyme,
isoelectric
the main
possible higher
subsidiary
as far
activity
from normal
liver
at about
Isoelectric
focusing
5.7
although
suggesting
at pH 6.0,
two sharp
peaks
However
the
peak was associated
of
‘neutral’
2).
gave clear
appearing
with
with
only
pI values
a white
that
the enzymic
showed activities
at this
peaks
with
Measured
to ‘acid’
form C was isoelectric liver
assayed
(Fig.
the
None of the peaks
the ratio
of mannasidasis
from
had a pI of 5.9-6.0
and 6.3.
that
and neutral
of the eluates
component
at pH 5.6,
the focusing
acid
at pH 4.0 and 6.0
at pH 6.0
latter
between
was assayed
was highest
during
as possible
columns
peaks
activities
RESEARCH COMMUNICATIONS
samples.
the a-mannosidase
focusing
at pH 4.0
C in normal
AND BIOPHYSKM
point.
when
of 5.6 and 4.9.
precipitate
that
formed
procedure.
Discussion From these is
a simultaneous
a-mannosidase
Gourlay
is
appears
of the
‘acidic’
normal.
have been reported (9)
‘neutral’ tempting
suggested activity
enzymes,
more neutral
activity
activity
appeared
in the soluble
fraction
of rat
that
cattle
of the affected
comparable
is
differently
that
closely
cattle
optimum
found
is still
isoelectric
focusing
an apparent
pI of 4.9
that
central, located
(lo),
have still
the residual
heterogeneous profile.
It
arises
from
is
and Marsh
and
in the lysosamal liver
with
In a deficiency
human mannoeidosis
although
the residual
5.5-6.0 separable
activity
be overlooked
the association
however
in contrast farms
in human
in view that
It
related
the C form,
of about
and
homogenates.
ta be reported.
possible
582
‘neutral’ a-mannosidase
the cell.
a-mannosidase cannot
(8)
while in
resembles
normally
the
A and B are closely
had a pH optimum
to the human enzymes
The possibility mannosidosis
human a-mannosidases
the same genetic
in mannasidosis
and ‘neutral’
‘acid’
under
ta the pH 4.0-4.5
‘acid’
tissues
pH optimum,
of Angus
Similar
in rat the
defect
A and B activities,
and rabbit
that
to suppose
lysasamal
disease
loss
it
C remaining
activities
the
experiments
of the
the peak with
of a-mannosidase
C of
BIOCHEMICAL
Vol. 49, No. 2, 1972
pI 5.6 with This and Mental
the aggregated work
was supported
Health
Research
material
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
by grants
eeparated
in this
from the Medical
region.
Research
Council
Fund.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
J. P. B. J. B. D. B. A. I. c. J.
Conchie and A. J. Hay, Biocham. J., 12, 327 (1959). A. &kerman, Clin. Chim. Acta, 23, 479 (1969). Kjellman, I. Gamstorp, A. Brun, P. A. Ackerman and B. Palmgren, Pediat., 3, 366 (1969). Hultberg, Stand. J. clin. Lab. Invest,, 26, 155 (1970). Robinson, R. G. Price and N. Dance, Biochem. J., 102, 525 (1967). G. Winchester, Biochem. J., 124. 929 (1971). Lundquiet and P. A. Ockerman~nzym. biol. clin,, l0, 300 (1969). Suzuki, H. Kuahida and H. Shida,, Seikagaku, 42-, 361 (1970). A. Marsh‘and G. C. Gourlay, Biochim. biophys. Acta, 235, 142 (1971). D. Hocking, R. D. Jolly and R. D. Batt, Biochem. J.,-i& 69 (1972).
583
Vol. 49, No. 2,1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HUMAN MANNOSIDOSIS -Carroll,
M.
N. Dance,
THE ENZYMIC DEFECT
P. K. Masson,
D. Robinson
and B. G. Winchester
Department of Biochemistry Queen Elizabeth College Campden Hill London Wg 7AH Received
August
31,1972
Summary Normal human liver a-mannosidase exists in at least 3 forms, separable by DEAE cellulose chromatography. The A and B forms are most active at pH 4.4 In two cases of mannosidosis, while activity of form C is maximal at pH 6.0. examined by ion exchange chromatography and isoelectric focusing, both A and B forms were absent and the residual a-mannosidase activity was due to the presence of the C form in normal amounts. The presence tissues rare
of a-mannosidase
including but
human (1,2).
Genetic
a case has been described
oligosaccharides activity
in the tissues Hultberg
(3).
and found optimum
it
to have
than
(4)
the normal
separated human liver of which
are absent
Materials
accumulation
the residual lower
molecular
has now
in liver
With
at approximately
but
they
normal
into
two patients
levels
in
were
stored
patient
and more neutral
of the existence could
the use of ion
from
a-mannosidase
in this
weight
been separated
samples
enzyme are
of mannose-rich
activity
The possibility
focusing.
this
by a decreased
humans was raised
in
a-mannosidase
Form C remained
examined
in many mammalian
involving
was accompanied
enzyme.
by isoelectric
disorders
in which
an apparently
one form of a-mannosidase
of more than
not
exchange
be clearly chromatography
at least with
pli
3 forms,
two
mannosidosis.
the affected
individuals.
and Methods
Post-mortem
samples
to Dr.
P. A. &kerman
Tissue
homogenates
pH 6.0
at 4O with
at 36,000
of normal
for
liver
the frozen
(10% w/v) a ground
g supernatants
(Whatman DE23, syringes
has been demonstrated
were glass
were
W. R. Balston,
and pre-equilibrated
Copynkht 0 1972 by Academic Press, Inc. AN rights of reproduction in any form reserved.
We are grateful
liver
samples
from mannosidosis
prepared
in 0.01
M sodium
homogeniser.
applied Maidstone, with
at -12O.
0.01
to ion
After exchange
Kent, M sodium 579
U.K.)
phosphate
centrifugation columns packed
phosphate
patients.
in
buffer, for
5 min
of DEAB cellulose 2 ml disposable
buffer,
pH 6.0.
Vol. 49, No. 2, 1972
Samples
were
chloride
BIOCHEMICAL
first
eluted
gradient
at a flow
(O-O.2
the same buffer
M) in that
buffer.
and then
and assayed
with
mannopyranoside
substrate
(Koch Light
Laboratories,
The final
(1 ml)
assay 0.05
pH 6.0.
M sodium
After
citrate
incubation
fluorescence
measured
Isoelectric was carried
out
(1.5
ml enzyme sample
for
of distilled
110 ml capacity
(6).
and assayed
M sodium
collected
water
homogenates,
40 h the
in
buffer, and
(5).
centrifuged
as above,
in an LKB column
column
that
U.K.).
phosphate
assays
a pH 3-8 gradient
After
Bucks.,
was stopped
glycosidase
as above except
at pH 4.0 and 6.0 were
ml) were
and 1.0 mM substrate
other
producing
sodium
Colnbrook,
pH 4.4 or 0.05
as described
a linear
4-methylumbelliferyl-o-D-
2-4 h at 30° the reaction
in ampholines
2.2 ml fractions
0.2
buffer, for
focusing
approximately
buffers
contained
with
Fractions
of 60 ml/h
either
rate
with
AND BIOPHYSICAL RESEARCH COMMUNJCATIONS
was drained
of
off
McIlvaine
phosphate-citrate
and shift
of pH optimum
into
used.
Results The large towards
decrease
in u-mannosidase
a more neutral
value
in mannosidosis
has been confirmed.
At pH 4.4 activities
nmoles/min/g
tissue,
within
mannosidosis
liver
samples
The loss
of activity
lo-73
the normal
in
On DEAE cellulose resolved
into
to 8.1
components
A and the two peaks
concentration
gradient
active
curves
at pH 4.4
optimum
pH of 6.0
In contrast eluted
with than
were
range
(7),
from
while
the
marked,
the a-mannosidase
70 to 80
tissue.
falling
of normal
The unabsorbed
1).
of activity
at pH 4.4
at pH 6.0.
varied
(4)
from
in mannosidosis.
named B and C.
maxima
liver
of 1.2 and 3.3 nmoles/min/g
and 31.4
(Fig.
to normal
liver
at pH 6.0 was much less
a-mannosidase
pH-activity
normal
had activities
chromatography
three
as compared of normal
the accepted
measured liver
activity
eluted
liver
activity
was called
by the chloride
Forms A and B had very
and were
about
Form C was about
was
twice
three
times
as active
similar more at its
as at pH 4.4. almost
all
as form C, the amount
the activity of this
present form being
580
in mannosidosis closely
comparable
liver
was
to the
BIOCHEMICAL
Vol. 49, No. 2, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IO
5 ‘; .C 5
0
t !
IO
L
f i P. .% 24
5
0 IO
20 Fraction
30
40
50
Number
Fig. 1. DEAE-cellulose column chromatography of supernatant from homogenates of (a) control human liver, (b) mannosidosis a-mannosidase activity at pH 6.0; o, u-mannosidase activity NaCl gradient.
fractions (2 ml) liver. l , at pH 4.4; -,
8-
6.
12 IO 3 6 4
2 PH ‘*.
b
Fraction Number
Fig. 2. Isoelectric focusing over the range pH 3-8 of supematant fractions (2.4 ml) from homogenates of (a) control human liver, (b) mannosidosis liver. a, a-mannosidase activity at pH 6.0; o, a-mannosidase activity at pH 4.0; ---, pH gradient.
581
Vol. 49, No. 2,1972
amount
BIOCHEMICAL
of a-mannosidase In order
forms
to differentiate
of the enzyme,
isoelectric
the main
possible higher
subsidiary
as far
activity
from normal
liver
at about
Isoelectric
focusing
5.7
although
suggesting
at pH 6.0,
two sharp
peaks
However
the
peak was associated
of
‘neutral’
2).
gave clear
appearing
with
with
only
pI values
a white
that
the enzymic
showed activities
at this
peaks
with
Measured
to ‘acid’
form C was isoelectric liver
assayed
(Fig.
the
None of the peaks
the ratio
of mannasidasis
from
had a pI of 5.9-6.0
and 6.3.
that
and neutral
of the eluates
component
at pH 5.6,
the focusing
acid
at pH 4.0 and 6.0
at pH 6.0
latter
between
was assayed
was highest
during
as possible
columns
peaks
activities
RESEARCH COMMUNICATIONS
samples.
the a-mannosidase
focusing
at pH 4.0
C in normal
AND BIOPHYSKM
point.
when
of 5.6 and 4.9.
precipitate
that
formed
procedure.
Discussion From these is
a simultaneous
a-mannosidase
Gourlay
is
appears
of the
‘acidic’
normal.
have been reported (9)
‘neutral’ tempting
suggested activity
enzymes,
more neutral
activity
activity
appeared
in the soluble
fraction
of rat
that
cattle
of the affected
comparable
is
differently
that
closely
cattle
optimum
found
is still
isoelectric
focusing
an apparent
pI of 4.9
that
central, located
(lo),
have still
the residual
heterogeneous profile.
It
arises
from
is
and Marsh
and
in the lysosamal liver
with
In a deficiency
human mannoeidosis
although
the residual
5.5-6.0 separable
activity
be overlooked
the association
however
in contrast farms
in human
in view that
It
related
the C form,
of about
and
homogenates.
ta be reported.
possible
582
‘neutral’ a-mannosidase
the cell.
a-mannosidase cannot
(8)
while in
resembles
normally
the
A and B are closely
had a pH optimum
to the human enzymes
The possibility mannosidosis
human a-mannosidases
the same genetic
in mannasidosis
and ‘neutral’
‘acid’
under
ta the pH 4.0-4.5
‘acid’
tissues
pH optimum,
of Angus
Similar
in rat the
defect
A and B activities,
and rabbit
that
to suppose
lysasamal
disease
loss
it
C remaining
activities
the
experiments
of the
the peak with
of a-mannosidase
C of
BIOCHEMICAL
Vol. 49, No. 2, 1972
pI 5.6 with This and Mental
the aggregated work
was supported
Health
Research
material
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
by grants
eeparated
in this
from the Medical
region.
Research
Council
Fund.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
J. P. B. J. B. D. B. A. I. c. J.
Conchie and A. J. Hay, Biocham. J., 12, 327 (1959). A. &kerman, Clin. Chim. Acta, 23, 479 (1969). Kjellman, I. Gamstorp, A. Brun, P. A. Ackerman and B. Palmgren, Pediat., 3, 366 (1969). Hultberg, Stand. J. clin. Lab. Invest,, 26, 155 (1970). Robinson, R. G. Price and N. Dance, Biochem. J., 102, 525 (1967). G. Winchester, Biochem. J., 124. 929 (1971). Lundquiet and P. A. Ockerman~nzym. biol. clin,, l0, 300 (1969). Suzuki, H. Kuahida and H. Shida,, Seikagaku, 42-, 361 (1970). A. Marsh‘and G. C. Gourlay, Biochim. biophys. Acta, 235, 142 (1971). D. Hocking, R. D. Jolly and R. D. Batt, Biochem. J.,-i& 69 (1972).
583