From the Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 1444 Budapest, Hungary and the § Dipartimento di Fisiopatologia e Medicina Sperimentale, Università di Siena, 53100 Siena, Italy
Received for publication, November 22, 2000
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ABSTRACT |
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The transport and intraluminal reduction
of dehydroascorbate was investigated in microsomal vesicles from
various tissues. The highest rates of transport and intraluminal
isotope accumulation (using radiolabeled compound and a rapid
filtration technique) were found in hepatic microsomes. These
microsomes contain the highest amount of protein-disulfide isomerase,
which is known to have a dehydroascorbate reductase activity. The
steady-state level of intraluminal isotope accumulation was more than
2-fold higher in hepatic microsomes prepared from spontaneously
diabetic BioBreeding/Worcester rats and was very low in fetal
hepatic microsomes although the initial rate of transport was not
changed. In these microsomes, the amount of protein-disulfide isomerase
was similar, but the availability of protein thiols was different and
correlated with dehydroascorbate uptake. The increased isotope
accumulation was accompanied by a higher rate of dehydroascorbate
reduction and increased protein thiol oxidation in microsomes from
diabetic animals. The results suggest that both the activity of
protein-disulfide isomerase and the availability of protein thiols as
reducing equivalents can play a crucial role in the accumulation of
ascorbate in the lumen of the endoplasmic reticulum. These findings
also support the fact that dehydroascorbate can act as an oxidant in
the protein-disulfide isomerase-catalyzed protein disulfide formation.
The lumen of the endoplasmic reticulum
(ER)1 and of the vesicular
structures of the whole secretory pathway is characterized by an
oxidizing environment reflected in a high ratio of glutathione disulfide versus glutathione (1, 2). The suitable redox properties of these organelles are necessary for the formation and the
maintenance of disulfide bonds in the secretory and plasma membrane
proteins. Oxidizing conditions can be generated by the import of an
oxidizing agent. It is apparently inconsistent with the above facts
that these compartments contain ascorbate, a reducing compound, at high
concentrations (3-5). The intraluminal accumulation of ascorbate can
theoretically be explained by an active transport process or by its
local generation from a membrane-permeable precursor. Whereas there are
no data supporting the first possibility in the ER, the facilitated
diffusion of dehydroascorbate (DHA), the oxidized form of ascorbate,
has been described in rat liver microsomes. DHA uptake is presumably
mediated by the glucose transporter T3 subunit of the
glucose-6-phosphatase system (6). Local ascorbate oxidation and DHA
formation have also been observed in microsomal vesicles (7).
Therefore, ascorbate accumulation can be attributed to the intraluminal
reduction of DHA taken up. However, enzyme(s) participating in the
process and the source(s) of the reducing equivalents are unknown.
Protein-disulfide isomerase (PDI), a major protein of the ER lumen, is
known to have DHA reductase activity (8, 9). The aim of the present
work was to explore the role of PDI in the intraluminal ascorbate
accumulation in the ER. To this end, DHA transport, DHA reduction, and
ascorbate accumulation were investigated in microsomes from various
organs/cells having different PDI activities.
Animals--
The BioBreeding/Worcester (BB/Wor) male rats were
provided by Møllegaard Breeding & Research Center A/S,
Copenhagen, Denmark. 80-day-old BB/Wor healthy and insulin-implanted
diabetic rats (180-200-g body weight) from the same colony were
delivered by Charles River Ltd., Budapest, Hungary. Animals were fed
ad libitum and housed in the rigidly controlled animal room
of our laboratory. Blood glucose concentrations were tested each
day until the exhaustion of the insulin implant. At that time, blood
glucose levels in diabetic rats exceeded the 20 mM value;
rats were kept for an additional 3 days in standard circumstances
without any insulin supplementation and were then sacrificed. Serum
glucose concentrations were 8.73 ± 0.68 and 28.24 ± 2.35 mM (means ± S.E.; n = 3;
p < 0.01) in control and diabetic rats, respectively.
Male Wistar and Harlan Sprague-Dawley rats were obtained from Charles
River Ltd., Budapest, Hungary.
Preparation of Microsomes--
Liver microsomal vesicles were
prepared from BB/Wor, Wistar, and Harlan Sprague-Dawley male rats
(180-230-g body weight) as previously described (10). Fetal rat liver
microsomes were prepared from 18-day-old fetuses (11). Nonhepatic
microsomes were prepared by the same procedure from rat brain (12),
rabbit skeletal muscle (13), fibroblasts, and J774 macrophages.
Microsomes were resuspended at a concentration of 50-70 mg of
protein/ml in Buffer A (20 mM MOPS containing 100 mM KCl, 20 mM NaCl, 1 mM
MgCl2, pH 7.2). The suspensions were frozen and maintained
under liquid nitrogen until use. Intactness of microsomal vesicles was
checked by measuring the latency of mannose-6-phosphatase (14) and
p-nitrophenol UDP-glucuronosyltransferase (15) (in the case
of liver microsomes) or by detection of the sustained light scattering
signal due to the shrinking of vesicles upon the addition of the
nonpermeant compound sucrose (in the case of nonhepatic and fetal liver
microsomes) (14). To measure intravesicular sucrose spaces, microsomes
were incubated overnight in Buffer A containing sucrose (1 mM) and its radiolabeled analogue (1 µCi/ml) at 4 °C
and then samples were filtered and washed as described in the next paragraph.
Uptake Measurements--
Microsomes (1 mg of protein/ml) were
incubated in Buffer A containing the indicated amount of ascorbate or
DHA and their radiolabeled analogues (1 µCi/ml) at 22 °C. At the
indicated time intervals, samples (0.1 ml) were rapidly filtered
through cellulose acetate/nitrate filter membranes (pore size 0.22 µm), and filters were washed with 1 ml of Hepes buffer (20 mM; pH 7.2) containing 300 mM sucrose and 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The total radioactivity retained by filters was measured by liquid scintillation counting. In each experiment, the pore-forming agent alamethicin (0.1 mg/mg protein) (6) was added to parallel incubates to
distinguish the intravesicular and bound radioactivity. The alamethicin-permeabilized microsomes were filtered and washed as above;
the portion of released radioactivity was regarded as intravesicular.
Western Blot--
Samples of liver microsomal fractions were
sonicated and dissolved in 0.1 M Tris buffer, pH 8.0, containing 20 mM dithiothreitol and 1% Triton X-100.
Microsomal proteins (15 µg) were separated by electrophoresis on 9%
sodium dodecyl sulfate polyacrylamide gels and electrophoretically
transferred to polyvinylidene difluoride (PVDF; Bio-Rad) membranes
(overnight; 30 mA) (16). The membranes were blocked for 1 h with
washing buffer (20 mM Tris-HCl, pH 7.6, 137 mM
NaCl, 0.1% Tween 20) containing 2% bovine serum albumin and then
probed with primary antibodies (SPA-890; StressGen) diluted in washing
buffer for 1 h. After 3 washes with washing buffer containing
0.3% gelatin, horseradish peroxidase-conjugated secondary antibodies
were added for 30 min. After 4 additional washes, the protein bands
were detected using the enhanced chemiluminescence (ECL) technique (16)
according to the manufacturer (Amersham Pharmacia Biotech).
PDI Activity--
In a turbidimetric assay (17) the catalytic
reduction and subsequent precipitation of reduced insulin DHA Reductase Activity--
Microsomal DHA reductase activity
was measured in Buffer A in the presence of 1 mM DHA and 2 mM glutathione as described (8), with the exception that
ascorbate formation was detected according to Omaye et al.
(19). The same experiments were also executed in conditions applied in
the transport assay, i.e. in the absence of added glutathione.
Measurement of Metabolites--
For the determination of protein
thiol oxidation and ascorbate production intact or permeabilized
microsomal vesicles (usually 1 mg of protein/ml) were incubated in
Buffer A containing 1 mM DHA at 37 °C. For
permeabilization microsomes were treated with alamethicin (0.1 mg/mg of
protein). Incubations were terminated by the addition of 0.05 volume of
100% trichloroacetic acid. Ascorbate content was measured in
trichloroacetic acid-soluble supernatants by the method of Omaye
et al. (19), based on the reduction of Fe3+ by
the oxidation of ascorbate and the subsequent determination of the
Fe2+- Chemicals--
Ascorbate, alamethicin,
diisothiocyanostilbene-2,2'-disulfonic acid, and glutathione were
obtained from Sigma.
L-[carboxyl-14C]Ascorbic acid
(13.7 µCi/mmol) and [U-14C]sucrose (612 µCi/mmol)
were from Amersham Pharmacia Biotech. DHA was produced by the
bromine oxidation method according to Ref. 6. Cellulose acetate/nitrate
filter membranes (pore size 0.22 µm) were from Millipore. All other
chemicals were of analytical grade.
DHA and Ascorbate Transport in Microsomal Vesicles--
Transport
of DHA and of ascorbate was compared in microsomal vesicles of
different origin. The highest DHA uptake was observed in adult rat
liver microsomes, whereas in vesicles from other sources the uptake
slightly exceeded or not exceed at all the level of the passive
equilibrium (Table I). The time
course of the uptake was also different; in nonhepatic microsomes the
uptake reached a steady-state level in the first minute of incubation (data not shown), whereas in adult rat liver microsomes a continuous accumulation could be observed (Fig. 1.)
Ascorbate uptake was similar in all kinds of the investigated
microsomal vesicles (Table I). Because the uptake can be influenced by
the dimensions of the intravesicular space, the intravesicular sucrose
space (which approaches the water space; see Ref. 11) was also
measured. The intravesicular sucrose space of the various microsomes
was similar (Table I) indicating that the difference in DHA transport was not because of the different size of the vesicles.
Effect of Diabetes on DHA Transport in Rat Liver Microsomal
Vesicles--
The time course of DHA uptake was investigated in
microsomal vesicles prepared from the liver of diabetic and control
BB/Wor and control Harlan Sprague-Dawley rats. DHA uptake exceeded the level of the passive equilibrium in all three different microsomes; no
difference was observed in the DHA uptake of microsomes from nondiabetic rats. However, microsomes from diabetic BB/Wor animals showed a 3-fold higher intravesicular accumulation of radioactivity (Fig. 1.) than those from healthy control BB/Wor rats. The difference could be explained by the increased intravesicular metabolism of DHA
rather than its faster uptake; the initial rate of uptake was similar
in microsomes from diabetic and control rats (Fig. 1.). The time course
and the steady-state level of ascorbate uptake were similar in all
three experimental groups (data not shown).
Microsomal DHA Reduction--
DHA reductase activity was measured
in rat liver microsomes in conditions similar to those of the transport
measurements, i.e. in the absence of added
glutathione. Ascorbate formation was about 2-fold higher in diabetic
microsomes (Table II). To clarify whether
this enhanced DHA reduction is because of a more active reductase
enzyme or an increased supply of reducing thiols, the experiments were
repeated in the presence of glutathione. Because glutathione slowly
enters microsomes (21), the vesicles were permeabilized with the
pore-forming compound alamethicin. In this case, the initial rate of
ascorbate formation was only slightly higher in microsomes from
diabetic animals (Table II). In the absence of GSH only the thiols of
microsomal proteins can provide the reducing power for the reaction.
Therefore, the increased DHA reduction could be because of a better
supply of protein thiols. In accordance with this assumption, the
initial protein thiol content was significantly higher in microsomes
from diabetic rats, and a more pronounced oxidation of protein thiols
could be observed in these microsomes upon DHA addition (Table II).
Microsomal PDI Level and Activity--
Western blot analysis of
liver microsomal samples from control and spontaneously diabetic rats
did not show any increase in PDI protein content in diabetes. Fetal rat
liver microsomes also contain similar amounts of PDI (Fig.
2). In accordance with the unchanged
protein levels, neither the turbidimetric nor the fluorescent assay
revealed a significant change of the oxidoreductase activity of PDI in
these conditions (data not shown).
The transport of ascorbate and DHA through the membrane of the ER
has been described in rat liver microsomal vesicles (6). The data
presented here suggest that these transports are general phenomena in
microsomal vesicles prepared from various tissues or cells. However,
liver microsomes only display a significant accumulation of
radiolabeled compounds upon DHA addition. This accumulation is likely
because of the intravesicular reduction of DHA to ascorbate. Because
microsomal preparations do not contain reducing equivalents in the form
of NADPH or glutathione, the main source of electrons for the reduction
of DHA must be protein thiols. The reaction between protein thiol
groups and DHA can even occur nonenzymatically or can be catalyzed by
PDI, an abundant enzyme of the (hepatic) ER (8, 9, 22-24). Our results
suggest that PDI activity might have a decisive role in the
intraluminal DHA reduction and ascorbate accumulation; the process was
the most intensive where the highest PDI activity can be found,
i.e. in liver microsomes (25, 26).
To further envisage the role of PDI, DHA transport was investigated in
liver microsomes from spontaneously diabetic BB/Wor rats. An increased
hepatic activity of PDI in experimental streptozotocin diabetes has
been reported (27), whereas others found a decrease in the same
conditions (28-30). We detected an increased accumulation of
radiolabeled compounds upon 14C DHA addition in microsomes
from diabetic animals, although neither the level nor the activity of
PDI was increased in these microsomes. Therefore, PDI activity
per se cannot explain the differences in ascorbate
accumulation in control and diabetic liver microsomes. The phenomenon
can be explained by a different supply of reducing agents. In fact, the
initial protein thiol level was higher, and the DHA-induced thiol
oxidation was faster in microsomes from diabetic rats. It should be
noted that streptozotocin diabetes also results in the elevation of
protein thiols in liver
microsomes.2 On the
other hand, fetal liver microsomes that contain low amounts of protein
thiols exhibited moderate ascorbate accumulation despite their high PDI
level. These findings suggest that at high PDI activity the
availability of protein thiols can also determine the rate of
intraluminal DHA reduction.
Ascorbate has been reported to promote protein disulfide formation in
secretory proteins in the ER lumen (31). This process is hindered in
diabetes; latent or intracellular scurvy appears because of the
competition for the uptake between DHA and glucose at the plasma
membrane (32), and metabolic hypoxia develops because of the excess of
reducing power (33). Both factors can result in decreased protein thiol
oxidation in the lumen of the hepatic ER. The increased production of
DHA is verified in diabetes (34), and its accelerated uptake
into the ER therefore can be regarded as a compensatory mechanism
supporting protein disulfide formation in this pathological state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chains
were followed in 0.2 ml of 0.1 mM sodium phosphate buffer
(pH 6.5) containing 2 mM dithiothreitol, 5 mM
EDTA, 8-80 µg of microsomal protein, and 80 µM insulin
at 37 °C. The aggregation was monitored at 650 nm using a Hitachi
F-4500 spectrophotometer. PDI activity was also measured by a more
sensitive fluorescent assay using the method of Heuck and Wolosiuk
(18). Briefly, 4-20 µg of microsomal protein was incubated in 0.2 ml
of 0.1 mM sodium phosphate buffer (pH 7.4) containing 75 µM dithiothreitol, 3 mM EDTA, and 0.7 µM difluoresceinthiocarbamyl-insulin (a gift from
A. P. Heuck and R. A. Wolosiuk) at 37 °C. Fluorescence was
monitored using a Hitachi F-4500 spectrophotometer; excitation and
emission wavelength were 495 and 520 nm, respectively.
,
'-dipyridyl complex. Protein thiols were
measured in the washed and resuspended pellets by the Ellman method
(20). Protein concentrations were measured with Bio-Rad protein assay
using bovine serum albumin as standard.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
DHA and ascorbate uptake in microsomes from various sources
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Fig. 1.
Time course of DHA uptake in rat liver
microsomal vesicles. Microsomes (1 mg of protein/ml) prepared from
BB/Wor diabetic ( ), BB/Wor control (
), or Harlan Sprague-Dawley
(
) rats were incubated in the presence of 1 mM DHA plus
the radioactive tracer at 22 °C. After the incubation, 0.1 ml of
sample was filtered as described under "Materials and Methods."
Parallel samples were incubated in the presence of the pore-forming
alamethicin; the alamethicin-releasable portion of DHA associated with
microsomes is shown. Data are mean ± S.D. of three measurements
or mean of two measurements.
Effect of diabetes on protein thiol content, DHA reductase
activity, and DHA-dependent protein thiol oxidation in
rat liver microsomal vesicles
View larger version (10K):
[in a new window]
Fig. 2.
Estimation of PDI level in the microsomal
fraction of control, diabetic, and fetal rat liver. Western
blotting of microsomal protein (15 µg) was performed as described
under "Materials and Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by a Ministry of Health grant, Ministry of Education Grant FKFP 0652/97, Országos Tudományos Kutatási Alap Grants T032873, F022495, and F25206, Hungarian Academy of Sciences Grant F-226/98, an Italian-Hungarian intergovernmental research and development project grant, and a NATO linkage grant.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.
International Research Scholar of the Howard Hughes Medical Institute.
¶ To whom correspondence should be addressed: Dept. of Medical Chemistry, Semmelweis University, P. O. Box 260, H-1444 Budapest, Hungary. Tel./Fax: 36-1-266-2615; E-mail: banhegyi@puskin.sote.hu.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M010563200
2 G. Nardai and P. Csermely, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; DHA, dehydroascorbate; PDI, protein-disulfide isomerase; BB/Wor, BioBreeding/Worcester; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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