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Regulation
of Glucose-6”Phosphate
Activity
in Red
Blood
Cells
Nonhemolytic
Because
of the strong
inhibition
dehydrogenase
concentrations
of NADPH
O.1#{176}/o-0.2#{176}/o
of Its potential
Is estimated
cells
NADP
contain
and
low
glucose
phogluconate
inhibited
diphosphoglycerate
red
under
ATP,
the
also
and
2,3-
Tripler,
T
are
with
HE DISCOVERY
ated with genetic
cose
to
Gd Alhambra,
associated
6-phospate:
many
NADP
reduction
of
pathway
to produce
From
the
by
University
Division
of
of
to
of
14,
Ph.D.
Center,
strongly
under
inhibited
physiologic
GM
: Director,
Duarte,
than
this
Biochemical
revised
Genetics,
Under
During
1972;
in red
1.1.1.49)
of
this
(G6PD)
catalyzes
pathway
of carbohydrate
(more
Grants
Washington,
Medical
are
variants
NADPH.
lactate.
USPHS
Yoshida,
Medical
cells,
E.C.
genetic
primarily
August
Supported
Akira
National
NADP
Department
Calif. 91010.
Submitted
the
dehydrogenase
oxidation
of
is metabolized
red
hemo-
oxidoreductase,
investigations
glucose
subjects
normal
City
12, 1972;
October
and
HL
no
of
University
blood
the
of
Washington
cells’
in
initial
step
metabolism
the
net
Medical
accepted
in the
caus-
conditions,
generation
Hope
led
man.
Embden-Meyerhof
October
of NADH
Center,
13,
Duarte,
1972.
15125.
of Biochemical
Genetics,
Formerly
Research
Professor
98195.
Mei Lin, M.S. : Research
Wash.
a
associ(D-glu-
enzyme
Department
Calif.
91010;
Seattle,
was
physiologic
via
90%)
process
Genetics,
15253
by
conditions.
that
primaquine-induced
hemolytic
anemia
deficiency
of glucose-6-phosphate
dehydrogenase
Glucose-6-phosphate
hexose
monophosphate
ing
and Gd
chronic
Lin
cells
to maintain
an adequate
level
of
reduced
glutathione.
(2) The nonhemolytic
variant
enzymes
are far less sensitive
to
the inhibition
by NADPH,
because
of their
low Km for NADP and high K5 for NADPH.
These
variants
are also more resistant
to
the inhibition
by ATP.
The
activities
of
these variant
enzymes
in red cells are estimated to be more than 3O/
of the normal
level. In black
people,
the common
variant
A-,
associated
with enzyme
deficiency,
is
less sensitive
to the inhibition
by NADPH,
and it IS probably
as active
as normal
in
physiologic
conditions,
and only
about
O.2#{176}/o
of Its
potential
activity
Is estimated
to be expressed
In red cells.
The very low shunt
pathway
activity
can be explained
by these
findings.
Some human
g!ucose-6-phosphate
dehydrogenase
variants
that are associated
with severe enzyme
deficiency,
such as Gd
Union,
Gd
Markham,
and
Gd
Mediterranean,
have no chronic
hemolytic
problem. On the other
hand,
several
variants
with less severe
enzyme
deficiency,
such
as Gd Manchester,
Mei
physiologic
concentration
of NADPH
due
to their high Km for NADP
or low
K1 for
NADPH.
Thus, these
vaniar.t
enzymes
prosumably
cannot
generate
NADPH
in red
6-Phosis
NADPH,
ant
of
6-phosphate.
by
only
in human
and
lytic
anemia.
Examination
of
the
enzymes
from
these
variant
subjects
under
simulated
physiologic
conditions
revealed
that: (1) Enzymes
from the hemolytic
van-
(Vmax)
concentrations
dehydrogenase
strongly
Hemolytic
Subjects
and
physiologic
and ATP,
activity
to be expressed
that
Yoshida
of glucoseby
From
Variant
By Akira
6-phosphate
Dehydrogenase
School
of
Medicine,
City
of Hope
of Medicine,
Technician,
Seattle,
Wash.
98195.
©
1973
Bl#{248}od,Vol.
by
Grune
& Stratton,
41, No. 6 (June),
Inc.
1973
877
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
878
YOSHIDA
occurs,
phate
since
1 mole
dehydrogenase,
dehydrogenase.
of NAD
while
Lactate
producing
NADH
However,
since
can
and
the
is reduced
1 mole
of
be
to NADH
by glyceraldehyde-3-phosNADH
is oxidized
to NAD
by
oxidized
NADPH
matured
in
human
by
the
red
the
aerobic
nucleated
cell
oxidative
cells
lacks
the
of
to maintain
to be
glutathione
necessary
perhaps
the
in
enzyme
with
is frequently
hemolytic
The
tissues.
enzymes
associated
determinant
X-chromosome
from
one
enzyme
deficiency
festation.
exogenous
Another
agents,
occur.
Other
nonspherocytic
problem
deficiency,
such
other
level
of
glutathione
reduced
of
characteristics
glucose-6-phosphate
appears
cell
of
and
this
glutathione
have
and
associated
enzyme
deficiency
anemia
even in the
some
Union7
variants
and
less
severe
Gd
Alhambra,9
disease
even
enzyme
these
variant
for the different
the
with
very
such
Tripler,’#{176}
of
any
or
clinical
mild
mani-
but requires
hemolysis
to
severe
no hemolytic
deficiency,
Cd
distin-
activity
associated
with
chronic
of exogenous
agents.
A
have
absence
are
deficiency
beans,
for
are
absence
Markham,8
and
in
with
associated
Gd
is located
which
normal
has severe
enzyme
infections,
or fava
with
of
variants,
of variants
as drugs,
with
not
80
group
such
is that
reason
dehydrogenase
about
variants
are
variants
the
a low
therefore,
as Gd
hemolytic
explain
with
Reduced
groups
within
the red
severe
genetic
deficiency
and,
(unpublished),
ter
chronic
state.
sulfhydryl
Thus,
in man.5
Among
another,6
about
40
variants
hemolytic
puzzling
while
reduced
of
of the
cells
has
a
by the red
anemia.
genetic
on the
guishable
in the
for maintaining
red cell surface.2
lactate
various
oxidative
LIN
pathway,
Krebs
cycle,
the hexose
monophosphate
shunt
pathway
in red
particular
importance
in generating
NADPH.
NADPH
is required
cell
AND
are
exogenous
enzymes
reported
in the
hemolytic
manifestations
enzyme
problem,
as Gd
Manches-
associated
with
agents.
Kinetic
literature
of these
cannot
variant
subjects.
In
this
paper,
we
variant
glucose-6-phosphate
ditions
associated
and
clinical
problem.
will
with
will
discuss
try to
hemolytic
elucidate
anemia,
MATERIALS
Normal
and
Variant
the
regulatory
dehydrogenase
Human
mechanism
activity
why
certain
while
other
AND
Glucose-6-Phosphate
under
of
normal
physiologic
mildly
deficient
variants
deficient
variants
have
and
conare
no
METHODS
Dehydrogenase
The normal
enzyme
(B+) was purified
and crystallized
as previously
described.11
The
crystalline
enzyme
was dissolved
in 0.01 M phosphate
buffer
(KH2PO4-Na2HPO4),
pH 7.0,
containing
bovine
serum
albumin
(10 mg/mI),
1 mM EDTA,
and 1 mM 2-mercaptoethanol
and was dialyzed
against
the same buffer
without
bovine
serum
albumin
for 3 hr at 4#{176}C.
Variant
enzymes
from hemizygous
variant
male subjects
were partially
purified
from red
cells by DEAE-cellulose
treatment
as previously
described.”
This
procedure
eliminated
most of the hemoglobin
and the 6-phosphogluconate
dehydrogenase
(6PCD)
activity
from
the hemolysate.
However,
when
measurable
6-phosphogluconate
dehydrogenase
activity
remained
after
the treatment
(Gd Union,
Gd Markham,
and
Gd Mediterranean),
the
enzyme
preparations
were
further
purified
by Gs1-Sephadex
column
chromatography
at
pH 5.8 as previously
described.’1
The partially
purified
variant
enzymes
were
dialyzed
against
0.01 M phosphate
buffer,
pH 7.0, contaIning
1 mM EDTA
and 1 mM 2-mercaptoethanol
for 3 hr at 4#{176}C
before
use.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
G-6-PD
ACTIVITY
Human
879
6-Phosphogluconate
6-Phosphogluconate
DEAE-cellulose
at
Dehydrogenase
dehydrogenase,
first
step
the
together
purification
of
with
of
hemoglobin,
was
g!ucose-6-phosphate
not
adsorbed
dehydrogenase
on
described
above.”
The eluate
(30 ml), which
contained
hemoglobin
and 6-phosphogluconate
dehydrogenase,
was dialyzed
against
0.01 M Tris-Cl
buffer,
pH 8.0, and placed
on a
DEAE-Sephadex
column
(2 X 30 cm) buffered
with the same Tris-Cl
buffer.
The column
was eluted with the same buffer. Hemoglobin
was first eluted,
and then 6-phosphogluconate
dehydrogenase
was
eluted
from
the
column.
The
fractions
with
6-phosphogluconate
dehydrogenase
activity
were treated
with ammonium
sulfate
(40 g/100
ml).
The precipitate
was dialyzed
against
0.01 M phosphate
buffer,
pH 7.0, containing
1 mM EDTA
and
1 mM 2-mercaptoethanol,
as described
above,
and was used
for enzyme
assay.
The partially
purified
preparation
had no glucose-6-phosphate
6-phosphate,
6-phosphogluconate,
NADP,
NADPH,
ATP
were
purchased
from
Sigma
Chemical
Co.
converted
to the sodium
salt before
use.
Measurement
of
Enzyme
dehydrogenase
2,3-diphosphoglycerate
2,3-Diphosphoglycerate
activity.
D-glucose
(Ba salt),
and
(2,3-DPC)
was
Activity
Glucose-6-phosphate
dehydrogenase
were measured
spectrophotometrically,
the buffer
used for the measurements
and 0.1 M KCI, and the temperature
enzyme
that catalyzes
the reduction
and 6-phosphogluconate
dehydrogenase
activities
as previously
described.”
Unless
otherwise
stated,
was 0.05 M Tris-Cl,
pH 7.3, containing
4 mM
MgCI2
was at 37#{176}C.
One unit is defined
as that amount
of
of 1 tmole
NADP
under
the assay
conditions.
RESULTS
Shunt
Pathway
and
Activity,
Glucose-6-Phosphate
6-Phosphogluconate
Physiologic
Dehydrogenase
with
the potential
and 6-phosphogluconate
actual
of the
activity
NADP
and
glucose
are
1 mm
ifl
by
AlT,
on the
activities
It has
and
Thus,
oxidize
whereas
7.3;
in
G6PD
inhibit
of these
NADPH
and
that
inhibition
was
1).
The
lower,
and
G6PD
compounds
and AlP,
the
saturation
about
2.2
moles
of
producing
only
Ringer’s
2-3
(or
that
or
1.5
nmoles
of
a similar
CO2
isotonic)
glucose-6-phosphate
both
enzymes,
Other
metabolites
in red
are
observed
inhibition
Km
pH
under
have
6PGD.
been
examined.’4
However,
consid-
could
have
major
for
only
inhibitors
6PGD
are
minor
effect
cells.’4
is a strong
at
6GPD
and
in red cells,
the
and the inhibitors
6PGD
NADPH
since
the
under
cells. To elucidate
the nature
of the regulation,
metabolites,
ATP,
coenzymes,
and
reduced
dehydrogenase
was
could
glucose-6-phosphate
in red blood
cells,
6-phosphogluconate,
suspended
of
2,3-DPG.
found
37#{176}C)(Fig.
concentration
are
of G6PD
cose-6-phosphate
competitive
activities
metabolites
and
been
of
low.
of
glucose
at 37#{176}C.’2”3 This implies
6-phosphogluconate
dehydrogenase,
concentrations
in red cells
NADPH,
cells
cells
strong
regulation
in red
of various
intermediate
the
G6PD
red
at 37#{176}Cat pH
red
1010
glutathione
in the
Several
intermediate
for
1.5
is very
imoles
1010
and
solution
containing
dehydrogenase
or
under
effects
Under
activities
(Vmax)
dehydrogenase
pathway
substrates,
of CO2
produced
ering
shunt
6-phosphate
jmoles
Activity
Activity,
Conditions
As compared
dehydrogenase
of
Dehydrogenase
8.0
competitive
and
inhibitor
at
physiologic
was
greater
when
for NADP
was 12
for
25#{176}C.’5 This
conditions
±
glucose
1.8 M,
glu-
strong
(pH
7.3
6-phosphate
and Ki for
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880
YOSHIDA
6
AND
LIN
0
6
,/‘
14
p
‘/
2
5.
‘/,
10
/
A
/
/
,
/
,
\
/
-
I
:
,,,
,
I [NADP]
Fig.
1 . Effect
tlon of glucose
,
of NADPH
1 [G6P]
IO)
cx
on rate
6-phosphate
of oxide
Fig.
.
by the enzyme.
0.05 M Tris-CI,
M KCI, 60 iM
was
whereas
60
zM
ATP
bition
itive
as
also
12.5
zM
2.1
±
on
rate
and
various
concentrations
6-phosphate.
Dots, absence
presence
of 1.5 mM ATP.
presence
and
of
oxidation
of
Ki was
glucose
800
9 ±
of glucose
of ATP; circles,
M
1.8
6-phosphate,
in
the
presence
of
6-phosphate.
glucose-6-phosphate
competitive
NADP.
4 M
±
in the
2.2
±
inhibited
was
with
44
24
was
of glucose
Km
of ATP
x I0
of glucose
6-phosphate
by the enzyme.
Reaction
mixture contained
0.05 M Tris-CI,
pH 7.3, 4 iM MgCI2, M KCI, 80 M NADP,
Reaction
mixture
contained
pH 7.3, 4 mM MgCl2,
0.1
glucose
6-phosphate,
and various
concentrations
of NADP. Dots, absence
of NADPH;
circles,
presence
of 45 M
NADPH.
NADPH
2. Effect
(
,
with
Km
and
for
6-phosphate
glucose
1.03
±
dehydrogenase.
glucose
6-phosphate
mM,
0.17
The
(Fig.
2) and
K for
and
respectively,
ATP
in the
of inhi-
mode
was
noncompet-
were
presence
estimated
of
M
80
NADP.
2,3-Diphosphoglycerate
genase
activity,
had
even
6-Phosphogluconate
inhibition
was competitive
were
estimated
as 18
6-phosphogluconate,
presence
NADPH
strate.
of
jM
effect
±
and
3
23
±
5
was
was
and
±
iM
and
for
M
25
Here
inhibited
inhibited
NADP
in the
±
5
by NADPH.
The
and Ki for NADPH
presence
of
10 M
of
in
the
respectively,
stronger
inhibition
by
of lower
concentrations
of the
dehydrogenase,
6-phosphogluconate
by
the
physiologic
(Fig. 3). The mode
of inhibition
(Fig.
4) and
was
noncompetitive
6-phosphogluconate
and
for
,M,
again,
2,3-diphosphoglycerate
6-phosphogluconate
Ki
dehydro-
to 4 mM).
also
Km
1.2
6-phosphogluconate.
strongly
glucose-6-phosphate
(up
7.7
was produced
in the presence
In contrast
to glucose-6-phosphate
dehydrogenase
on
concentrations
dehydrogenase
with NADP,
and
800
little
at higher
2,3-diphosphoglycerate
concentration
was
with
competitive
NADP.
were
estimated
subof
with
Km for
as
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
G-6-PD
ACTIVITY
881
0
16
4
/
2
III’
.‘
:
I0
8
%.-
6
q)
6
q)4
2
Fig.
,
I
2
- Diphosphoglycerate,
3
3. InhIbitIon
3
0
4
of 6-phosphogluconate
inhibition
8 zM
in the
the
Fig.
M
tlvlty.
of 1.5
absence
in the red
incubated
reported
as
lower
value
5 ± 3 nmoles
and 35
for NADP
(1 nmole/ml
concentration
value.
of
Assuming
concentration
presumably
in
NADP
that
of NADPH
lower
than
the
in
the
in the
reaction
45
zM
above,
ac-
of 80 i&M
i.e.,
40%
mixture
containing
NADPH,
an attempt
red
water
±
as compared
made
complete
of NADP
containing
protection
from
process
is very
is presumably
content
of
and
should
be about
50
2 M.
The
concentrations
±
cells
5
the
NADPH
glucose
than
is
70%,
and that
of glucose
in
were
red cells.’6
was reported
oxidation
difficult,
lower
red
to estimate
6-phosphogluconate
4 nmoles/ml
of packed
of washed
fresh
red cells)
extraction
cells
was
and
The concentrations
Ringer’s
solution
Since
during
dehydrogenase
in the presence
dehydrogenase,
dehydrogenase
cell.
investigators.
to NADP
of 2,3-dlphosphoglycerate
mixture
contained
0.05 M
pH 7.3, 4 M MgCl2, 0.1 M KCI, 80
and
described
cells
same
0
of AlT.
analysis
dehydrogenase
human
red
the
8
Reaction
TrIs-CI,
AlP
mM
glucose-6-phosphate
NADPH
6
(xl04)
iM
NADP, and various
concentrations
of
6-phosphoglycerate.
Dots, absence
of 2.3diphosphoglycerate
circles,
presence
of 0.8
mM 2,3-diphosphoglycerate.
6-phosphogluconate,
in the
kinetic
4. Effect
mM,
respectively,
6-phosphogluconate
0.022
of
by
activity
±
inhibited
presence
10
activity
From
the
also
NADP,
to the
0.12
and
ATP
4
on 6-phosphogluconate
Tris-CI,
pH 7.3 4 mM MgCl2, 0.1 MM KCI, 1.5
mM ATP, 45 tM NADPH,
8 M NADP, 12.7
uM 6-phosphogluconate,
and various
concentnatlons
of 2,3-diphosphoglycerate.
NADP.
2
l/[6PG],
dehydrogenase
activity
by 2,3-diphosphoglycerate.
Reaction mixture contained
0.5 M
10 ± 2 ,M
0
mM
the
the
A
of
actual
reported
the
molar
of NADP
6-phosphate.
is
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
882
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AND
LIN
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G-6-PD
ACTIVITY
Al?,
and
27
±
5.7
mM,
883
2,3-diphosphoglycerate
nmoles,
2.3
1.1
respectively,
that
a 70%
human
water
is associated
70%
of
membrane
tion
fresh
4 moles/m1,’7
with
about
30%
of 6-phosphogluconate
cells
40
content
(1.5
mM)
in fresh
and
with
human
red
cells
is too
in the
presence
and
in
M
40
of the
contain
1). This
measured
by
C02
strong
of substrate,
for
Vmax
6-phosphate,
(Table
Because
the
ing
cells
glucose
phoglycerate
activity
red
concentrations
of
that
only
value
is also
logic
conditions.
Regulation
As
genase
0.15%
mM
1.5
AlT,
of
of Activity
and
is comparable
Vmax
by NADPH
the
Of
to that
Table 2. 6-Phosphogluconate
enzyme
of the
of Variant
described
above,
the
is strongly
regulated
(1LM)
6PG.
No Inhibitor
and
by
can
shunt
be
and
inhibitis
2,3-disphos-
shunt
pathway
Dehydrogenase
in red cells.
cells is several
expressed
pathway
activity
of normal
by the concentrations
150
80
40
(Table
activity
Assummicro2).
under
glucose-6-phosphate
of NADP,
Activity
Under Various
dehydroNADPH,
glu-
Conditions
6PG, 5 M; AlP,
NADPH 45
2,3-DPO
1
mM
1.5 mM;
1.5 mM
80
60
50
40
32
20
8
26
14
6
22
12
5
17
10
4.4
4
2
This
physio-
Dehydrogenases
10 M;
AlP, 1.5 mM;
NADPH 45 M
2.3-DPG
1 mM
1.5 mM
600
340
I
0.5
deter-
2,3-diphosphoglycerate,
suppressed
in red
Glucose-6-Phosphate
1500(Vm)
00
10
5
2
to be
free
actual
6PG.
NADP
small
dehydrogenase
45
M NADPH,
mM
to the
red
concentra-
generation.’3”4
inhibition
comparable
the
with
and
The
coenzyme,
NADP,
activity
of 6-phosphogluconate
is strongly
that the concentration
of 6-phosphogluconate
molar,
of
B. Bartlett,
personal
of glucose-6-phosin 1010 red cells
glucose-6-phosphate
about
1-2
jM
1.5
value
Part
and
presumably
state.
phate
of various
cells.
as
mM,
hemoglobin
of it is in free
column
chromatographic
method
(Dr.
C.
Table
1 and Table
2 show
the activities
dehydrogenase
and 6-phosphogluconate
dehydrogenase
0.1%-0.2%
reported
1.5
of free 2,3-diphosphoglycerate
on the ratio
of oxygenated
to
not be far from
reality
to assume
mined
by the
communication).
ors.
Only
expressed
were
M,
in red
is bound
2,3-diphosphoglycerate
and
red
i.e.,
hemoglobin
membrane.’#{176}
The concentration
red cells
fluctuates
depending
hemoglobins.
However,
it may
about
cell
and
assuming
2,3-diphosphoglycerate
the red cell
in circulating
deoxygenated
in
moles,
3
1.5
2.5
1.3
2.2
1.1
Activities
are expressed
as nmole of NADPH
produced
In 1 mm by 100 red cells at 37#{176}C
and pH 7.3. Values listed are obtained from data of enzyme activities
measured in presence
of given concentrations
of 6-phosphogluconate,
ATP, NADPH, 2,3-diphosphoglycerate,
and
various concentrations
of NADP, making use of Interpolation
whenever
necessary.
Value
of Vmaj is obtained
from
enzyme
activity
measured
In absence
of inhibitors,
extrapolating
to Infinite
substrate
and NADP
concentratIons.
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884
YOSHIDA
cose 6-phosphate,
glucose-6-phosphate
activity
and
of the
ATP
in red
dehydrogenase
enzyme
in intact
analysis
of the variant
Variants
Associated
Manchester,
Gd
were
examined.
Alha.mbra
are
are
variants
constants
of particular
no
low
NADPH
inhibition
Fig.
5B.
variant
NADP)
by
NADPH
in
NADPH
(Table
the
60
M
absence
of AT?.
of
of
1.5
deduced
of the
of
also
Therefore,
in
enzymes
the
variant
of this
group
enzyme
are
variant
range,
enzyme
is shown
(competitive
and
a very
i.e.,
mixture
strongly
with
low
55%
Ki for
inhibition
containing
NADPH,
M
8
as compared
to the
functioning
in red
is scarcely
also
Michaelis
normal
coenzyme.
inhibited
enzyme,
Gd
of these
and
the
and
NADP
M
45
Gd
activities
normal)
substrate
reaction
and
the
of
within
variant
kinetic
Anemia:
enzyme
are
Km for
the
and Gd Bangkok
Gd Tripler,
and
20%
strongly
the
AT?
cell
Manchester
a high
inhibits
mM
red
the
from
Hemolytic
8.0
for
6-phosphate,
inhibited
by
NADPH
AT!’.
The inhibition
by NADPH
is competitive
wth
NAD?.
Kinetic
(Km for NAD?
and
Ki for NADPH)
and
the activities
in the
of various
concentrations
of NAD?,
and physiologic
concentrations
NADPH,
AT?,
and
Table
3. It is clear
dehydrogenases
should
Variants
Associated
Hemolytic
have
was
than
pH
is very
because
glucose
cells (Table
1).
Other
variant
and by
parameters
presence
profile
ATP
presence
NAD?,
at
affinity
enzyme
3).
the
(more
25#{176}Cand
unusually
The
cells
Gd Torrance,2’
Gd Manchester,
since
decreased
at
The
in
red
Alhambra,9
variants,
interest,
severely
measured
suggesting
variant
LIN
the correlation
between
hemolytic
symptoms,
the
enzymes
in vitro.
With
Chronic
Nonspherocytic
Tripler,’#{176} Gd
Among
these
not
cells.
To elucidate
deficiency
and
AND
2,3-diphosphoglycerate
Anemia:
less
than
associated
jects
have
Gd
5%
are
shown
of
Union,7
Gd
red
normal
Markham,8
cell
and
enzyme
Gd Mediterranean
subjects
factors24
or heterogeneity
Thus
fair,
Markham.
are associated
within
the Gd
no hemolytic
These
three
Gd
activity,
with
chronic
hemolytic
symptoms.
Some
hemolytic
problems
induced
by fava
beans
suggested.
and
Gd
in
Table
1
and
that
all these
hemolytic
variant
glucose-6-phosphate
have
very
low activity
in red cells in vivo.
With
Severe
Enzyme
Deficiency
But Without
Chronic
has
are
they
are
Gd Mediterranean
(favism).
Since
with
favism,
Mediterranean
abnormality
variants
Mediterranean23
yet
not
subnot all
certain
other
genetic
variant
have
been
been
found
polymorphic
in Gd Union
in
certain
populations.
These
three
the
inhibition
5C).
They
NAD?
glucose-6-phosphate
by
NADPH,
have
either
high
(Mediterranean
variants
are
Markham
compared
more
dehydrogenase
as
is shown
K
for
variant)
resistant
to
specified
condition.
The
expected
in vivo
more
than
30%
of the
activities
normal
their
activities”
“red
cell
enzyme
NAD?H
or both
22%,
and
Mediterranean
to 50%
inhibition
of
by
the
the
of
shunt
variants
the
Union
(Union
(Markham
inhibition
20%
normal
by
Al?,
resistant
or
i.e.,
under
(Fig.
low
(Table
Km
3).
for
These
Union
17%,
by
1.5 mM
AlT
under
the previously
these
variant
enzymes
in
pathway
activity
(Table
estimated
to
example
variant)
variant)
inhibition
enzyme
are
variant
the
conditions
red
1),
cells
as
are
although
of
near
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G-6-PD
ACTIVITY
885
Table 3. Michaelis
Constant
Normal and Variant
for NADP and Inhibition
Constant for NADPH
Glucose-6-Phosphate
Dehydrogenases
KM for NADP
Enzyme
K1 for NADPH
(/LM)
B
A
A-
(1hM)
12.5
7.2
8
Union
9
6.7
13
8.2
Markham
Mediterranean
Manchester
Alhambra
Trlpler
Torrance
Michaelis
containing
of
37
6.0
3.8
18.5
16
9.5
0.8
19.5
3.3
80
9.3
2.6
3.0
constant
and inhibition
constant
were measured
4mM MgCI2, 0.1 M KCI, 60 pM glucose-fl-phosphate,
in 0.05
M Tnis-CI,
pH
7.3
and various concentrations
of NADP and NADPH at 37#{176}C.
Note that these values are different from the values of KM
given in Table 4, which were determined
at 25#{176}C,
pH 8.0, and near saturation
of
glucose-fl-phosphate.
substrate
and
NADPH
The
are less
Common
males
are
zygotes
of
GdA.
with
the
This
8.0
of
GdA+
red
cell
and
that
and
enzyme
of
subjects
shunt
was
6-phosphate
activity
GdA
is
have
pathway
no
attributed
to
the
lished
observation).
enzymes
Michaelis
were
6
±
reported
constant
was
concentration
for
fore,
the
variant
5D).
The
A
inhibition
for
by
and
and
kinetic
properties
and
that
of
by other
glucose
the
55
±
and
enzyme
enzyme
1.5
mM
was
Al?
a high
the
Ki value
and
normal
GdA
is
oxidative
is
It has been
as active
as
unusual
a lower
Km
buffer,
glucose-
relationships
(Yoshida,
activities
of the
examined.
in Tris-Ci,
pH
unpubA
and
be
for
glucose
However,
37#{176}Cin the
8.0,
at 25#{176}C)
6-phosphate.
the
A4
variant
but
are
in
These
enzymes,
contrast
to
as
the
when
the Michaelis
presence
of a lower
A- enzyme
had a low Km (8 ± 1.2
(13 ± 2 &M) for NAD?H;
there-
more
resistant
to
also
more
resistant
to inhibition
the
to
had
activity
was
under
black
hemi-
without
enzyme
enzyme
7 M
of
NADPH
in Tris-borate
that borate
inhibites
normal
and
laboratories,252#{176}
6-phosphate,
NADP
A-
of
are
normal.
but
manifestations.
from
GdA
conditions
should
enzyme
(measured
NADP
is close
of
produces
NADP
by Luzzatto
and Afolayan.’3
determined
at pH 7.3 and at
of
value
pM)
physiologic
of A-
close
to
reported
previously
values
M
0.5
of
Therefore,
under
constants
are
values
activity
concentrations
by
found
males
GdA+
anemia,1
that
absence
20%
black
10%-20%
fact
the
About
of
of
hematologic
red cells
in
25#{176}Cin
12%
hemolytic
dehydrogenase
the
and
4).
GdA:
about
NAD?
and was less severely
inhibited
8.0, at 27#{176}C.’4However,
it has been
between
A
pH
at
primaquine-induced
variant
that
the
normal.’3
for
pH
The
normal)
associated
stress,
reported
saturation
than
5% of normal
(Table
Black
Variants
GdA
and
hemizygotes
of
(80%
NAD?
previously
inhibition
described
by
by
NADPH
AlT,
condition.
(Fig.
i.e.,
30%
Thus,
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YOSHIDA
886
AND
LIN
70
80
30
.
0
20
C
4._A9.!.4
B
A
20
30 NADP, 40 ,M
50
60
0
C
10
#{176} ‘#{176} 20
30NADP, 40 M
50
60
70
80
20
30
40
NADP,
M
0
Fig. 5. Inhibition
of normal
and variant
glucose-6-phosphpate
NADPH.
Reaction
mixture
contained
0.05 M Tnis-CI, pH 7.3, 4 mM
glucose
Activity
6-phosphate,
and
is expressed
by glucose
enzyme;
Union
concentrations
of NADP with and without
NADPH.
that could be obtained
by saturation
of enzymes
NADP. (A), normal
B enzyme;
(B), Manchester
variant
of Vm
enzyme;
conditions,
the
normal
enzyme
Kinetic
characteristics
physiologic
conditions
and
and
variant
under
physiologic
as actively
as the
by
M
60i
various
as per cent
6-phosphate
(C),
dehydrogenases
MgCl2, 0.1 M KCL,
are
(D), A- variant
variant
(Table
A
1).
and
expected
similar
to that
enzyme.
enzyme
activity
of the
in red
cells
can
function
of the
A
enzyme
under
normal
B enzyme
(Tables
1
3).
DISCUSSION
As
compared
dehydrogenase
actual
activity
Vmax for
unknown.
pathway
these
to
the
enzymes.
In the present
can be explained
dehydrogenase
presence
of the
6-Phosphogluconate
potential
activity
and 6-phosphogluconate
of the shunt
pathway
Thus
far,
exact
of
(Vmax)
dehydrogenase
is very
low,
mechanism
work,
it is shown
that the
by the strong
suppression
glucose-6-phosphate
in red
i.e., only
for
blood
this
was
also
strongly
suppressed
the
of
problem
low activity
of the
of glucose-6-phosphate
by the physiologic
concentrations
of NADPH
and
physiologic
concentrations
of glucose
6-phosphate
dehydrogenase
cells,
0.1%-0.2%
is
shunt
AT?,
in the
and NADP.
by
the
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G-6-PD
ACTIVITY
physiologic
the
concentrations
presence
Kinetic
of
887
analysis
high
that
to be
accumulation
of
these
rate-limiting
be easily
variants
Gd
and
Thus,
under
correlated
(Table
4).
to
As
to have
little
nation
of
these
anemia
are
Km value
the
factors
the
and
with
enzyme
have
no
the
the
(more
lion.
inhibition
by
than
of normal
30%
It has
been
in
generation
normal
in
red
Gd
activity
red
cells
cells
the
on
Gd
can
be
variant
variant
Gd
Gd
nor
and
these
variant
maintain
an
enzymes
far
can
maintain
relatively
under
the
simulated
physiologic
shunt
pathway
activity
is
black
of the
of
and
Gd
to underanemia.
less
sensitive
high
activity
concentra-
30%
by
of
Mediterranean
The
normal,
and
are
Km
cannot
level
of
measured
about
populations.
10%-20%
high
with
enzymes
due to high
they
Mediterranean
unusually
associated
enzymes
adequate
are
(Gd
hemolytic
hemolytic
variant
Mil-
combi-
variants
Gd
Markham,
it was difficult
Hemolytic
concentration
variant
the
chronic
variants
deficiency
the
is
by
with
and
Gd
explained
deficiency
Thus,
cells
to
among
can
the
substrate
are expected
Albuquerque,3’
Union,
problem,
Gd
subjects
by
the
Ashdod,27
Km for
enzymes
several
The expected
enzyme
activity
1) agrees
with
this finding.
is a common
of
that
from
Gd
enzyme
and
level)
reported
red
cells.35
(Table
A-
NAD?H,
Gd
high
variant
many
following
: (1)
of physiologic
for NAD?
or low
Ki for NADPH.
generate
sufficient
NAD?H
in red
reduced
glutathione.
(2) Nonhemolytic
to
or
become
dehydrogenase
Worcester,28
Gd
Oklahoma,
severe
(e.g.,
hemolytic
revealed
the
by NAD?H
reagents
could
associated
Since
4).
between
analysis
inhibited
Tripler)
very
(Table
deficiency
chronic
dehydroconcentrations
exogenous
However,
4).
Gd
neither
substrate
relationships
Kinetic
strongly
Gd
Strasbourg34
(Table
Aihambra,
associated
for
more
severe
Mediterranean)
stand
two
Gd
as
Hemolysis
Gd
cells
under
pathway
is
low red cell enzyme
activity
of these
by Kirkman
and
Riley
in the case
of
such
cells.
and
accumulation
conditions.
with
unusually
at 25#{176}C). Therefore,
these
in red
a low
some
glucose-6-phosphate
Gd Ramat-Gam,27
and
variants,
Clichy,33
Manchester,
by
associated
activity
Gd
of
A relatively
normal
red
the
shunt
dehydrogenase
the extremely
pointed
out
several
Gd
Freiburg,3#{176} are
( measured
at pH 8.0
waukee,32
and
in
in
0.1%-0.2%
cells.
6-phosphogluconate
changes
in the
induced
certain
only
red
jIM)
(40
in
AT?
6-phosphogluconate.
human
rather
than
by slight
coenzymes
and
and
dehydrogenases
normal
6-phosphate
manifestations
as Gd Bat-Yam,27
Oklahoma,29
two
in
6-phosphogluconate
enzyme
Hemolytic
variants,
such
of NADP
these
dehydrogenase
may be altered
metabolites
of blood.
2,3-diphosphoglycerate,
in red cells
suggests
that
the
rate-limiting
enzyme
glucose-6-phosphate
genase.
The situation
storage
for
expressed
glucose
of 6-phosphogluconate
physiologic
conditions
of
NAD?H,
concentrations
indicated
is expected
Vma
of
of physiologic
C02
that
of
variant
red
cell
no
enzyme
hemolytic
manifestation
occurs
unless
the subjects
are
type
antimalarial
drugs.
Luzzatto
and Afolayan
way activity
in red cells from
Gd A
subjects
administered
with
primaquinereported
that the shunt
pathwas close
to normal.’
They
observed
a sigmoidal
the
concentration
of
rate of
that
the
molecule
of
NADP
relationship
NADP
and
increased
the
between
suggested
affinity
the
of
another
enzyme
binding
NADP
reaction
of the
binding
and
first
to
the
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888
YOSHIDA
Table
4. Characteristics
of Normal
Activity’
KM
(#{176}/o
Not associated
with chronic
Heat Stability
pH Optima
hem olytic anemia
50-70
2.9-4.4
Normal
Normal
Normal
Normal
Normal
Normal
Normal
8’..-9
8’.-9
8-9
<3
8-12
4.4-6.3
3.6-5.2
Low
Biphasic
19-26
1.2-1.6
Low
Low
Biphasic
Biphasic
Slightly
low
Biphasic
Normal
Biphasic
Very low
Very low
Very low
Low
Slightly
low
Biphasic
Biphasic
Markham
Mediterranean
1.5-10
< 5
Taiwan-Hakka
2-9
chronic
-
25-52
hemolytic
0
Ramat-Gan
-
10.7-12.2
< 5
Indonesia
with
for NADPt
(i.)
Dehydrogenase
80
8-20
Union
Associated
Bat-yan
KM
LIN
100
Normal
A
A-
Glucose-fl-Phosphate
for G6Pt
(MM)
Normal)
Enzyme
B
and Variant
AND
4-9
anemia
27
-
35
-
0
Worcester
Oklahoma
Ashdod
0
4-10
10
11.6
127-200
100
61
20
Freiburg
Albuquerque
10-20
1
87-118
115
4
11
Milwaukee
0.5
2
224
178
6
5
2.4
96
60
48-60
Clichy
Strasbourg
Bangkok
Torrance
-
8.0
8.2
Biphasic
Biphasic
8.2
8.0
9-10
9.0
8-8.5
8-8.5
-
Very
-
low
-
-
-
13
5.3
2.4
Low
Very
Very
low
low
Manchester
25-30
64
6
Low
Biphasic
Alhambra
Tripler
9-20
35
55
30
2.6
Low
Very
Truncate
Data are collected
with
chronic
from references
nonspherocytic
ten variants
with
chronic
hemolytic
anemia)
eRelative
enzyme
activity
phospha’e
given in the text. Another
hemolytic
associated
severe
anemia
have
red cell
been
enzyme
have been reported.5
In red blood
cells
several
reported,
deficiency
measured
low
Biphasic
variants
associated
and another
more
(but not associated
at near
saturation
than
with
of glucose-fl-
and NADP at 25#{176}C
and pH 8.0.
tMichaells
6-phosphate
normal
constant
was measured
in absence
of inhibitors
at near
(for KM of NADP) or of NADP (for KM of glucose-6-phosphate).
and
variant
enzymes.36
Thus,
they
postulated
saturation
two
of glucose-
dissociation
con-
i.e., 21 .tM for the first binding
and 12 iM
for the second
binding
of
with
the normal
B enzyme,
and
23
zM for the first
binding
and
for the second
binding
of NAD?
with
the A- enzyme.
They
estimated
stants,
NADP
1.3 ,M
also
that
were
16
the
inhibition
and
constants
210
M,
(Ki)
variant
proposed
characteristic
from
unfortunate
of
that
at pH
8.0
red
cells
all their
in the
for
respectively.
high
Ki for NAD?H
of the
as normal
in red cells.
The
buffer
-
kinetic
presence
NADPH
Because
A-
Gd
studies
of high
enzyme,
mechanism
Awere
of
of
low
these
Km
two
for
it could
can
subjects.
carried
concentrations
function
explain
the
However,
out
by
of glucose
and
as actively
physiologic
it
using
enzymes
NAD?
was
Tris-borate
6-phosphate
very
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
G-6-PD
ACTIVITY
mM)
(4
effect
at
on
27#{176}C.As
the
the
in
Kinetic
(pH
the
activity
and
7.3
that
NAD?H
tion
the
of
NAD?
thione
is maintained
the
case
of
red
cells
pathway
activity
is not
resulting
decrease
of
hemolysis
of
various
oxidative
The
in
with
G6?D
reduced
glutathione
the problem
work
made
it
determined
under
present
characteristics
at 25#{176}Cin the
in the
the
between
of
enzyme
activity
could
the
could
inhibition
physiologic
easily
elevate
(Table
1).
to the concentraunder
level
physiologic
of reduced
under
gluta-
oxidative
stimulation
clear
stress.38
of
activity,
by
in detail.
enzyme
the
of
shunt
red
cells
glucose-6-phosphate
analysis
insight
into
hemolytic
simulated
under
the
problem
anemia
of
induced
G61’D
and
(at
kinetic
optimal
substrate
and
pH
NAD?
information
and
for
investiga-
activity
misleading
variant
of
several
conditions
provide
in the
variant
and
vitro
concentrations
often
Kinetic
deficiency
in
expressed
been
studied
that
red cell
of high
the
not
been
unphysiologic
activity
of
provide
exogenous
not
clear
inhibitors)
manifestation.
ditions
has
had
presence
actual
dehydrogenase
other
severity
absence
the
hemolytic
It is
the
oxidative
stress.14’35
The
red
cells38
would
explain
the
with
enzyme
deficiency
under
in
activity,
tors,26’394#{176} but
cerning
and
8.0
the
associated
of physiologic
hemolytic
and
cells
overcome
variants
understanding
8-8.8
the
deficiency,
to
to
level
related
NAD?H
of
(pH
6-phosphate).
level
of
red
physiologic
concentration
under
and
observed
stresses.
importance
The
that
normal
sufficient
G6PD
several
than
to a normal
is directly
to
under
sensitive
of NAD?
cells
6?GD
inhibitory
relationship
was
a lower
less
is stimulated37
unchanged
NAD?
an
conditions
condition
related
pathway
of
of glucose
was
increase
inversely
shunt
of
concentration
a slight
sigmoidal
unphysiologic
enzyme
had
a
reexamined
presence
as under
A-
borate
and
concentration
the
activity
in red
of G6?D
and
and
the
the
unphysiologic
Nevertheless,
conditions,
In
well
variant
variant
enzyme
Since
the activity
above,
in the buffer.
A- enzyme
were
of a higher
the
under
condition.
and
37#{176}Cin
as
presence
interesting
the
described
of borate
of the
6-phosphate),
25#{176}Cin
by
been
dehydrogenase,
enzyme
presence
characteristics
conditions
glucose
has
glucose-6-phosphate
between
only
889
the
con-
relationship
dehydrogenase
physiologic
and
con-
glucose-6-phosphate
by
food,
drugs,
and
agents.
ACKNOWLEDGMENT
We
are
grateful
Gd Torrance,
Gd
to Dr.
E. Beutler
Bangkok)
and
for
the
providing
partially
the
purified
variant
blood
enzyme
samples
preparation
(Gd
from
Alhambra,
Gd Tripler.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1973 41: 877-891
Regulation of Glucose-6-Phosphate Dehydrogenase Activity in Red Blood
Cells From Hemolytic and Nonhemolytic Variant Subjects
Akira Yoshida and Mei Lin
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