Ability
Dehyd N
NADH h
Dehyd
Leonard ka
Tatyana
*Departm ity
1300 Univ
Received O
At the n
presence
the V
max
o
than the
erol-3-ph
tion. Und
els of cy
inhibited
on oxidat
dehydrog
increased the ratio of NADPH to NADH oxidation of
glycerol-
the meas
drogenas
and their
nase reac
hydrogen
oxidation
more tha
the level
due to th
zymes w
complexe
or NADP
phospha
NADH is
plex with
ced
nd
d a
fec
t
a
rog
© 1
Wo
H/N
1
This re
Grant DK 4
2
To who
dressed. Fa
3
Present
mouth Med
0003-9861/99
Copyright ©
All rights of
Archives of Biochemistry and Biophysics
Vol. 364, N
Article ID a3-phosphate dehydrogenase. On the basis of
ured K
D
of complexes between malate dehy-
e and these reduced pyridine nucleotides,
K
m
in the glycerol-3-phosphate dehydroge-
tions, it could be concluded that malate de-
ase would have markedly inhibited NADPH
and inhibited NADH oxidation considerably
n observed if its only effect were to decrease
of free NADH or NADPH. This indicates that
e opposite chiral specificity of the two en-
ith respect to reduced pyridine nucleotides,
s between malate dehydrogenase and NADH
H can function as substrates for glycerol-3-
te dehydrogenase, but the complex with
less active than free NADH, while the com-
NADPH is as active as free NADPH. Mg
21
Dihydroxyacetone phosphate is reduced by NADPH
in muscle and liver cytosolic fractions in the presence
of high levels of Mg
21
and a pH of 7.4 (1–3). However,
pure muscle cytosolic glycerol-3-phosphate dehydroge-
nase (E.C. 1.1.1.8), while reactive with NADH, is
rather inactive with NADPH under these conditions.
The pure muscle enzyme is about equally reactive with
NADPH as with NADH at a pH of 7.1 but even then 1.0
mM levels of Mg
21
inhibit NADPH oxidation and acti-
vate NADH oxidation (4). This suggests that there are
factors in the cytosol not completely present when the
pure enzyme is assayed which enhance NADPH oxida-
tion. Furthermore, although there is one gene for glyc-
erol-3-phosphate dehydrogenase (5) the kinetic proper-of Cytosolic Malate Dehydrogenase
rogenase to Increase the Ratio of
Oxidation by Cytosolic Glycerol-3-p
rogenase
1
A. Fahien,*
,2
Jose´ I. Laboy,* Zafeer Z. Din,* Pra
Budker,† and Michael Chobanian*
,3
ent of Pharmacology and †Department of Pediatrics, Univers
ersity Avenue, Madison, Wisconsin 53706
ctober 22, 1998, and in revised form January 6, 1999
ormal pH of the cytosol (7.0 to 7.1) and in the
of physiological (1.0 mM) levels of free Mg
21
,
f the NADPH oxidation is only slightly lower
V
max
of NADH oxidation in the cytosolic glyc-
osphate dehydrogenase (E.C. 1.1.1.8) reac-
er these conditions physiological (30 mM) lev-
tosolic malate dehydrogenase (E.C. 1.1.1.37)
oxidation of 20 mM NADH but had no effect
ion of 20 mM NADPH by glycerol-3-phosphate
enase. Consequently malate dehydrogenase
enhan
nase a
scribe
had ef
only in
there w
dehyd
nase.
Key
NADP
o. 2, April 15, pp. 185–194, 1999
bbi.1999.1117, available online at http://www.idealibrary.com onties of thi
in the sa
specific d
to NADH
ing amou
sociated w
(1, 3) oxid
search was supported by National Institutes of Health
701.
m correspondence and reprint requests should be ad-
x: (608) 262-9300. E-mail: lafahien@facstaff.wisc.edu.
address: Department of Medicine and Pediatrics, Dart-
ical School, Hanover, NH 03755.
$30.00
1999 by Academic Press
reproduction in any form reserved.and Lactate
ADPH to
osphate
sh Prabhakar,*
of Wisconsin Medical School,
the interactions between malate dehydroge-
glycerol-3-phosphate dehydrogenase de-
bove. Lactate dehydrogenase (E.C. 1.1.1.27)
ts similar to those of malate dehydrogenase
he presence of Mg
21
. In the absence of Mg
21
,
s no evidence of interaction between lactate
enase and glycerol-3-phosphate dehydroge-
999 Academic Press
rds: glycerol-3-phosphate dehydrogenase;
ADH activity; heteroenzyme interaction.s enzyme can differ from one organ to another
me animal (6). Thus it is possible that organ
ifferences with respect to the ratio of NADPH
activity of this enzyme could be due to vary-
nts of different factors which are tightly as-
ith the enzyme in vivo. As discussed previously
ation of NADPH by glycerol-3-phosphate de-
185

hydrogen
ical sign
paper is
oxidation
result of
ing with
ity sugge
between
A-sided s
tosolic m
dehydrog
strates fo
genase (9
free NAD
can inhib
than exp
the level
NADPH
affinity f
examined
EXPERIM
Enzymes
phate dehy
method (12
NAD to an
Boehringer
hydrogenas
(Type II), p
liver malic
liver glutam
cytosolic fra
Substrates
Sigma Che
the pH of th
buffer used
electrophor
only one ba
rabbit mus
as with glu
and pig he
97%, respec
Resolutio
bose, NAD,
crystallize
phosphate
tallized by u
with norit a
Enzyme
phate dehy
by measuri
A
280 nm
0.1%
and
ment, its co
A
260 nm
0.1%
of 0.
concentrat
malate deh
pig heart l
(21), 0.93 (
With the ex
fractions, t
molar conc
(17, 19), 5.
d 3
f th
glu
nas
deh
cs o
red
dro
be
d m
en
abl
E
a
.
re t
is
pl
^
r, w
s E
he
ciat
qu
, d
ste
r s
, v i
en
coe
he
pyr
ion
S]/
a
S
ordi
][E
ubs
an
es o
ly a
sp
n o
or m
red
city
es s
186ase in the cytosol is of considerable physiolog-
ificance. Therefore a major objective of this
to determine if the ratio of NADPH to NADH
could be enhanced in cytosolic fractions as a
glycerol-3-phosphate dehydrogenase interact-
cytosolic enzymes. For example, one possibil-
sted by previous results (7, 8) is complexes
NADH and dehydrogenases which have
pecificity with respect to NADH, such as cy-
alate dehydrogenase (E.C. 1.1.1.37) or lactate
enase (E.C. 1.1.1.27) can function as sub-
r the B-specific glycerol-3-phosphate dehydro-
–11) but can be less active substrates than
H. Consequently the A-sided dehydrogenases
it, but inhibit to a considerably lesser extent
ected on the basis of their ability to decrease
of free NADH. However, they might not alter
oxidation because they might have a quite low
or NADPH. These and other possibilities are
in this paper.
ENTAL PROCEDURES
and reagents. Rabbit muscle cytosolic glycerol-3-phos-
drogenase was prepared by us with a previously described
) or obtained from Boehringer-Mannheim. We did not add
y of the purification steps or to facilitate crystallization.
-Mannheim was also the source of pig heart lactate de-
e. Rabbit muscle aldolase and lactate dehydrogenase
ig heart cytosolic malate dehydrogenase, and chicken
enzyme were obtained from Sigma Chemical Co. Bovine
ate dehydrogenase, pig heart homogenates and rat liver
ctions were prepared as described previously (1, 13–15).
, coenzymes, and other reagents were obtained from
mical Co. Stock solution of all reagents were adjusted to
e assay. Enzymes were extensively dialyzed against the
in the assays prior to use in the experiments. Upon
esis on 10% sodium dodecyl sulfate polyacrylamide gels,
nd was found with the commercial and our preparation of
cle cytosolic glycerol-3-phosphate dehydrogenase, as well
tamate dehydrogenase. Cytosolic malate dehydrogenase
art lactate dehydrogenase were essentially pure (96 to
tively).
n of glycerol-3-phosphate dehydrogenase. The ADP-ri-
and NADH-X, which are tightly bound to the purified,
d commercial skeletal muscle cytosolic glycerol-3-
dehydrogenase as well as the enzyme purified and crys-
s, were removed from the enzyme by treating the enzyme
s described previously (16, 17).
concentration. Prior to treating the glycerol-30phos-
drogenase with norit, its concentration was determined
ng the absorbance at 280 and 260 nm using values of
A
260 nm
0.1%
of 1.0 0.833, respectively (18). After norit treat-
ncentration was determined using values of A
280 nm
0.1%
and
97 and 0.60, respectively (17, 19). For determining the
ion of glutamate dehydrogenase, aldolase, cytosolic
ydrogenase, rabbit muscle lactate dehydrogenase, and
actate dehydrogenase values of A
280 nm
0.1%
of 0.9 (20), 0.73
22), 1.6 (23), and 1.4 (24), respectively, were employed.
ception of assays performed with enzymes in cytosolic
he concentration of all enzymes is expressed in terms of
(23), an
weight o
genase,
hydroge
lactate
Kineti
tween a
ing dehy
complex
describe
levels of
consider
enzyme
ered we
(where P
can take
E
D
1 S
Howeve
genase i
can be t
E
D
asso
E
a
-E
D
. E
anism 1
yielding
the othe
In Eq. 1
catalytic
reduced
tion of t
reduced
dissociat
K
1
5 [E
a
S][E
D
]/[E
E
D
]. Acc
5 [E
a
][S
can be s
[S], [E
D
]
the valu
metrical
In these
alteratio
quired f
The p
the velo
complex
FAHIEN ET AL.entration of enzyme subunit, using values of 3.9 3 10
4
6 3 10
4
(25), 3.7 3 10
4
(22), 3.5 3 10
4
(26), 3.3 3 10
4
level of S. T
so that the.3 3 10
4
(24), respectively, were used for the molecular
e monomeric subunit of glycerol-3-phosphate dehydro-
tamate dehydrogenase, aldolase, cytosolic malate de-
e, rabbit muscle lactate dehydrogenase, and pig heart
ydrogenase.
f enzyme–enzyme interactions. Kinetic interactions be-
uced pyridine nucleotide (S) a pyridine nucleotide donat-
genase (E
D
) and a catalytic dehydrogenase (E
a
) within a
tween the two enzymes was studied with a previously
ethod (7, 8). In these experiments it is determined how
zyme E
D
, which are in the range of the level of S and
y higher than the level of E
a
, alter the catalytic activity of
In most previous studies the only reaction steps consid-
he reaction which takes place in the absence of E
D
:
E
a
1 S ^ E
a
2 S ^ E
a
1 P,
a product) plus some of the additional reactions which
ace in the presence of E
D
:
E
D
2 S and
E
D
2 S 1 E
a
^ E
D
2 S 2 E
a
^ E
D
1 P 1 E
a
.
e (27) have demonstrated that when glutamate dehydro-
D
and mitochondrial malate dehydrogenase is E
a
, there
additional steps shown in Mechanism 1. These steps are
es with both E
a
and E
a
-S and S also associates with
ation 1 is the rapid equilibrium rate equation for Mech-
erived by assuming the rate constants of the product
ps k
1
and k
2
are slow compared with the rate constants of
teps of the mechanism.
v
@E
aT
#
5
k
1
1
k
2
@E
D
S#K
1
@S#K
5
1 1
K
1
@S#
S
1 1
@E
D
#
K
2
1
@E
D
S#
K
5
D
[1]
s the initial velocity, [E
aT
] is the total concentration of the
zyme, [E
D
S] is the concentration of the complex between
nzyme and the noncatalytic enzyme, [E
D
] is the concentra-
free noncatalytic enzyme, [S] is the concentration of free
idine nucleotide. The dissociation constants K
2
to K
6
are the
constants for the reactions shown in Mechanism 1. Thus:
[E
a
S]; K
2
5 [E
a
][E
D
]/[E
a
E
D
]; K
3
5 [E
D
][S]/[E
D
S]; K
4
5 [E
a
]
E
D
]; K
5
5 [E
a
][E
D
S]/[E
a
SE
D
] and K
6
5 [E
a
E
D
][S]/[E
a
S
ng to the rapid equilibrium derivation K
1
K
4
5 K
3
K
5
5 K
2
K
6
D
]/[E
a
SE
D
]. Consequently, the terms [E
D
]/K
4
or [E
D
]K
1
/K
2
K
6
tituted for the term [E
D
S]K
1
/[S]K
5
in Eq. 1. The values of
d [E
D
S] in Eq. 1 were calculated with the use of Eq. 2 and
f K
3
or the K
D
of the E
D
-S complex measured spectrofluoro-
s described previously (27).
K
3
5
@E
D
#@S#
@E
D
S#
[2]
ectrofluorometric experiments there was no chemical
f the reduced pyridine nucleotides during the time re-
easurements.
icted velocity of the reaction catalyzed by E
a
is defined as
observed if there were negligible E
a
-E
D
and E
a
-S-E
D
o that the only effect of E would be to decrease the free
D
his would be the case if K
2
and K
5
of Eq. 1 are quite high
terms in this equation containing these constants are