Vol.71,No.4,
1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RAT LIVER 6-PHOSPHOGLIJCONOLACTONASE: A LOW Km ENZYME P.J. Instituto
Schofield
de Enzimologia
de la Universidad
Received
and A. Sols
de1 CSIC, Autonoma,
Facultad
de Medicina
Madrid-34,
Spain
June 28,1976
SUMMARY. By specific enzymic synthesis of the substrate 6-phosphogluconolactone ti bLtu, the Km for rat liver 6-phosphogluconolactonase was found to be 80 PM. This value is approximately 100 fold lower than the previously determined value, and is compatible with the kinetic parameters of both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and hence with the flux through the oxidative segment of the pentose phosphate pathway. The initial, involves that
catalytic
be modulated
aX.
(1)
in the
specific
in a completely
activities
activity
of the G6PDH at free
been
found
activity
activity
the pathway 6PG, viz,
site.
the
the
free
by the
action
acid
is
generally
of a specific
specific
(6PGL), assumed
6-phosphogluconolactonase.
Sapag-Hagar
is an apparent
imbalance
of conditions,
than
the
activators
corresponding
of G6PDH is
6PGDH/G6PDH
of flux
through
the lactone
hydrolysis
by spontaneous
range of 6PGDH have
or calculated
and the
to occur
dehydrogenases
However,
of an impairment product
be expected
in the physiological
low measured
The irnnediate
thus
a variety
much smaller
possibility
pathway
NADP-specific
there
liver,
occurring,
6-phosphoglucono-6-lactone
to the
rat
might
manner.
NADP+/NADPH ratios
results),
may suggest at that
for
of 6PGDH is
no naturally
(unpublished ratio
these
phosphate
It
of the two enzymesunder
the specific
Since
of both synchronous
that,
and that
- 0.01).
of the pentose
G6PDH* and 6PGDH.
activity
have demonstrated
(0.1
phase
two dehydrogenases,
the maximal
would ti
the
and oxidative,
of the hydrolysis
However,
little
*Abbreviations: G6PDH, glucose 6-phosphate dehydrogenase (EC 1.1.1.49); 6PGDH, 6-phosphogluconate dehydrogenase (EC 1.1.1.44); G6P, glucose 6phosphate; 6PG, 6-phosphogluconate; 6PGL, 6-phosphoglucono-b-lactone.
Copyrighr 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
1313
of lactone and/or is
Vol.71,No.4,
1976
known about lactonase
BIOCHEMICAL
this
lactonase
provides
in mamnalian
the link
between
6-phosphogluconolactonase, lactone
of approx.
In view
of the
This
the
communication
of the
Km value reports
RESEARCH COMMUNICATIONS
despite
the fact
two dehydrogenases.
&.
7 mM, and a total specific
systems
the
Kagawa ti
respective
dehydrogenases,
AND BIOPHYSICAL
(2)
have obtained
activity
of approx.
activities
of 7 mM for
the
For rat
liver
a Km value
for
35 U/g tissue
and Km values the lactonase
a re-investigation
that
the
(3).
of the two
seems anomalously
of the basic
kinetic
high.
parameters
lactonase.
Experimental. The livers of adult rats fed ud lib were homogenised with 4 vols of 5 mM K2HP0~-1 mM dithioerythritol-150 mM KCl-1 NM MgC12-0.5 reel CaC12, adjusted to pH 7.0. The supematant resulting from centrifugation at 30,000 g x 30 min at 4O was used itnnediately or stored overnight in a frozen state. For kinetic studies the reaction mixture contained, in a total vol of 1 ml, 5 mM NADP+, 100 mM imidazole pH 7.0, approx. 10 U G6PDH and 5 mM MgC12. The reaction giving 6PGL formation was started by the addition of G6P (cont. range: 30 - 200 pM) and after 30 set, approx. 2.5 U 6PGDH was added. After a further 30 set, tissue extract as a source of 6-phosphogluconolactonase was added. The change in extinction at 340 nm was recorded (path length: 1 cm). For measurement of the spontaneous hydrolysis of 6PGL at 25' and and pH 7.8 by the method of Horecker and Smyrniotis (4), the reaction contained, in a total vol of 1 ml, 1.5 mM NADP+, 50 mM Tris (pH 7.0 or approx. 5 U G6PDH, 1 U 6PGDH and 5 mM MgCl*. The reaction was started addition of 50 pM G6P, and the extinction at 340 nm determined. Values are quoted as the mean f S.E.M. computed by the method of least squares. Results
and discussion.
Major
the unavailability
and lability
method
and Smyrniotis
of Horecker
enzyme studies.
In our
low and unreliable
yields
G6P which
is similar
The major
product
experimental only
in btiu amount
by the
(4) this
proved
method,
of 6PGL, despite obtained
was 6PG, the yield
method
in the
of the substrate.
to the value
manipulations
feasible
hands,
difficulties
The lines
to routinely
use of the appropriate
of G6PDH, and at a relatively
u&z,
of the
Preparation
bromine
by Horecker being proved the
lactone
1314
were
lactonase
routine
oxidation
are
kinetic
of G6P, gave
80% utilisation and Smymiotis less
than
of (4).
20%, and
unsuccessful.
Thus the
was to synthesise
mass of G6P and a relatively low pH to reduce
fit
of 6PGL by the for
approximately
the yield
synthesise
study
unsatisfactory
of lactone
to improve
of best
pH 7 mixture 7.8), by the
spontaneous
large hydrolysis
it
BIOCHEMICAL
Vol. 71, No. 4,1976
of the subsequent varied,
ranging
lactone.
Smyrniotis
(4),
presence follow
The values
from approx.
and 24 min at pH 6.4
(6).
By application
in which
order
G6P is
kinetics,
of the
&
with
hydrolysis
the half-life (4)
of 6PGL are
to 20 min at pH 7.0
a large
of the
half-life
Thus,
lactone
for
of the methodology
reacted
the
7 and 3.5 min respectively. preparation
cited
1.5 min at pH 7.4
of 6PGDH, spontaneous first
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
at pH 7.0
at room temp.
A&!& proved
of Horecker
excess
lactone
(5) and
of G6PDH in
at 25'
was found
and pH 7.8 being
and at pH 7.0,
the to
approx.
the
feasible.
8
.t 6 E
Fig.1. The rate of spontaneous hydrolysis of the 6-phosphoglucono-&lactone formed &I b.Ltu at varying concentrations of lactone. The values are expressed The regression line was computed by the method as the mean * S.E.M. (n = 3). of least squares (r = 0.96, p
In the presence (cont.
range:
GPGDH after lactone This giving half-life
of a large
30 - 200 uM) was rapidly 30 set
demonstrated
at pH 7.0 was 5.5 rate
excess
of spontaneous
a half-life
that
of G6PDH, 69.8 converted
the
+ 0.3% per min over hydrolysis
exhibited
of 12 min at pH 7.0,
as determined
rate
by the method
which described
1315
+ 1.4% of the G6P
to the
lactone.
of spontaneous the first is
hydrolysis
concentration order
kinetics
in good agreement above.
Addition
Subsequent
range
of of the
used.
(Fig.1) with addition
the
Vol.71,No.4,
BIOCHEMICAL
1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3 3 0.2 _ ; 9
I
Q 0.1 -: I I I
I 1 TIME (min)
I 0.5
L 2
Typical 6-phosphogluconolactonase assay. The reaction mixture Fig.2. contained 5 mM NADP, 100 IIM imidazole pH 7.0, approx. 10 U GLPDH and 5 mM MgC12. The reaction was started by the addition of G6P (65 PM). After 30 set approx. 2.5 U 6PGDH was added (A) to determine the rate of spontaneous hydrolysis of the lactone. After a further 30 set tissue extract (10 ul) was added (B) and the initial velocity of the lactonase determined.
of tissue
extract
absorption
of the 6PG formed
was routinely
used for
Fresh the
extract
only
extracts
as manifested
thus
normal to that
of magnitude
less
be noted
by the non-specific
in the millimolar
range;
that
in turn,
lactonase
resulted
showed
increase
in
was due to
(Fig.2).
animal
values
was quite in less
less
the
lactone
than
for low.
than
variability
This
method
Kagawa oxamate the
1316
determined et uL.
method
(2)
pH of the
of the
in
to be 80 yM, with (Fig.3).
The total but
by Kagawa ti measured
and this
Km value
Storage
10% loss
by Kawada et (Le. (3), that
the
of the Km value.
was found
of 10 U/g tissue
found
that
erratic
the value
state
similar
should
to give
the Km for
in the
of NADP+ which,
that
extract
conditions,
activity
it
thawed
in the
studies.
tended
in a frozen
two orders
of lactone
kinetic
overnight
is
However,
the
but suggested
these
activity is
tissue
and the
maximal
hydrolysis
by the endogenous
lactone,
activity, Under
rapid
at 340 nm due to reduction
oxidation
for
caused
method
incubation
the Km value a&
(2).
the disappearance is sensitive medium was not
BIOCHEMICAL
Vol. 71, No. 4, 1976
/ / / / /
AND BIOPHYSICAL
I .Ol
I .02
RESEARCH COMMUNICATIONS
I .03
'%PGL
(pM I-'
Fig.3. Lineweaver-Burk plot with 6-phosphogluconolactone as the substrate synthesised &I &Au. The reciprocal of the initial velocity is expressed as (pmole/min)-'. The regression line was computed by the method of least squares (r = 0.99, p
specified,
and that
lineality that
of the
the
mental
high
the
reaction
Km value
artifact
at 61'
reduction
in lactonase
for
above,
we have
5 min caused
30% loss
loss
obtained
to keep the
(2)
Hence, is
and relatively
of rat
the oxamate
liver
With that
heat whereas
insensitive
method
method,
found
resulted
for
that
of "heat
in a marked
of analysis
of the
treatment
appear
of experi-
method
treatment
would
a result
supernatant
the specific
it
exact
extract
out-
at 60'
5 min at 65'
for
caused
activity. within
at higher
the
importance,
from G6P by G6PDH is of the
physiological
pH values
lactone
oxidation
was difficult the assay".
using
in activity,
of lactonase
since
(3),
determined
much greater
the
during
activity".
assumes
as is
"it
by Kagawa et &.
3 minutes
The use of pH 7.0, compromise,
velocity
Kawada et uX.
treatment
complete
stated
due to the non-specific
determination.
lined
authors
the spontaneous
while hindered
free
range,
at lower by the
6PG by 6PGDH.
1317
hydrolysis
pH values
displacement
is a satisfactory
the
of lactone formation
of
of the equilibrium,
The determined
K,,, value
of
BIOCHEMICAL
Vol.71,No.4,1976
80 UM for closely
the
the kinetic
previously for
lactone,
the
and the total parameters
determined
Km value
phosphate
activity
in the
range
since
both
Hence,
are
of the
same orders
is
presumably
not
that
the
&I uivo,
of 7 mM.
hexose
conditions.
lactonase
itself
whereas
was truly
the
would
total
of 10 U/g, than
in the range
may prevail
30-70
the
PM, and
lactonase
and the
of NADPH, it
between if
the
Km of the
two dehydrogenases
flux
does
more
of physiological
and the
accumulation the
fit
of the two dehydrogenases
a variety
activity
as the
impair
of magnitude
are
U/g over
by the normal
such an impairment
two orders
activity
The Km values
substrates
1.5-30
of magnitude
hindered
lactonase
of the two dehydrogenases
respective
the total
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
lactonase unlikely
the two dehydrogenases Km for
the
lactonase
greater.
Acknowledgements. We wish to thank Dr. R. Lagunas for helpful discussion and Miss A. Nieto for valuable technical assistance. P.J.S. was on leave from the School of Biochemistry, University of New South Wales, Kensington, N.S.W., Australia. References 1.
Sapag-Hagar,
M.,
Lagunas,
R. and Sols,
A. (1973)
Biochem.
Biophys.
Res.
Corn. 5&l, 179-185. 2. 3.
Kagawa, 364-371. Kawada,
Y.,
Kagawa,
A. and Shimazono,
N. (1964) J. Biochem.
(Tokyo)
56-,
M., Takiguchi,
H., Kagawa, Y., Suzuki, K. and Shimazono, N. (Tokyo) 51, 405-415. and SmyrniotE, P.Z. (1953) Biochim. Biophys. Acta 12,
(1962) J. Biochem. 4.
Horecker,
B.L.
98-102. 5. 6.
Kawada, Biophys. Glaser,
M., Kagawa, Y., Takiguchi, H. and Shimazono, N. (1962) Biochim. Acta 2, 404-407. L. and Brown, D.H. (1957) J. Biol. Chem. 216, 67-79.
1318