Download Glycogen Metabolism in the Normal Red Blood Cell

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Glycogen
Metabolism
By Shimon
Evidence
for
active
ism in normal
in the Normal
W. Moses,
glycogen
mature
Nava
Bashan,
metabol-
red
blood
related to RBC
HE
MATURE
glucose
(RBC)
has
meet
no
synthesis
contrast,
glycogen
tients
type
affected
with
VI).9’#{176}Sidbury
cells
is
is
not
broken
possible
metabolism
to
glycogen
study
the
mature
blood
tion
under
of radioactivity
active
glucose
Prom
Beer
the
Hadassa,
and
Submitted
accepted
10,
10,
by
W. Moses,
Research
1972;
USPHS
first
Grant
M.D.:
Lecturer,
revision
investigator,
Am
Head
and
the
either
and
in
degrada-
distribution
incorporation
Hospital
is also
metabolism
synthesis
University,
the
of
Negev
Hadassa
radio-
University,
Medical
School,
May
19,
1972;
second
revision
July
7,
1972;
12672-03.
of
Negev
Pediatric
Alisa
Gutman,
M.D.:
Senior
Hebrew
University,
Hadassa
836
Central
pa-
glycogen
enzyme
unless
glycogen
after
It
active
of normal
determining
molecule
Hebrew
cell.9
an
accumulates
and
In
of
development
of
glycogen
metabolism,
mature
of glycogen
Negev
Biochemistry,
present.48
7972.
Shimon
Senior
glycogen
be
Israel.
March
July
rates
Laboratory,
of
the
is missing.
investigate
conditions
cell
catalyzing
erythrocytes
maintains
in the presence
glycogen
to
the
active
in
blood
disease
(type
III and
in affected
red blood
in
an
degree
whereas
measuring
to
in
of
environmental
red
enzymes
shown
stage
of
significant
Research
Supported
Center;
cell,
Department
Jerusalem,
early
mature
erythrocyte
steady
state
favors,
within
the
into glycogen.
Pediatric
Sheva,
although
been
absence
incubation
on
normal
glycogen-storage
that
the glycogen
or phosphorylase
was undertaken
varying
14C-Uglycogen
substantial
deposition
be observed.
demonstrated
an
the
breakdown,
amylo-1,6-glucosidase
The present
red
of
any
the normal
in which
the
of
from
view
down
that
activities,
in
been
types
assumed
remnant
that,
may
depends
have
has
pH for
into
accumulation
of glycoin
erythrocytes
with
affecting
glycogen
The
stores,3
breakdown
certain
et al.
vestigial
erythrocyte
breakdown,
glycogen
glycogen
and
optimum
any significant
gen,
whereas
enzyme
defects
requirements.’2
deposition
Gutman
incorporation
ERYTHROCYTE
energy
significant
glycogen
a
its
Cell
7.6. Replacing
the radioactive
glucose
employed
for
incorporation
after 1 hr of incubation
with non labeled
glucose
resulted
in a gradual
loss of
radioactivity
from
erythrocyte
glycogen.
In normal
cells,
glycogen
synthesis
and breakdown
do not result
in
concentration
HUMAN
to
The
glucose
was pH
in the medium.
The major
part of the
incorporated
radioactivity
resided
in
the outer
branches
of the glycogen
T
Blood
and Alisa
molecule.
cells
(RBC)
is presented.
Initial
rates
of
‘4C-U-glucose
incorporation
into erythrocyte
glycogen
were
found
to be
independent
of substrate
concentration over a range
of 3.3-16.6
mM.
Incorporation
of label into glycogen
was
initially
linear
but reached
a plateau
after a variable
period
of time that was
Inversely
Red
Pediatrics
University,
Research,
Lecturer,
Medical
Clinical
School,
“B”
and
Pediatric
heva,
Beer-S
Soroka
Research,
israel.
Medical
Biochemistry,
Jerusalem,
Blood,
Nava
Soroka
Bashan,
Center,
Beer-Sheva,
Department
of
Medical
M.Sc.:
Israel.
Biochemistry,
Israel.
Vol. 40, No. 6 (December),
1972
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GLYCOGEN
METABOLISM
IN RED BLOOD
MATERIALS
The
various
enzymes,
Boehringer,
Mannheim,
Diazyme
reagent
was
glucose
was
obtained
Preparation
Fresh
drawn
fugation
After
pressed
the red
0.15 M
originally
The
before
CELL
AND
837
METHODS
coenzymes,
and
glycolytic
Germany
or Sigma
Chemicals,
obtained
from
Miles
Chemicals,
from
the
Radiochemical
intermediates
St. Louis,
Elkhart,
Center,
were
Mo.
Ind.
Amersham,
obtained
from
Radioactive
14C-U-
England.
of Eryfhrocytes
blood
was obtained
from
healthy
laboratory
personnel.
The blood
samples
were
into heparinized
tubes,
and the erythrocytes
(RBC)
were
sedimented
by centriin the cold.
aspiration
of the
supernatant
and
the buffy
coat,
the remaining
blood
was
through
a cotton-wool
sieve as described
by Busch
and Pelz.11
Two passages
of
blood
cells suspension
through
the syringe
containing
the cotton
wool
soaked
in
NaCl solution
were found
to remove
99% of leukocytes
and 98% of thrombocytes
present.
erythrocytes
subsequently
were washed
three times with 10 vol of cold 0.15 M NaCl
their use in the various
studies.
Analytical
Methods
Glucose
was determined
by the glucose
oxidase
method.12.13
Maltose
was measured
by
the Somogy
and Nelson
method,14
and glycogen
was measured
with diazyme
reagent.’5
‘4C-U-Clucose
incorporation
into glycogen
was measured
in an incubation
mixture
contaming
30% RBC suspension,
6.6 mM glucose,
2.5 Ci
14C-U-glucose,
15
mM
NaHPO4,
120
mM
glycylglycine
buffer,
in a shaking
acetic
acid
described.’8
and
scintillation
spectrometer.
Analysis
water
bath
carrier
the
7.8,
in
glycogen,
Radioactivity
of
pH
at 37#{176}C.
The
and
a final
volume
reaction
was
the
measurements
distribution
of
of
6 ml.
stopped
polysaccharide
were
14C-U-glycosyl
made
units
This
by
mixture
the
was
isolated
a
Packard
with
between
was
addition
the
incubated
of
as
trichloropreviously
Tricarb
outer
liquid
branches
and
the
limit dextrin
fraction
of the glycogen
was carried
out after p-amylolysis.17
Maltose,
liberated
by fl-amylase,
was separated
from
other
compounds
by descending
paper
chromatography
utilizing
Whatman
No. 1 paper.
The solvent
used
was butanolpyridine-water
(3:2,5:1,5
by v/v).
Separation
of glycogen
from
other
polysaccharides
was
performed
as follows:
after
14C-U-glucose
was incorporated
into intact
RBC as described
above,
an equal
volume
of
6%
HgC12 was added
to precipitate
the protein.
Nonlabeled
glycogen
was added
to the
supernatant.
The total glycogen
was subsequently
extracted
by repeated
alternate
precipitations
with
alcohol
and
resuspension
in water
as previously
described.7
The polysaccharide
suspension
was subsequently
run through
a Biogel-200
column.
Eluting
phase
was water.
Glycogen
and radioactivity
were determined
in each of the 2-ml fractions
collected
by a
fraction
collector.
RESULTS
Incorporation
of Glucose
Incubation
of
normal
of a radioactive
appearance
of
material.
radioactivity
concentration.
This
Biogel-200
column
polysaccharide
of
the
with
This
in
material
extracted
could
Glycogen
RBC
(Fig.
nonlabeled
incorporated
Into
1),
be recovered
material
a fraction
was
which
from
glycogen
‘4C-U-glucose
further
showed
erythrocytes
added.
In
as maltose
resulted
was
identified
that
sedimented
in
as
the
appearance
glycogen
at 60%
by
the
ethanol
characterized
by separation
on a
that the peaks
of the radioactive
were
addition,
identical
most
on f3-amylolysis
with
of
the
(Fig.
the
peaks
radioactivity
2).
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838
MOSES,
The rate of incorporation
cyte glycogen
was 0.04 ±
This
rate
could
not
have
of radioactivity
of
moles
glucose
0.01
been
due
to
BASHAN,
‘4C-U-glucose
incorporated/g
contamination
of
AND GUTMAN
into erythroHb per hr.
the
RBC
by
leuko-
cytes or thrombocytes.
The maximum
rate of glucose
incorporation
attributable
to the number
of leukocytes
and thrombocytes
remaining
in the cell suspension after the removal
procedure
would
have been 0.05 m&moles’8
and 0.03
m&moles,
the
respectively.’9
incorporation
The
reaction.
initial
As
linear
slope
shown
represents
in Fig.
the
the
2,
curve
net
rate
of
approaches
a
plateau
after 2 hr. indicating
a steady
state
that is the result
of an equilibrium
state between
the rates of incorporation
into and loss of radioactivity
from
glycogen.
On examining
the distribution
of radioactivity
within
the glycogen
molecule,
an Increase
of radioactivity
is observed
mainly
in the outer branches
and to a small
extent
(about
10%)
in the limit dextrin
fraction
(core).
To
exclude
the
from
physical
possibility
trapping
chromatographic
with
cold
similar
gen
separating
glycogen,
and
glycogen
was observed
16.6 mM
(Fig.
figure),
glucose
and
noted.
Further
No
for
the
resulted
during
the
first
carried
mixed
through
was
found
in the
spot.
of
‘4C-U-glucose
into
from
nature
of
erythrocyte
glycogen
glycogen
metabolism
in
blood
cells
was
obtained
in experiments
that
showed
that
replacement
radioactive
glucose
by nonlabeled
glucose
after
1 hr of incubation
resulted
a gradual
decrease
of radioactivity
from
RBC glycogen
(Fig. 4).
The
incorporation
maximum
maximum
of glucose
activity
being
for glycolytic
into
reached
activity.
glycogen
at
pH
was
7.6,
shown
which
is
to be pH
very
near
red
of
in
dependent,
to
the
pH
Fig.
1. Comparison
of peaks
of
added
cold glycogen
with radioactive
erythrocyte
glycogen
after
passage
through
Biogel-200
column.
Incubation of ABC
‘4C-U-glucose
was performed
as described.
After
denaturation with cold TCA, 20 mg of glycogen
were added
to supernatant.
Glycogen
0.
U
was
separated
by
cose
alcohol.
resuspended
plied
a
glyco-
concentration
was varied
from
in the low glucose
medium
shown
in the lower
part of the
in radioactivity
dynamic
core
was
was
in the maltose
incorporation
medium
glucose
3). After
2-hr
incubation
had almost
disappeared
(as
decrease
maltose
radioactivity
of
in the
molecule
mixture
when
a progressive
evidence
the
was detected
initial
rate
detected
glycogen
the
radioactive
subsequently
procedure.
all radioactivity
difference
in the
3.3 to
(3.3 mM),
radioactivity
within
procedure,
and
chromatographic
No
was
that
the
of maltose
to
collected
a Biogel-200
in which
column
radioactivity
60 X 3 elution
and
glycogen
phase-water.
were
from
radioactive alu-
repeated
precipitations
with
Precipitated
glycogen
was
in 5 ml of water
and ap-
Samples
determined.
of 2 ml were
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GLYCOGEN
METABOLISM
It is apparent
tion
medium
from
the
IN RED BLOOD
Fig. 5 that
incorporation
at low
rate
with
ing fresh cells
been incubated
a fresh
one
had
839
concentrations
of ‘4C-U-glucose
with time up to 2 hr. However,
when
the rate of incorporation
tended
to
phenomenon
could not be ascribed
to
in the composition
of the incubation
mixture
CELL
no effect
of RBC
into
in the
glycogen
the concentration
of cells
decrease
as a function
depletion
of substrate
or
medium,
since replacing
on
in an incubation
medium
resulted
in a normal
rate
this
phenomenon,
in which
other
of incorporation.
Fig. 2. Distribution
of incorporated
‘4C-U-gtucose
between
outer tiers and
limit dextrin
fraction.
Aliquots
of glycogen
were
exposed
to /3-amylolysis.
Subsequently,
separation
of
outer
branches
from
the limit
dextrin
fractions
(core)
was performed
by paper
chromotography,
and radioactivity
of
the various
fractions
was determined.
incuba-
was
linear
was increased,
of time.
This
other
changes
the incubation
whereas
incubat-
had
previously
cells
2
0
3
Hours
20
U
15
E
10
.
5
Gkicose- Cncorporohon
___________________________
2
Glucose
utihsotion
0
IS
0
E
----
0
Hours
Fig. 3. ‘4C-U-Glucose
incorporation
into glycogen
and glucose
utilization
with two substrate
concentrations.
One
milliliter
of
ABC
was
suspended
in
ml of incubation
medium
containing
3.3 (open
circles)
or 16.6 (closed
cirdes)
mM glucose.
Samples
of medium
were
removed
at specified
intervals
and were
analyzed
for the radioactivity
glycogen.
for glucose
incorporated
and
into
2
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840
MOSES,
BASHAN,
AND GUTMAN
Fig. 4. Pulse
studies
of glucose
incorporation
into
glycogen
in normal
erythrocytes.
Initial
incubation
condition as in Methods.
After
1 hr of incubation,
red blood
cells were washed
in 0.15 M NaCI and reincubated
in an
incubation
mixture
as before,
but without added
radioactivity.
Open
circles
represent
cells
reincubated
in nonradioactive
incubation
mixture.
Black
dots represent
controls.
25
Fig. 5. Effect
of ABC concentration
on rate of glucose
incorporation
into
glycogen.
RBC suspensions
of different
concentrations
were
incubated
with
‘4C-U-glucose.
After
2 hr, cells
from
tube
containing
highest
ABC
concentration
were
separated
from
medium
and washed
with
cold
0.9#{176}/o
NaCI
solution.
(A) Cells
were
subseE
quently
reincubated
in fresh
incuba.
tion
medium.
(B)
Separated
incubation
medium
was used for incubation
with
fresh
ABC. Black
circles,
0.5 ml ABC;
open
circles,
1.0
ml
ABC;
black
squares;
2.0 ml ABC. (A),
(B)
Final
volume
of
incubation
mixture
was 4 ml. Composition of incubation
mixture
as described
---‘-#{149}-.
in methods.
flme
(hours)
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GLYCOGEN
METABOLISM
To establish
of the mature
blood
cell
suspension
the
younger
older
cell
than
that the glucose
erythrocyte
and
suspension,
cell
counts
IN RED BLOOD
the
was
exposed
were
observed
higher
density
last
fraction
ing
that
that
but
of mature
this
conclusion
the
blood
was
stored
for
24
stored
red
blood
hr
the
cells,
as well
by
ACD
in
suspension
into
did
show
is not
only
(Table
1).
no
the
: red
order
to
blood
separate
density,
from
high
glycogen
cells.
incorporation
which
had
in
in which
older
of reticulocytes
presented
in
cell
cells
a function
in the red
performed
hr
1
‘4C-U-glucose
of incorporation
is also
present
to be of lower
of younger
comprising
devoid
for
are known
incorporated
was
were
centrifugation
fraction
RBCs
capacity
red
to
into glycogen
of reticulocytes
experiments
which
The
841
incorporation
not only
following
erythrocytes,
population.
CELL
more
However,
readily
even
incorporation,
a function
Further
evidence
performed
reticulocytes
were
capacity
to incorporate
this
indicat-
of
studies
the
reticulocyte
reticulocytes
to support
on
found;
blood
yet
glucose
this
into
glycogen.
DISCUSSION
This
report
tains
an
formed
active
Most
the
radioactivity.
metabolism
present
limit
The
glycogen
experiment
in the
steady
that
blood.
Both
to
the
can
mature
not
column
erythrocyte
be
main-
attributed
separation
to
state,
reached
into
glycogen,
and
indicate
that
the incorporation
of radioactive
and not into
other
components
of the cell.
in the outer
tiers
of the glycogen
molecule,
the
rate
after
of
contains
only
small
amounts
of
of radioactivity
from
erythro-
incorporation,
mm
30
units
of
initial
as
12.5%
for
2 hr.
from
shown
increase
equilibrium
erythrocyte
in
RBC
The
linear
an
incubation
with
time
an
represents
glycosyl
whereas
by
the
the
glycogen.
presence
concentration
factors
of
pulse
‘4C-U-glucose
between
entry
The
of
50%
incorporation
responsible
for
this
and
plateau
is
RBC
conis
a function
of
Table
of Incorporation
of 14C-U-Glucose
Into Glycogen
in Red Blood
Separated
According
to Their Relative
Densities
or Stored
1. Rates
other
experiments
4).
exit of radioactive
approached
after
centration,
that
dextrin
fraction
(core)
initial
rate of disappearance
is similar
(Fig.
incorporation
as
indicating
breakdown
studies
is indeed
into
glycogen
of the
label
is found
whereas
The
evidence
glycogen
elements
enzymatic
glucose
cyte
presents
linear
phenomenon
Rates
Cells
of
Incorporation
Retlculocytes
(#{176}/o)
‘Centrif
by careful
obtained
Activity
(mmole/mI
RBC/hr)
Unspun RBC
Upper layer of spun RBC’
Lower layer of spun ABC’
1.3
3.9
0
20.1
42
12.5
RBC
0
12.1
stored
for24
hr in ACD
ugatlon
was performed
at 4#{176}C,
25,000 g for 60 nihi. Upper
layer was obtained
aspiration
of approximately
10#{176}/o
of top fraction of spun ABC. Lower layer was
by free flow of lowest fraction
from bottom
of punctured
centrifugation
tube.
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842
MOSES,
are not immediately
reach
a plateau
was
BASHAN,
AND GUTMAN
The observation
that the time required
to
of RBC concentration
does not support
the
possibility
that
a time-dependent
running
down
of the synthetic
mechanism
of the cells was responsible
for the observed
phenomenon.
The fact that most of the radioactivity
resided
in the outer
tiers of the
glycogen
molecule
suggests
that
under
the conditions
of the experiment
very little branching
of the newly
formed
chains
took
place.
The
limitation
imposed
on de novo
contribute
to the
glycogen
slowing
of this phenomenon
The accumulation
not be implicated
by
suspension.
environment
increasing
cell
RBC
hemoglobin
important
factor
of
a function
special
of
interest
metabolism
by
the
rate
of branching
but
can
not
of synthesis
It has,
therefore,
takes
place
concentration.
could
possibly
regulating
the
glycogen
metabolism.
The separation
and
only
synthesis
down
may
explain
the
possibly
dependence
on the concentration
of RBC in the incubation
mixture.
of an inhibitory
factor
in the incubation
medium
could
in the observed
decrease
in the rate of incorporation
in
concentrated
cell
in the intracellular
rated
apparent.
a function
aging
in view
the
of
in
that
mature
that
that
oxygen
glycogen
presence
be
an
an
involved
as
of
of
might
metabolism
erythrocytes,
well-known
a change
is accele-
saturation
pH and
of enzymes
intracellular
activities
establish
but
of
be assumed
incubation
Differences
change
relative
studies
reticulocytes
to
during
is not
well.
active
in
This
is
glycogen
in young
nucleated
red cell precursors
that seems
to be retained
form in the mature
red blood
cell.
The steady
state present
in the normal
red blood
cell does not lead to
glycogen
accumulation.
This may be related
to the minimal
functional
activity
of glycogen
synthetase,2#{176} in contrast
to markedly
higher
activities
of the
enzymes
catalyzing
glycogen
breakdown.8
In abnormal
conditions,
namely
in the absence
of erythrocyte
amylo-1,6-glucosidase
and of phosphorylase,
in a vestigial
yet
the
normal
resulting
in
steady
glycogen
the
normal
functional
state
of glycogen
accumulation
that
synthesis
may
and
breakdown
is
reach
several
hundred
upset,
times
level.
REFERENCES
1. Murphy,
J. R.: Erythrocyte
metabolism.
H. Glucose
metabolism
and
pathways.
J.
Lab. Clin. Med. 55:286,
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1972 40: 836-843
Glycogen Metabolism in the Normal Red Blood Cell
Shimon W. Moses, Nava Bashan and Alisa Gutman
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