From the Department of Biochemistry and the
§ Department of Physiology, Michigan State University,
East Lansing, Michigan 48824
Received for publication, August 10, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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Glycerol-3-phosphate dehydrogenase from pig brain
mitochondria was stimulated 2.2-fold by the addition of 50 µM L-ascorbic acid. Enzyme activity,
dependent upon the presence of L-ascorbic acid, was
inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl
ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial
glycerol-3-phosphate dehydrogenase was activated by either 150 µM L-ascorbic acid (56%) or 300 µM iron (Fe2+ or Fe3+ (62%)) and
2.6-fold by the addition of both L-ascorbic acid and iron.
The addition of L-ascorbic acid and iron resulted in a
significant increase of kcat from 21.1 to 64.1 s Mitochondrial glycerol-3-phosphate dehydrogenase
(mGPDH)1 (EC 1.1.99.5) plays
a critical role in the shuttle of glycolytically generated reducing
equivalents into mitochondrial electron transport and oxidative
phosphorylation in numerous tissues (1-4). In pancreatic islet In confirmation of the original observations of Sigal and King that
scorbutic guinea pigs demonstrated abnormal glucose tolerance (15),
L-ascorbic acid was shown to be essential for the release of insulin from scorbutic guinea pig pancreatic islets (16, 17).
Further studies have demonstrated that L-ascorbic acid is
an essential cofactor for the activation of mGPDH (oxidase) in
mitochondria from guinea pig tissues and rat liver (18). In purified
preparations of mGPDH from a variety of sources, both iron and acid
extractable sulfur have been reported (2, 19-21), suggesting that an
iron/sulfur center is involved in the catalytic mechanism of this
mitochondrial inner membrane bound enzyme. No further evidence,
however, was obtained to support this suggestion, and the iron center
of mGPDH has remained uncharacterized.
In addition to the need to clarify the iron/L-ascorbic acid
relationship of mGPDH in intact mitochondria, it was essential to
examine the effect of L-ascorbic acid, iron and specific
di-iron metalloenzyme inhibitors on homogeneous mGPDH. In the present study, the effects of propyl gallate and other related
polyhydroxybenzoate inhibitors on pig brain mGPDH in intact
mitochondria and on preparations of pure mGPDH were examined. Because
of the potential role of mGPDH for shuttling reducing equivalents into
the mitochondria during glucose-induced insulin release from pancreatic
Materials
DL- Methods
Isolation of Mitochondria--
Frozen pig cerebrum was
homogenized in an all glass Dounce homogenizer with a 10× volume of
homogenizing fluid described by Greenawalt (22) consisting of 200 mM mannitol, 80 mM sucrose, and 10 mM potassium HEPES, pH 7.4. The mitochondrial fraction was
isolated by differential centrifugation following the sedimentation protocol reported by Lai and Clark (23). Briefly, homogenization was
performed on ice with eight thrusts of the Dounce plunger. All
subsequent steps were conducted on ice or in a refrigerated centrifuge
(Sorval-RC2-B) at 4 °C. The homogenate was first centrifuged at
1,000 × g for 10 min, and the supernatant was removed.
The supernatant fraction was then centrifuged at 15,000 × g for 10 min. The upper layer was decanted off, and the
pellet was gently rinsed with small amounts of homogenizing fluid to
remove broken mitochondrial remnants. The pellet was gently resuspended
in 5 volumes of homogenizing fluid using a Dounce homogenizer and
centrifuged at 12,000 × g for 10 min. The supernatant
was discarded, and the pellet was gently washed with homogenizing fluid
to remove light weight fluffy mitochondrial fragments. The pellet was
resuspended in 2.5 volumes of homogenizing fluid in a Dounce
homogenizer, and the suspension was centrifuged at 12,000 × g for 10 min. The supernatant was decanted off, and the
pellet was resuspended in ~1 ml of 250 mM sucrose, 10 mM Tris-HCl, pH 7.5, per g original tissue weight.
Electron Transport Particles--
Mitochondria were isolated as
described above and placed in a freezer at Mitochondrial Enzyme Assays--
mGPDH activity in intact
mitochondria was measured by oxygen uptake using a Clark oxygen
electrode. mGPDH from freshly prepared mitochondrial suspensions of
5-7 g protein/ml in 250 mM sucrose, Tris-HCl, pH 7.5, from
frozen pig brain was assayed as described previously (18). The Clark
oxygen electrode chamber of 3.0 ml (model 53 oxygen monitor, Yellow
Springs Instrument Co. Yellow Springs, Ohio) contained 250 mM sucrose, 10 mM Tris-HCl, pH 7.5, 1 mM GSH, 0.5-0.8 mg of mitochondrial protein, with or
without 50 µM L-ascorbic acid. Typically,
oxygen uptake was measured for 3-6 min followed by the addition of 50 mM DL-glycerol-3-phosphate, pH 7.5, at
37 °C. The rate of oxygen uptake was measured with a Linseis
recorder, and the rate, in nmol oxygen uptake/min, was corrected for
the basal oxygen uptake rate before addition of the substrate. Values
were expressed as nmol oxygen uptake/min/mg protein. Stock solutions of
the inhibitor compounds were made up in absolute ethanol. When added to
the assay mixture, ethanol aliquots of 30 µl or less were
preincubated with the mitochondria for 3 min followed by the sequential
addition of 50 µM L-ascorbic acid (3 min of
incubation) and 50 mM DL-glycerol-3-phosphate. Controls of up to 30 µl of ethanol in the 3.0-ml reaction volume had
no effect on the oxygen uptake rates (data not shown).
NADH dehydrogenase (oxidase) from pig brain ETPs was assayed
spectrophotometrically by a modification of the method of Singer (25).
The reaction mixture contained 100 mM potassium phosphate, pH 7.4, 1 mM GSH, 0.26 mM NADH, 0.1-0.12 mg
ETP protein, with or without 3 min of preincubation with various levels
of inhibitors or 50 µM L-ascorbic acid at
30 °C in a total of 500 µl. The reaction was initiated by the
addition of NADH, and the decrease in absorbance at 340 nm was recorded
in a Gilford Response II recording spectrophotometer. NADH
dehydrogenase activity (nmol/min/mg protein) were calculated from the
extinction coefficient of NADH of 6.22 × 103
M
Succinoxidase activity in ETP preparations was assayed by a
modification of the method of Green and Ziegler (24). The rate of
succinate oxidation at 37 °C was measured by oxygen uptake analysis
using a Clark oxygen electrode. The 3.0-ml reaction chamber contained
100 mM potassium phosphate, pH 7.4, 1 mM GSH,
0.3 mg of ETP protein, 50 mM potassium succinate, and with
or without various concentrations of inhibitors or 50 µM
L-ascorbic acid. When inhibitors or L-ascorbic
acid were included, ETPs were preincubated for 3 min. Ethanol, up to 30 µl/3.0 ml reaction volume, did not affect the succinoxidase activity.
mGPDH Purification--
All steps in the purification of mGPDH
were monitored for activity spectrophotometrically by the method of
Garrib and McMurray (26). The assay cuvettes contained in a final
volume of 0.5 ml, 90 mM potassium phosphate, pH 7.5, 1 mM KCN, 3 mM INT, 0.6 mM menadione,
and various enzyme fractions. The reaction was initiated by the
addition of 30 mM DL-glycerol-3-phosphate and
was monitored at 500 nm. The extinction coefficient of reduced INT was
taken as 11.5 × 103 M
The purification of mGPDH was a modification of the method of Garrib
and McMurray (27). In a typical preparation, 65-100 g of frozen pig
brain were thawed and homogenized at 4 °C in 650-1000 ml of
homogenizing buffer containing 200 mM mannitol, 80 mM sucrose, 0.2 mM L-ascorbic acid,
1 mM DTT, and 10 mM HEPES, pH 7.4, in a Waring
blender for 1 min at low speed. The suspension was centrifuged at
1000 × g for 10 min in a Sorval RC2-B refrigerated
centrifuge at 4 °C. The supernatant was stored on ice or kept at
4 °C for all subsequent steps, and the pellet was resuspended in 300 ml of homogenizing buffer and homogenized with an all glass Dounce homogenizer using five strokes of the pestle. The homogenate was diluted with 300 ml of homogenizing buffer, stirred for 5 min, and then
centrifuged at 1000 × g for 10 min. The supernatant
fractions of the previous steps were combined and centrifuged at
15,000 × g for 10 min. The supernatant was discarded,
and the pellets were resuspended in 300 ml of 0.15 M KCl
containing 1 mM DTT and centrifuged at 15,000 × g for 10 min. The pellets were resuspended in 40 ml of 10 mM Tris-HCl, pH 7.5, containing 250 mM sucrose followed by dilution 10-fold with 1 mM DTT. The slurry was
stirred for 60 min on ice, and the diluted suspension was centrifuged at 15,000 × g for 15 min. The pellet was washed with
300 ml of 50 mM potassium phosphate, pH 7.5, 0.1 mM DTT, and 0.02% NaN3 followed by
centrifugation at 15,000 × g for 15 min. The pellets were resuspended in 50 mM potassium phosphate, 0.1 mM DTT, and 0.02% NaN3, and the protein
content was determined by the BCA method described previously. The
suspension was stored at DEAE-Sepharose Chromatography--
Washed and stored
mitochondria were thawed at 37 °C, chilled, and kept at 4 °C
throughout each subsequent purification step. The mGPDH was solubilized
by addition of appropriate amounts of 10% Triton X-100 (Triton
X-100/protein = 0.25 mg/mg protein) and was stirred for 60 min.
The suspension was centrifuged at 60,000 × g for 30 min, and the supernatant fraction was saved for further purification. A
2.5 × 15 cm column of DEAE-Sepharose (fast flow) was equilibrated
with buffer A consisting of 50 mM potassium phosphate, pH
7.5, 0.1% Triton X-100, 0.02% NaN3, 0.1 mM
DTT, and 10% glycerol. After loading the sample, the column was washed
with buffer A, and the enzyme was eluted with a linear NaCl gradient
consisting of 150 ml of buffer A and 150 ml of buffer A containing 0.6 M NaCl at a flow rate of 1 ml/min. The fractions with
enzyme activity were pooled and diluted with triple distilled water to
10 mM potassium phosphate, pH 7.5, 0.1 mM DTT,
0.1% Triton X-100, 2% glycerol, and 0.02% NaN3.
Hydroxylapatite Chromatography--
A column of hydroxylapatite
(2.5 × 6.0 or 12.0 cm) was equilibrated with buffer B consisting
of 10 mM potassium phosphate, pH 7.5, 0.1 mM
DTT, 0.1% Triton X-100, 100 mM NaCl, 5% glycerol, and
0.02% NaN3. The diluted enzyme sample from the DEAE column was applied to the hydroxylapatite and washed with buffer B. The enzyme
was eluted by a linear sodium phosphate gradient consisting of 50 ml of
buffer B and 50 ml of buffer B with 150 mM sodium phosphate, pH 7.5. The enzyme containing fractions were subjected to
LDS-PAGE to monitor purity. The fractions with highest activity and
purity were pooled and concentrated to 1.5 ml by an Amicon Centiprep
YM-10 apparatus.
Sephacryl S-300-HR Chromatography--
The concentrated enzyme
fraction was applied to a Sephacryl S-300 (fast flow) column (2.5 × 92 cm) equilibrated with buffer C (50 mM potassium
phosphate, pH 7.5, 0.05% Triton X-100, 0.02% NaN3, 0.1 mM DTT, 20% glycerol, and 0.2 M NaCl at a flow
rate of 0.25 ml/min. The fractions containing mGPDH activity were
pooled, concentrated as described above, and subjected to LDS-PAGE
analysis to assess purity.
LDS-PAGE--
LDS-PAGE was performed on samples of purified
mGPDH by a modification (28) of the basic method of Laemmli (29). The
stacking and separating gels (0.75 mm) had polyacrylamide
concentrations of 5 and 8%, respectively. The gels were run at a
constant 100V in a Bio-Rad minigel apparatus, and the gel was stained
with Coomassie Brilliant Blue R-250.
Homogeneous mGPDH Assay--
For measurement of the mGPDH
specific activity and kinetic properties, preparations of the pure
enzyme were assayed by oxygen uptake using the Clark electrode
following a modification of the assay described by Beleznai et
al. (30). Homogeneous mGPDH (2-5 µg), stabilized in a solution
of 50 mM HEPES, pH 7.5, 0.1 mM DTT, 0.05%
Triton X-100, 0.02% NaN3, and 20% glycerol, was placed in a 3.0-ml chamber at 37 °C containing 100 mM potassium
phosphate, pH 7.6, 0.6 mM GSH, 0.05% Triton X-100, and
0.15 mM menadione (freshly prepared as a 10 mM
stock solution in absolute ethanol) as electron acceptor. When
L-ascorbic acid was added, stock solutions of 10 mM crystalline L-ascorbic acid were freshly
prepared in Chelex treated triple distilled water and stored on ice.
For the addition of Fe2+ or Fe3+, freshly
prepared stock solutions of 10 mM FeSO4 or
FeCl3 were added prior to the preincubation period. After a
2-min preincubation, followed by a period (3-5 min) of linear
recording for blank oxygen consumption, rates for mGPDH were initiated
by the addition of 75 mM
DL-glycerol-3-phosphate (stock solution of 1.5 M substrate dissolved in 30 mM potassium
phosphate, pH 7.6). In the absence of enzyme, the addition of substrate
caused no change in background oxygen uptake rate. For inhibition
analysis, stock solutions of 60 mM propyl gallate in
absolute ethanol were stored at Enzyme Kinetics--
Oxygen uptake measurements were carried out
as described above using varying concentrations of
L-glycerol-3-phosphate (as the DL mixture) with 4.4 µg of
protein in a 3.0-ml oxygen electrode chamber. GSH (0.6 mM),
which had no effect on the oxygen uptake reaction rates, was added to
each sample to maintain the L-ascorbic acid, when added, at
the level designated, 150 µM. When iron was added to the
reaction mixture, it was provided as 150-300 µM
FeSO4 or 300 µM FeCl3. Reaction
rates after substrate addition were corrected by rates measured in the
absence of substrate. Other controls were run in the absence of enzyme
when appropriate. Values were expressed as nmol/min and kinetic
constants, Km(app) and
Vmax(app), were determined using the advanced
kinetics software provided by Gilford for the Response II
spectrophotometer. The values for kcat were
calculated by dividing Vmax(app) by the molar concentration of pig brain mGPDH with the molecular weight taken as
75,000 (21).
INS-1 Cell Culture--
INS-1 cells (kindly provided by Dr.
C. B. Wollheim (31)) were routinely cultured in
CO2/air (1:19) at 37 °C in RPMI 1640 medium containing
11.1 mM glucose and supplemented with 10% fetal bovine
serum, 1 mM pyruvate, 10 mM HEPES, pH 7.4, 50 µM 2-mercaptoethanol, 100 units penicillin/ml, and 100 µg streptomycin/ml. Cells were passed weekly by trypsin-EDTA
detachment. All experiments were performed on INS-1 cells between
passages 70-85.
Insulin Secretion Studies--
For static secretion studies,
INS-1 cells were plated onto 12-well plates at a density of 1.5 × 106 cells/well in RPMI 1640 medium plus supplements and
grown for 24 h. The growth medium was then changed to RPMI 1640 containing 4 mM glucose plus the supplements described
above, and cells were cultured for an additional 30 h. Cells were
then incubated for 60 min at 37 °C in Krebs Ringer bicarbonate
buffer (KRB buffer) (118.5 mM NaCl, 2.54 mM
CaCl2, 1.19 mM KH2PO4,
4.74 mM KCl, 25 mM NaHCO3, 1.19 mM MgCl2, 10 mM HEPES, pH 7.4, 0.1% bovine serum albumin) containing 4.0 mM glucose.
Cells were then incubated for 20 min at 37 °C in KRB buffer
containing 100 µM 3-isobutyl-1-methylxanthine and either
4 or 16.7 mM glucose in the absence or presence of metabolic inhibitors as indicated in the figure legends. Propyl gallate
was dissolved in absolute ethanol, and therefore all controls contained
equal amounts of ethanol. Concentrations of insulin released into the
medium were determined by insulin enzyme-linked immunosorbent assay
using a modification of the procedure described by Kekow et
al. (32). Insulin released into the medium was normalized to
cellular protein concentrations determined according to Lowry et
al. (33).
Glucose Utilization Studies--
Glucose usage was measured
using a modification of the method of Zawalich and Matschinsky (34,
35). INS-1 cells were plated onto 12-well plates at a density of
1.5 × 106 cells/well and grown for 24 h. The
growth medium was then changed to RPMI 1640 medium containing 4 mM glucose and supplements described above and incubated
for an additional 30 h. Cells were then incubated for 60 min at
37 °C in KRB buffer containing 4.0 mM glucose. Glucose utilization was then measured by incubating cells for 30 min at 37 °C in 1 ml of KRB buffer containing 100 µM
3-isobutyl-1-methylxanthine and either 4 mM or 16.7 mM glucose and [5-3H]glucose at a final
specific activity of 2.2 dpm/pmol. Vehicle controls or metabolic
inhibitors were added as indicated in the figure legends. Background
controls were determined by incubating the medium in the absence of
cells. After incubation, duplicate 50-µl samples of the incubation
medium were added to Eppendorf tubes containing 5 µl of 1 N HCl. The Eppendorf tubes were then placed in
scintillation vials containing 0.5 ml of H2O, and the scintillation vials were sealed tightly and incubated at 50 °C for
18 h. After cooling, the Eppendorf tubes were removed, 10 ml of
Safety-Solve scintillation mixture were added to the vials, and the
samples were counted in a Beckman scintillation counter. Glucose
utilization was then determined from the following formula and
expressed as picomoles of glucose metabolized per min per mg protein.
The equilibration coefficient (EQC) was determined with
3[H]H2O following the procedure outlined
above. Glucose usage = (dpm Pig Brain mGPDH--
Pig brain mGPDH activity in isolated intact
mitochondria was 28.6 ± 6.6 nmol/min/mg protein
(n = 11). Addition of 50 µM
L-ascorbic acid increase mGPDH activity to 62.9 ± 10.7 nmol/min/mg protein (n = 11). These activities
were completely inhibited by 10 mM KCN (data not shown),
indicating that a functional cytochrome c oxidase was
required to complete the reaction with oxygen whether L-ascorbic acid was present or not. SHAM, PCAEE, propyl
gallate, and lauryl gallate were potent inhibitors of pig brain mGPDH
activity in intact mitochondria stimulated by L-ascorbic
acid (Fig. 1). In contrast, these agents
were without effect on the basal activity, i.e. activity in
the absence of L-ascorbic acid (data not shown). The
concentration of each compound calculated to cause 50% inhibition of
the L-ascorbic acid stimulated activities were: SHAM,
27.7 ± 6.9 µM; PCAEE, 585 ± 203 nM; propyl gallate, 305 ± 113 nM; and lauryl gallate, 111 ± 42 nM.
NADH dehydrogenase and succinoxidase activities from pig brain ETPs
were compared in the presence and absence of the four hydroxybenzoic
acid derivatives. Minimal concentrations of each agent, previously
found to completely inhibit the L-ascorbic acid-stimulated mGPDH activity, had no effect on these two well established iron/sulfur enzymes (data not shown). In addition, L-ascorbic acid had
no stimulatory effect on the activity of either NADH dehydrogenase or
succinoxidase (data not shown).
Purification of mGPDH--
The purification of mGPDH from pig
brain was accomplished by a series of steps including Triton X-100
extraction of washed mitochondria, DEAE-Sepharose chromatography,
Bio-Gel HT hydroxylapatite chromatography, and Sephacryl S-300 gel
chromatography (Table I). mGPDH was
judged to be homogeneous based on LDS polyacrylamide gel
electrophoresis (Fig. 2) with a molecular
weight of 75,000. This value is in good agreement with that reported by
Cottingham and Ragan (21).
mGPDH Activity--
The basal activity of purified mGPDH was
14.0 ± 2.2 µmol/min/mg protein (Table
II). This activity could be stimulated
56% to 21.9 ± 3.3 µmol/min/mg (p = <0.001) by
the addition of 150 µM L-ascorbic acid. The
addition of 300 µM Fe2+ (optimal level based
on dose-response curve; data not shown) also activated the purified
enzyme by 62% to a value of 22.7 ± 4.2 µmol/min/mg
(p = <0.005), suggesting that there was a partial loss
of iron from the enzyme binding centers during the purification steps.
The addition of both L-ascorbic acid and Fe2+
resulted in a 2.6-fold increase in activity over that of the control
(36.7 ± 3.0 µmol/min/mg, p = <0.001).
Preincubation of the enzyme with 200 µM propyl gallate
caused a decrease in activity from 14.0 to 9.1 ± 1.6 µmol/min/mg (p = <0.005), which we attribute to the
complete blockage of the iron pathway of electron transport between
substrate and the acceptor, menadione. From these data, it can be
estimated that 35% of the basal activity utilized the iron-mediated
pathway, whereas 65% followed the FAD/FADH2 linked pathway
to menadione. In contrast, in the presence of 150 µM
L-ascorbic acid, 52% of the substrate oxidation passed
along the iron-dependent pathway, whereas 48% followed the
FAD/FADH2-dependent pathway. The difference
between control samples with inhibitor and L-ascorbic acid
supplemented samples with inhibitor (9.1 versus 11.4 µmol/min/mg) were not significantly different by statistical
analysis. Propyl gallate also completely inhibited the Fe2+
and Fe2+ plus L-ascorbic acid activity
stoichiometrically (data not shown). When Fe3+ was provided
as 300 µM FeCl3 instead of 300 µM Fe2+, comparable results were obtained
(data not shown).
mGPDH Kinetics--
The kinetic constants for purified mGPDH are
reported in Table III. In these studies,
the values for basal control are compared with those obtained for
enzyme supplemented with 150 µM L-ascorbic acid and 150 µM Fe2+. The
Km(app) for basal enzyme was 10.0 ± 1.2 mM L-glycerol-3-phosphate (in good
agreement with reference number 3), whereas that for the stimulated
enzyme was 14.5 ± 4.9 mM (not statistically
different). L-Ascorbic acid and Fe2+
supplementation increased the Vmax(app) from
56.9 ± 13.2 nmol/min to 161.9 ± 24,7 nm/min
(p = <0.0001). Expressed as
kcat values, the controls were 21.1 ± 9.2 compared with 64.1 ± 25.3 s Effect of Propyl Gallate on Glucose Utilization and Insulin
Secretion from INS-1 Cells--
In pancreatic
Incubation of INS-1 cells in 16.7 mM glucose led to a
4.83 ± 0.11-fold (n = 4) increase in the rate of
conversion of 5-[3H]glucose to
[3H]H2O compared with cells incubated in 4.0 mM glucose (Fig. 3). The
addition of 500 µM propyl gallate led to a 23.64 ± 0.57% (n = 4, p < 0.006) and
13.61 ± 3.42% (n = 4, p < 0.008) reduction in glucose usage in cells incubated in 4 or 16.7 mM glucose, respectively. The addition of 1 mM
propyl gallate led to a 75.20 ± 2.32% (n = 4, p < 0.0001) and 80.75 ± 1.23%
(n = 4, p < 0.0001) reduction in cells
incubated in 4 or 16.7 mM glucose, respectively. Treatment of cells with propyl gallate concentrations lower than 250 µM had no significant effects on glucose usage in cells
incubated in 4.0 or 16.7 mM glucose. Next the ability of
aminooxyacetate (AOA), an inhibitor of aspartate aminotransferases in
the malate-aspartate shuttle (37), to potentiate the propyl
gallate-mediated inhibition of glucose usage was determined. Incubation
of cells in 5 mM AOA led to a 27.08 ± 1.47%
(n = 4, p < 0.004) and 23.77 ± 3.10% (n = 4, p < 0.005) reduction in
glucose utilization in cells cultured in 4.0 mM or 16.7 mM glucose, respectively. Combined treatment of cells with
5 mM AOA and 500 µM propyl gallate led to a
further reduction in glucose usage in cells incubated in 16.7 mM glucose. Nevertheless, combined treatment of 5 mM AOA and 1 mM propyl gallate were not able to
reduce glucose usage below that observed with 1 mM
propyl gallate alone.
The ability of propyl gallate to inhibit both purified mGPDH activity
and glucose usage in INS-1 cells suggests that propyl gallate may also
inhibit glucose-induced-insulin release. Incubation of INS-1 cells in
16.7 mM glucose led to a 3.15 ± 0.34-fold
(n = 4, p < 0.0006) increase in
insulin release compared with cells incubated in 4.0 mM
glucose (Fig. 4). The addition of 250 µM, 500 µM and 1 mM propyl
gallate led to a 65.93 ± 2.59% (n = 4, p < 0.003), 66.16 ± 7.19% (n = 4, p < 0.004), and 74.11 ± 4.06% (n = 4, p < 0.002) reduction in
insulin secretion, respectively, from cells incubated in 4 mM. Importantly, the addition of 500 µM and 1 mM propyl gallate led to a 89.22 ± 1.67%
(n = 4, p < 0.0001) and 91.18 ± 3.66% (n = 4, p < 0.0001) reduction
in insulin release, respectively, from cells incubated in 16.7 mM glucose.
Treatment of cells with 250 µM propyl gallate had no
significant effects on the ability of 16.7 mM to induce
insulin release. Next the ability of AOA to potentiate the propyl
gallate-mediated inhibition of insulin release was determined.
Incubation of cells in 5 mM AOA led to a 36.62 ± 3.35% (n = 4, p < 0.03) and
58.75 ± 3.78% (n = 4, p < 0.001) reduction in insulin release from cells cultured in 4.0 or 16.7 mM glucose, respectively. Combined treatment of cells with
5 mM AOA and 500 µM or 1 mM
propyl gallate were not able to further reduce insulin release from
cells incubated in either 4.0 or 16.7 mM glucose when
compared with cells incubated with 500 µM or 1 mM propyl gallate alone.
mGPDH of mammalian origin has been purified to homogeneity by
other laboratories with results, suggesting that the inner
mitochondrial membrane bound enzyme contains iron and acid releasable
sulfur (2, 19-21). However, the amount of iron found after
purification ranged between 1 mol of iron/100,000-350,000 × g of enzyme protein. Because pig brain mGPDH is a monomer of
75 kDa, these iron level estimations suggested that significant loss of
iron occurred during the purifications previously reported. In
agreement with this suggestion, the activity of our pure enzyme
preparations was stimulated by the addition of iron. It is difficult to
account for the origin of the acid releasable sulfur from homogeneous
mGPDH reported by earlier workers (2, 3, 19-21). Little further
evidence has been published to support the existence of a typical
iron/sulfur center (38, 39). Moreover, NADH dehydrogenase and
succinoxidase, key enzymes in mitochondrial electron transport and well
established iron/sulfur metalloenzymes (4), were not activated by
L-ascorbic acid nor inhibited by propyl gallate or its
homologues in the present study.
The finding of potent inhibition of the L-ascorbic
acid-stimulated activity of mGPDH by propyl gallate and related
compounds in intact mitochondria or that inhibition by propyl gallate
of combined iron and L-ascorbic acid activation of
homogeneous mGPDH is consistent with results expected for a di-iron
metalloenzyme (40). For example, Schonbaum et al. (41)
reported that substituted benzohydroxamic acids specifically inhibited
the cyanide-insensitive "alternative oxidase" electron transport
pathway in isolated plant mitochondria. Siedow et al. (42)
identified conserved amino acids, including two copies of the
iron-binding motif, EXXH, in the C-terminal domain of the
alternative oxidase that suggested the presence of a hydroxo-bridged
binuclear iron center. Recently, mGPDH was cloned and sequenced from
rat (9), human (43), and Drosophila melanogaster (44)
sources. Inspection of the sequences of the mammalian enzymes revealed
the presence of conserved sequences at 139EAL142H and another sequence
at 360DVY363H (D instead of E) that could function as a di-iron binding
site (9, 43). Future studies are needed to elucidate the possible site or sites of iron binding in mGPDH and the action of
L-ascorbic acid in maximizing the flow of electrons into
the ubiquinone pool.
Trypanosomes, isolated from the blood of hosts, exhibit a
SHAM-sensitive, cyanide-insensitive terminal oxidase that utilizes glycerol-3-phosphate as substrate by participating in a
NAD+ regenerating cycle (45). This glycerol phosphate
oxidase system has been the subject of studies aimed at designing
drugs, such as homologues of SHAM and the inhibitors selected in the
present work, that specifically kill trypanosomes in vivo
without producing severe toxic side effects in the host (46-49). The
metal chelation properties of these drugs have been implicated in their
inhibitory action (46). Alternatively, the effect of hydroxamic acids
on the glycerol phosphate oxidase system of trypanosomes was proposed to be due to their ability to competitively displace ubiquinol, the
putative electron carrier, from the dehydrogenase to the terminal oxidase of the glycerol phosphate oxidase complex (50). Whether either
of these two unique systems, plant alternative oxidase and trypanosome
terminal oxidase, are activated by L-ascorbic acid remains
to be explored.
If mGPDH is indeed an essential enzyme in the proximal events linked to
glucose-stimulated glucose metabolism and insulin release as previously
reported (5, 8, 11, 51), specific binuclear iron inhibitors such as
propyl gallate should significantly inhibit the process. This
hypothesis was tested in the INS-1 cell line because they have been
previously reported to have high levels of mGPDH activity similar to
that observed in rat pancreatic Our previous studies have shown L-ascorbic acid is
essential for insulin release from scorbutic guinea pig islets (16, 17) and that L-ascorbic acid serves as an essential cofactor
for mGPDH (oxidase) activation in mitochondria isolated from guinea pig tissues and rat liver (18). However, the requirement for ascorbic acid
in glucose-induced insulin release from INS-1 cells has been difficult
to directly access. In unpublished studies, we have established that
INS-1 cells are capable of dephosphorylating exogenously added
ascorbic acid 2-phosphate, thus releasing ample amounts of vitamin C. Addition of 1 mM ascorbic acid 2-phosphate, however, did
not enhance glucose utilization or glucose-induced insulin release from
INS-1 cells.2 The inability
of exogenously added ascorbic acid 2-phosphate to stimulate both
glucose utilization and insulin release is most likely due to the
difficulty of establishing scorbutic INS-1 cells because these cells
are capable of scavenging trace amounts of ascorbic acid present in our
commercial source of fetal bovine serum.2 Nevertheless, the
ability of propyl gallate to block ascorbic acid-activated mGPDH
activity in vitro and to reduce both glucose utilization and
insulin release from INS-1 cells suggests that ascorbic acid plays an
essential role in glucose-induced insulin release.
Overall we conclude that mGPDH is activated by L-ascorbic
acid via a potential di-iron reactive center and is effectively inhibited by propyl gallate and other polyhydroxybenzoic acid derivates. Furthermore, the glycerol phosphate shuttle, in comparison with the malate-aspartate shuttle, is crucial to the release of insulin
from INS-1 cells in response to elevated glucose levels. Use of propyl
gallate and other related polyhydroxybenzoate inhibitors may serve as
effective tools for studying the role of mGPDH in glucose-induced
insulin secretion.
1, without significantly increasing the
Km of L-glycerol-3-phosphate (10.0-14.5 mM). The activation of pure
glycerol-3-phosphate dehydrogenase by either L-ascorbic
acid or iron or its combination could be totally inhibited by 200 µM propyl gallate. The metabolism of [5-3H]glucose and the glucose-stimulated insulin
secretion from rat insulinoma cells, INS-1, were effectively inhibited
by 500 µM or 1 mM propyl gallate and to a
lesser extent by 5 mM aminooxyacetate, a potent
malate-aspartate shuttle inhibitor. The combined data support the
conclusion that L-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the
enzyme is potently inhibited by agents that specifically inhibit
certain classes of di-iron metalloenzymes, and that the enzyme is
chiefly responsible for the proximal signal events in INS-1 cell
glucose-stimulated insulin release.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells, many studies support the significant participation of mGPDH and
the L-
-glycerol-3-phosphate shuttle in the proximal events that signal the release of insulin in response to increased glucose (5-12). Recently, particular importance has been attributed to
the role of NADH in the glucose-induced activation of mitochondrial metabolism and insulin secretion (13, 14). These studies emphasized the
essential roles played by both the glycerol-3-phosphate and the
malate-aspartate shuttles in modulating the cytosolic NADH pool.
cells, we also investigated the effects of a di-iron metalloenzyme
inhibitor, propyl gallate, on glucose usage and glucose-induced insulin
release from the rat insulinoma cell line, INS-1.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Glycerol phosphate, mannitol, MOPS,
NaN3, Triton X-100, SHAM, PCAEE, DEAE-Sepharose (fast
flow), and Sephacryl S-300 (fast flow) were purchased from Sigma.
Sucrose, HEPES, Tris-base, glutathione, and NADH were from Roche
Molecular Biochemicals. L-Ascorbic acid and glycerol were
from J. T. Baker, Inc. L-ascorbic acid-2-phosphate
(Mg2+) was purchased from Wako Pure Chemical Industries,
Ltd. Menadione was a product of Nutritional Biochemicals Corp. Lauryl
gallate, propyl gallate, and
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride were
purchased from Aldrich. Hydroxylapatite, 40% acrylamide/bis solution,
and low molecular weight protein standards were obtained from Bio-Rad.
Succinic acid was a product of Mallinckrodt Chemical Works.
[5-3H]D-Glucose (10-20 Ci/mmol) was
purchased from PerkinElmer Life Sciences. Rat insulin antibodies and
rat insulin standards were purchased from Linco (St. Charles, MO). All
other chemicals used were of A.C.S. reagent grade. Pig brains were
kindly provided by Thomas Fortan from Michigan State University Meats
laboratory. Cerebra were removed shortly after slaughter, chilled on
ice, washed in 0.25 M sucrose to remove excess blood, and
frozen (
70 °C) for storage.
20 °C until needed.
ETPs were prepared according the method of Green and Ziegler (24). The
thawed mitochondrial preparations were centrifuged at 12,000 × g for 10 min, and the pellet was resuspended to a
concentration of ~20 mg protein/ml in a solution of 250 mM sucrose, 10 mM Tris-HCl, pH 7.5, 15 mM MgCl2, and 1 mM ATP at 4 °C.
The suspension was sonicated by a Branson Sonifier 450 with 3 × 30 s bursts of energy (40 watts). The suspension was centrifuged
at 1000 × g for 10 min, and the supernatant fraction
was further centrifuged at 100,000 × g for 40 min in a
Spinco Ultracentrifuge 40K rotor. The pellet was resuspended in 250 mM sucrose, 10 mM Tris-HCl, pH 7.5, containing
10 mM MgCl2 and 1 mM ATP. The
suspended ETPs at ~5-6 mg protein/ml were stored at
20 °C until
assayed. Protein concentrations of mitochondria or ETP preparations
were determined by the BCA protein assay protocol according to the
manufacturer's direction (Pierce) using bovine serum albumin as standard.
1 cm
1. Because 5 µl of
ethanol were without effect on enzyme activity, all additions of
inhibitors were made with less than 5 µl of ethanol.
1
cm
1 at 500 nm (26). Rates were expressed as nmol/min, and
protein was determined by the bicinchoninic acid protocol described above.
70 °C until required.
20 °C. When propyl gallate was
included, it was added to the incubation mixture at 37 °C for 2 min
prior to measurement of control oxygen uptake rates and those following
addition of DL-glycerol-3-phosphate.
blank)/(specific activity × EQC × min).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
Inhibition of the L-ascorbic
acid-dependent mGPDH activity of pig brain mitochondria by
lauryl gallate (A), propyl gallate
(B), SHAM (C), and PCAEE
(D). The rates are expressed as nmol oxygen
consumed/min/mg protein in the presence of 50 µM
L-ascorbic acid as described under "Methods." Each
value is the mean ± S.E. error for 4-11 separate
experiments.
Purification of pig brain mGPDH
View larger version (53K):
[in a new window]
Fig. 2.
LDS-PAGE analysis of pig brain mitochondrial
glycerol-3-phosphate dehydrogenase. Lane 1, Bio-Rad low
molecular weight standards; lane 2, 5.6 µg of protein
taken from the reactive fraction isolated via the Sephacryl S-300 gel
chromatography purification step. The band indicated by an
arrow migrated with an estimated molecular weight of 75,000. The gel was stained with Coomassie Brilliant Blue R-250.
Effect of L-ascorbic acid, iron, and propyl gallate on
homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase
1. Taken together,
kcat/Km values were 2.1 × 103 and 4.4 × 103
M
1 s
1, for control and
supplemented samples, respectively.
Homogeneous mitochondrial glycerol-3-phosphate dehydrogenase kinetic
parameters
cells, it is well
established that there is a relationship between glucose metabolism
through mGPDH and insulin release (5-9, 11, 12, 36). Therefore, agents
that effectively inhibit L-ascorbic acid-induced mGPDH
activity may have profound effects on glucose utilization and insulin
release from pancreatic
cells. To examine this possibility, the
effects of propyl gallate on glucose utilization and insulin secretion
from INS-1 cells were determined.
View larger version (43K):
[in a new window]
Fig. 3.
Effect of propyl gallate and/or AOA on
glucose utilization in INS-1 cells. Glucose utilization was
measured by incubating cells for 30 min at 37 °C in KRB buffer
containing either 4 or 16.7 mM glucose and
[5-3H]D-glucose (final specific activity, 2.2 dpm/pmol) in the presence or absence of metabolic inhibitors.
Conversion of [5-3H]glucose to
[3H]H2O was determined as described under
"Experimental Procedures." Data represent the means ± S.E. of
four independent experiments.
View larger version (32K):
[in a new window]
Fig. 4.
Effect of propyl gallate and/or AOA on
glucose-induced insulin release from INS-1 cells. Static insulin
release was determined by incubating cells for 20 min at 37 °C in
KRB buffer containing 4.0 or 16.7 mM glucose in the
presence or absence of metabolic inhibitors. Insulin levels were
determined as described under "Experimental Procedures." Data
represent the means ± S.E. of four independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (12, 53). Incubation of INS-1
cells with propyl gallate at concentrations of 500 µM and
1 mM effectively reduced
[5-3H]D-glucose metabolism and
glucose-induced insulin release. The ability of propyl gallate to
reduce glucose utilization is consistent with the hypothesis that mGPDH
inhibition would reduce the reoxidation of cytosolic NADH and thereby
inhibit glycolysis at the level of triose phosphates. Blockage of mGPDH
would also cause a reduction in shuttling of cytosolic NADH generated
from glycolysis and pyruvate into the mitochondria, thus leading to an
overall reduction in ATP generation and thereby markedly reducing
insulin release. Under similar culture conditions, 5 mM AOA
was only partially effective in inhibiting glucose utilization and led
to a 50% reduction in glucose-induced insulin release. The ability of
AOA to inhibit glucose-induced insulin release in INS-1 cells is
consistent with previous reports showing that millimolar concentrations
of AOA inhibit insulin secretion from rat islets by 50% (37, 54). Our
results suggest that in INS-1 cells the glycerol phosphate shuttle is
more active than the malate-aspartate shuttle in the regeneration of
NAD+ consumed during glycolysis because propyl gallate is
more effective than AOA at suppressing both glucose utilization and
glucose-induced insulin release. This conclusion, however, directly
contradicts Ishihara et al. (52) results that suggest that
the malate-aspartate shuttle is more active than the glycerol phosphate
shuttle in INS-1 cells. Our observation also contradict results from
Eto et al. (14) demonstrating that in mouse islets
glucose-induced insulin release is only markedly suppressed when
activities of both the glycerol phosphate and malate/aspartate shuttles
are impaired. Nevertheless, our results are in general agreement with those of Sekine et al. (12), Dukes et al. (13),
and Eto et al. (14) regarding the importance of cytosolic
NADH and its subsequent oxidation through the glycerol-3-phosphate and
malate-aspartate shuttles.
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FOOTNOTES |
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* This work was supported by grants from the American Diabetes Association (to W. W. W. and L. K. O.) and by a grant from the Juvenile Diabetes Foundation (to L. K. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 517-355-6475, Ext. 1287; Fax: 517-355-5125; E-mail: olsonla@msu.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M007268200
2 W. W. Wells, H. K. Cirrito, and L. K. Olson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: mGPDH, mitochondrial glycerol-3-phosphate dehydrogenase; AOA, aminooxyacetate; SHAM, salicylhydroxamic acid (N,2-dihydroxybenzamide); PCAEE, protocatechuic acid ethyl ester(ethyl 3,4-dihydroxybenzoate); propyl gallate, 3,4,5-trihydroxybenzoic acid propyl ester; lauryl gallate, 3,4,5-trihydroxybenzoic acid lauryl ester; ETP, electron transport particle; INT, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride; GSH, glutathione; LDS-PAGE, lithium dodecyl sulfate-polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol.
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---|
1. |
Ringler, R. L.,
and Singer, T. P.
(1959)
J. Biol. Chem.
234,
2211-2217 |
2. | Dawson, A. P., and Thorne, C. J. R. (1969) Biochem. J. 111, 27-34[Medline] [Order article via Infotrieve] |
3. | Dawson, A. P., and Thorne, C. J. R. (1975) Methods Enzymol. 41B, 254-259[Medline] [Order article via Infotrieve] |
4. | Hatefi, Y., and Stiggall, D. L. (1976) in The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. XIII , pp. 175-295, Academic Press, San Diego, CA |
5. |
MacDonald, M. J.
(1981)
J. Biol. Chem.
256,
8287-8290 |
6. | Rasschaert, J., and Malaisse, W. J. (1991) Biochem. J. 278, 335-340[Medline] [Order article via Infotrieve] |
7. | Giroix, M.-H., Rasschaert, J., Baile, D., Leclercq-Meyer, V., Sener, A., Portha, B., and Malaisse, W. J. (1991) Diabetes 40, 227-232[Abstract] |
8. | Rasschaert, J., Ling, Z., and Malaisse, W. J. (1993) Mol. Cell. Biochem. 120, 135-140[Medline] [Order article via Infotrieve] |
9. |
Brown, L.,
MacDonald, M. J.,
Lehn, D. A.,
and Moran, S. M.
(1994)
J. Biol. Chem.
269,
14363-14366 |
10. | Fernandez-Alvarez, J., Conget, I., Rasschaert, J., Sener, A., Gomis, R., and Malaisse, W. J. (1994) Diabetologia 37, 177-181[CrossRef][Medline] [Order article via Infotrieve] |
11. | Malaisse, W. J. (1995) Diabetic Med. 12, 479-481[Medline] [Order article via Infotrieve] |
12. |
Sekine, N.,
Cirulli, V.,
Regazzi, R.,
Brown, L. J.,
Gine, E.,
Tamarit-Rodriguez, J.,
Girotti, M.,
Marie, S.,
MacDonald, M. J.,
Wollheim, C. B.,
and Rutter, G. A.
(1994)
J. Biol. Chem.
269,
4895-4902 |
13. |
Dukes, I. D.,
McIntyre, M. S.,
Mertz, R. J.,
Philipson, L. H.,
Roe, M. W.,
Spencer, B.,
and Worley, J. F., III
(1994)
J. Biol. Chem.
269,
10979-10982 |
14. |
Eto, K.,
Tsubamoto, Y.,
Terauchi, Y.,
Sugiyama, T.,
Kishimoto, T.,
Takashi, N.,
Yamauchi, N.,
Kubota, N.,
Murayama, S.,
Aizawa, T.,
Akuanuma, Y.,
Aizawa, S.,
Kasai, H.,
Yazaki, Y.,
and Kadowaki, T.
(1999)
Science
283,
981-985 |
15. |
Sigal, A.,
and King, C. G.
(1936)
J. Biol. Chem.
116,
489-492 |
16. | Wells, W. W., Dou, C.-Z., Dybas, L. N., Jung, C.-H., Kalbach, H. L., and Xu, D. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11869-11873[Abstract] |
17. | Dou, C.-Z., Xu, D. P., and Wells, W. W. (1997) Biochem. Biophys. Res. Commun. 231, 820-822[CrossRef][Medline] [Order article via Infotrieve] |
18. | Jung, C.-H., and Wells, W. W. (1997) Biochem. Biophys. Res. Commun. 239, 457-462[CrossRef][Medline] [Order article via Infotrieve] |
19. | Ringler, R. L., and Singer, T. P. (1962) Methods Enzymol. V, 432-439 |
20. | Cole, E. S., Lepp, C., Holohan, P. D., and Fondy, T. P. (1978) J. Biol. Chem. 253, 7952-7959[Abstract] |
21. | Cottingham, I. R., and Ragan, C. I. (1980) Biochem. J. 192, 9-18[Medline] [Order article via Infotrieve] |
22. | Greenawalt, J. W. (1974) Methods Enzymol. 31, 310-323[Medline] [Order article via Infotrieve] |
23. | Lai, J. C. K., and Clark, J. B. (1979) Methods Enzymol. LV, 51-59 |
24. | Green, D. E., and Ziegler, D. M. (1963) Methods Enzymol. 58, 416-425 |
25. | Singer, T. P. (1974) in Methods of Biochemical Analysis (Glick, D., ed), Vol. 22 , pp. 123-175, Academic Press, New York |
26. | Garrib, A., and McMurray, W. C. (1984) Anal. Biochem. 139, 319-321[Medline] [Order article via Infotrieve] |
27. |
Garrib, A.,
and McMurray, W. C.
(1986)
J. Biol. Chem.
261,
8042-8048 |
28. | Hagopian, K. (1999) Anal. Biochem. 273, 240-251[CrossRef][Medline] [Order article via Infotrieve] |
29. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
30. | Beleznai, Z., Szalay, L., and Jancsik, V. (1987) Eur. J. Biochem. 170, 631-636[Abstract] |
31. | Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P. A., and Wollheim, C. B. (1992) Endocrinology 130, 167-178[Abstract] |
32. | Kekow, J., Ulrichs, K., Muller-Ruchholtz, W., and Gross, W. L. (1988) Diabetes 37, 321-326[Abstract] |
33. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, J. R.
(1951)
J. Biol. Chem.
193,
265-275 |
34. | Zawalich, W. S., and Matschinsky, F. M. (1977) Endocrinology 100, 1-8[Abstract] |
35. | Zawalich, W. S., Pagliara, A. S., and Matschinsky, F. M. (1977) Endocrinology 100, 1276-1283[Abstract] |
36. | Malaisse, W. J. (1992) Int. J. Biochem. 24, 693-701[CrossRef][Medline] [Order article via Infotrieve] |
37. | MacDonald, M. J. (1982) Arch. Biochem. Biophys. 213, 643-649[Medline] [Order article via Infotrieve] |
38. | Beinert, H. (1973) in Iron-sulfur proteins (Lovenberg, W., ed) , pp. 1-36, Academic Press, New York |
39. | Rouault, T. A., and Klausner, R. D. (1996) Trends Biochem. Sci. 21, 174-177[CrossRef][Medline] [Order article via Infotrieve] |
40. | Fox, B. G. (1997) in Comprehensive Biological Catalysis (Sinnott, M., ed) , pp. 261-348, Academic Press, London |
41. | Schonbaum, G. R., Bonner, W. D., Storey, B. T., and Bahr, J. T. (1971) Plant Physiol. 47, 124-128[Medline] [Order article via Infotrieve] |
42. | Siedow, J. N., Umbach, A. L., and Moore, A. L. (1995) FEBS Lett. 362, 10-14[CrossRef][Medline] [Order article via Infotrieve] |
43. | Lehn, D. A., Brown, L. J., Simonson, G. D., Moran, S. M., and MacDonald, M. J. (1994) Gene (Amst.) 150, 417-418[Medline] [Order article via Infotrieve] |
44. | Ross, J. L., Davis, M. B., MacIntyre, R. J., and McKechnie, S. W. (1994) Gene (Amst.) 139, 219-221[Medline] [Order article via Infotrieve] |
45. | Grant, P. T., and Sargent, J. R. (1960) Biochem. J. 76, 229-237[Medline] [Order article via Infotrieve] |
46. | Fairlamb, A. H., and Bowman, J. B. R. (1977) Int. J. Biochem. 8, 669-675 |
47. | Amole, B. O., and Clarkson, A. B., Jr. (1981) Ex. Parasitol. 51, 133-140 |
48. | Grady, R. W., Bienen, E. J., and Clarkson, A. B., Jr. (1986) Mol. Biochem. Parasitol. 19, 231-240[Medline] [Order article via Infotrieve] |
49. | Grady, R. W., Bienen, E. J., and Clarkson Jr, A. B. (1986) Mol. Biochem. Parasitol. 21, 55-64[Medline] [Order article via Infotrieve] |
50. | Clarkson Jr, A. B., Grady, R. W., Grossman, S. A., McCallum, R. J., and Brohn, F. H. (1981) Mol. Biochem. Parisitol. 3, 271-292[Medline] [Order article via Infotrieve] |
51. | MacDonald, M. J., Warner, T. F., and Pellet, J. R. (1983) J. Clin. Endocrinol. Metab. 57, 662-664[Abstract] |
52. |
Ishihara, H.,
Wang, H.,
Drewes, L. R.,
and Wollheim, C. B.
(1999)
J. Clin. Invest.
104,
1621-1629 |
53. | Rutter, G. A., Pralong, W.-F., and Wollheim, C. B. (1992) Biochim. Biophys. Acta 1175, 107-113[Medline] [Order article via Infotrieve] |
54. | Malaisse, W. J., Malaisse-Lagae, F., and Sener, A. (1982) Endocrinology 111, 392-397[Abstract] |