From the Department of Obstetrics and Gynaecology, Ninewells Hospital and Medical School, Dundee University, Dundee, DD1 9SY, Scotland
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ABSTRACT |
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Liver microsomal glucose-6-phosphatase (Glc-6-Pase) is a multicomponent system involving both substrate and product carriers and a catalytic subunit. We have investigated the inhibitory effect of N-ethylmaleimide (NEM), a rather specific sulfhydryl reagent, on rat liver Glc-6-Pase activity. Three thiol groups are important for Glc-6-Pase system activity. Two of them are located in the glucose-6-phosphate (Glc-6-P) translocase, and one is located in the catalytic subunit. The other transporters (phosphate and glucose) are not affected by NEM treatment. The NEM alkylation of the catalytic subunit sulfhydryl residue is prevented by preincubating the disrupted microsomes with saturating concentrations of substrate or product. This suggests either that the modified cysteine is located in the protein active site or that substrate binding hides the thiol group via a conformational change in the enzyme structure. Two other thiols important for the Glc-6-Pase system activity are located in the Glc-6-P translocase and are more reactive than the one located in the catalytic subunit. The study of the NEM inhibition of the translocase has provided evidence of the existence of two distinct areas in the protein that can behave independently, with conformational changes occurring during Glc-6-P binding to the transporter. The recent cloning of a human putative Glc-6-P carrier exhibiting homologies with bacterial phosphoester transporters, such as Escherichia coli UhpT (a Glc-6-P translocase), is compatible with the fact that two cysteine residues are important for the bacterial Glc-6-P transport.
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INTRODUCTION |
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The strategic position of Glc-6-Pase1 (EC 3.1.3.9) in carbohydrate metabolism, at the connection between gluconeogenesis and glycogenolysis, makes it a key enzyme in blood glucose homeostasis (1). The enzyme is a nonspecific phosphohydrolase tightly associated with the endoplasmic reticulum and nuclear membranes of liver and kidney cells (1). It has been identified as a 38-kDa protein (2, 3) and has been cloned in several animal species (4-7).2 The active site of the enzyme is located in the endoplasmic reticulum lumen (8). In vitro, with the use of microsomes (small endoplasmic reticulum vesicles), the protein activity exhibits a phenomenon termed latency, with part of its activity expressed only when the membrane is disrupted, increasing both activity and affinity (9). To explain the role of the membrane in the function of Glc-6-Pase, two models have been proposed, the conformational model (10, 11) and the transport model (12, 13).
The conformational model (10, 11, 14-18) explains all of the kinetic observations by assuming that the protein can be found in several active conformations, each one possessing particular properties. The transport model involves specific permeases termed T1 (Glc-6-P), T2 (phosphate), and T3 (glucose). These carriers allow the translocation of the substrate molecules (Glc-6-P) through the membrane into the endoplasmic reticulum lumen, followed by its hydrolysis by the Glc-6-Pase catalytic subunit and then the elimination of the reaction products (phosphate and Glc) from the endoplasmic reticulum cisternae (Fig. 1; for review see Ref. 19). However, direct evidence, such as the unequivocal identification of one of the transporters, has to be produced. Recently, an elegant study using a very specific Glc-6-Pase inhibitor has provided compelling evidence for the existence of T1 (20). The first indications for the existence of such a Glc-6-P translocase were provided using chemical modification of thiol groups of the Glc-6-Pase system. Although the presence of an accessible sulfhydryl residue(s) necessary for the transport activity of the Glc-6-P translocase T1 is clearly established (21-24), the number of such residues is still to be elucidated. The difficulty with determining this is that the catalytic subunit can also be inhibited by thiol reagents (25, 11).
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In this paper, we demonstrate that two thiol groups of the Glc-6-P translocase and one in the Glc-6-Pase catalytic subunit are important for the activity of the system. We also show that conformational changes in the translocase protein are triggered by Glc-6-P binding.
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EXPERIMENTAL PROCEDURES |
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Chemicals--
Glc-6-P (sodium salt), mannose 6-phosphate
(disodium salt), glucose, alamethicin, histone IIA, NEM, bovine serum
albumin (free fatty acid), and orthovanadate were from Sigma. Mannose,
potassium phosphate, PIPES, sucrose, EDTA, -mercaptoethanol,
ascorbic acid, ammonium molybdate, HEPES, and sodium dodecylsulfate
were from Merck.
Preparation of Rat Liver Microsomes--
Fed Wistar rats
(220-250 g) were used. Liver microsomes were made as described
elsewhere (26). The microsomes were resuspended in a 0.25 M
sucrose, 5 mM HEPES buffer, pH 7.4; quickly frozen; and
kept at 70 °C until used. The protein concentrations were determined using the Lowry method (27) as modified by Peterson (28)
using bovine serum albumin as standard. The intactness of the
microsomal vesicles was estimated using the latency of mannose
6-phosphate (29) and was greater than 90% in all the microsomal
preparations.
Activity Measurements-- A 96-well microplate assay derived from the colorimetric technique previously described was used (30). The substrates were dissolved at various concentrations in a 24 mM HEPES, 3 mM EDTA buffer, pH 6.5. In some substrate sets, histone IIA at a 1 mg/ml final concentration was added in order to measure the Glc-6-Pase activity in disrupted vesicles (31). In the assay, 5 µl of microsomal suspension were incubated with 25 µl of substrate stock solutions for time periods between 10 and 30 min (depending on the substrate concentration) at 30 °C. Then, 250 µl of a stop solution (0.28% ammonium molybdate, 2.2% SDS, 1.1% ascorbic acid in 0.33 M sulfuric acid) were added in order to measure colorimetrically the amount of phosphate formed. Under our conditions, at least three identical measurements were made, and blanks, in which stop solution was added before the substrates, were used to correct the assay values. A standard curve with known amounts of phosphate was made under identical conditions. The microplates were then incubated at 46 °C for 20 min and read in an ELISA plate reader at 820 nm. The microsomal intactness was measured using 1.67 mM mannose 6-phosphate ± histones. The activity was corrected for intactness using the following equation (32, 33),
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(Eq. 1) |
NEM Chemical Modification--
A 0.25 M stock
solution of NEM in sucrose/HEPES buffer, pH 7.4, was prepared by
warming up the mixture at 46 °C for 5 min. This solution or
dilutions of this stock solution were used to modify rat liver
microsomes. In a typical experiment, a pool of microsomal vesicles (1 mg/ml) was incubated in sucrose/HEPES buffer, pH 7.4, at room
temperature, in the presence of various concentrations of NEM. Then, at
different times, aliquots were withdrawn, and a 2-fold molar excess
(with respect to NEM concentration) of -mercaptoethanol was added in
order to prevent further modification by the alkylating reagent. The
samples were kept on ice until assayed for activity or for
transport.
Calculations of Inhibition Kinetics-- With nondisrupted microsomes (chemical modification performed in absence of histones), the Glc-6-Pase activity inhibition curves observed were fitted to the following equation (34),
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
Light Scattering Experiments-- Changes in microsomal vesicle size and shape induced by osmotic modification in the medium were monitored using a Hitachi F-4500 spectrofluorometer equipped with a temperature-controlled cuvette holder (30 °C) and a magnetic stirrer. The mV output signals were acquired at 0.1-s intervals. Both excitation and emission wavelength were 400 nm; the slits were 1 nm for excitation and 5 nm for emission. Stock solutions of sucrose (1 M), Glc-6-P (0.3 M), phosphate (1 M), and glucose (1 M) were prepared in 4 mM PIPES, pH 7.1. NEM-treated and untreated rat liver microsomes were diluted in the same PIPES buffer to a final protein concentration of 0.2 mg/ml. Then, 500 µl of the microsomal vesicle suspension were placed in a cuvette to equilibrate at 30 °C until a stable baseline was obtained. With the use of a Hamilton syringe, 25 µl of substrate stock solutions were added to the cuvette through a hole in the cuvette holder lid. After the trace recovered a baseline level, 5 µl of a 1 mg/ml solution of alamethicin (in ethanol) were added in order to fully permeabilize the microsomes (36-38).
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RESULTS |
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Determination of the NEM Concentration Suitable for Glc-6-Pase
Activity Inhibition in "Untreated" (Absence of Histones)
Microsomes--
Liver microsomes were incubated in a sucrose/HEPES
buffer with different NEM concentrations at room temperature for 1 h and then blocked with -mercaptoethanol. Glc-6-Pase activity
assayed in nondisrupting conditions started to be inhibited at a NEM
concentration higher than 100 µM; the IC50
was 500 µM (not shown). When the activity was measured in
NEM-modified vesicles in the presence of histones (permeabilized
membranes), the activity loss was very low; the residual activity
slightly decreased after 1-2 mM NEM. In the presence or
absence of 0.1 mM vanadate, a competitive inhibitor of the
Glc-6-Pase catalytic subunit (39), the inhibition in intact vesicles
was identical to the control (without vanadate), and when the activity
was assayed in the presence of histones, no significant loss of
activity was observed (with 1-2 mM NEM concentrations).
NEM Kinetic Inhibition in Untreated Microsomes--
Nondisrupted
microsomes in sucrose/HEPES buffer, pH 7.4 (Fig.
2), were incubated at room temperature
with various concentrations of NEM (between 0.1 and 1 mM).
At different times, an aliquot was withdrawn, and a 2-fold molar excess
of -mercaptoethanol (with respect to NEM concentration) was added.
The microsomal intactness remained unchanged and was between 90 and
95% whatever the NEM concentration and incubation time used (not
shown). A logarithmic representation of Glc-6-Pase activity in intact
microsomes, modified with 0, 0.25, 0.5, or 1 mM NEM and
assayed with 15 mM Glc-6-P as substrate (Fig.
2A), shows two inhibition phases (chemical modification of
two different sites), one faster than the other, both of which are
dependent on NEM concentration (second order inhibition mechanism).
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Kinetic Constants of NEM Modification in Untreated
Microsomes--
We calculated the k1app and
k2 values for various NEM concentrations. Because
k1app values are dependent on the Glc-6-P
concentration used to measure the residual activity, we plotted the
logarithm base 10 of the k1app values against
the Glc-6-P concentration for various NEM concentrations (Fig.
3A). The straight lines
obtained allowed us to extrapolate the value of
k1app for 0 mM Glc-6-P (k1extrapolated). On the contrary, k2
values were not sensitive to the Glc-6-P concentration (Fig.
3B). The plots of rate constant as a function of inhibitor
concentration gave a linear relationship (Fig. 3, C and
D), consistent with the idea that NEM was reacting (at each
site) with one or a small number of cysteines (if more than one, the
modification rates must be very similar). The values of the second
order kinetic constant for the quickly (kI) and the slowly
(kII) modified sites are 2360 and 33 min1
M
1, respectively.
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Dissociation Constant Value of Glc-6-P for the First NEM-modified Site-- From the data shown in Fig. 3A, we calculated the dissociation constant between Glc-6-P and the quickly modified site using the relation described under "Experimental Procedures." A saturation curve was obtained (Fig. 4, inset). The double reciprocal representation of the data gives a straight line that allows the calculation of the KD value of Glc-6-P for the fast NEM-modified site. The affinity constant is 2.02 ± 0.26 mM.
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Protection by Glc-6-P and Vanadate-- NEM inhibition experiments were performed in the presence of 50 mM Glc-6-P or 100 µM vanadate as described in Table I (legend). The rate constant of the second (slow) NEM-modified site is not dependent on the Glc-6-P concentration used to assay the Glc-6-Pase residual activity. However, when the microsomes were incubated in the presence of both 0.5 mM NEM and 50 mM Glc-6-P, then there was a 2-fold decrease of k2 (p < 0.005 compared with control), showing a protection from NEM (Table I). The k2 was unchanged (not significantly different compared with control) when 100 µM vanadate was added to the microsomes. The first (fast) NEM-modified site rate constant (k1extrapolated) was doubled when the microsomes were incubated with 50 mM Glc-6-P but unchanged (compared with control) when 100 µM vanadate was used (Table I).
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Effect of NEM on Microsomal Transport of Glc-6-P, Phosphate, and
Glucose--
We investigated the transport of both substrate (Glc-6-P)
and products (phosphate and glucose) from Glc-6-Pase reaction, in untreated microsomes incubated for various time periods with 1 mM NEM (Fig. 5). Typical
traces are presented for sucrose (Fig. 5a), a nonpermeating
compound, Glc-6-P (Fig. 5b), phosphate (Fig. 5c),
and glucose (Fig. 5d) with microsomes incubated with 2 mM -mercaptoethanol. The same type of traces are shown
for microsomes treated for 1 h with 1 mM NEM and in
which 2 mM
-mercaptoethanol was added in order to stop
the NEM alkylation reaction (Fig. 5, e-h). Traces for
sucrose (Fig. 5e), phosphate (Fig. 5g), and
glucose (Fig. 5h) were identical to those obtained in normal
(no NEM) microsomes (Fig. 5, a, c, and
d). The Glc-6-P transport traces obtained with 1 mM NEM-treated microsomes for 2, 20, 30, and 60 min are
shown in Fig. 5f. In that case, both half-lives and light scattering variations (from the top of the trace to the recovered baseline; see Fig. 5h, double arrow line) are
modified. To monitor the NEM modification of the different transport
systems, we chose to use the light scattering variation (as shown in
Fig. 5h) as a parameter reflecting the percentage of
microsomal vesicles still exhibiting transport. Then we plotted the
logarithm of these values against time for the three different
transport systems (Fig. 6). The phosphate
and glucose transport were not affected by NEM-microsome alkylation.
However, Glc-6-P transport was reduced in a first order manner. The
value of the inhibition rate constant (0.025 min
1) is not
significantly different from the value of the rate constant obtained
from Glc-6-Pase activity measurements with the same NEM-treated microsomes: 0.028 min
1 (corresponds to the second
inhibition phase rate constant, k2).
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Determination of the NEM Concentration Suitable for Glc-6-Pase
Inhibition in Disrupted Microsomes--
Liver microsomes (1 mg/ml)
previously permeabilized for 30 min with 1 mg/ml of histones were
incubated at room temperature with various amounts of NEM. After 1 h, a 2-fold excess (over NEM) of -mercaptoethanol was added to
prevent further modification. The residual Glc-6-Pase specific activity
was assayed using 20 mM Glc-6-P and plotted against NEM
concentration (not shown). Inhibition of Glc-6-Pase activity was
observed for NEM concentration values higher than 1-2 mM
and is maximal for concentrations over 20-25 mM. The
IC50 measured in these conditions was 7-8
mM.
NEM Kinetic Inhibition in "Disrupted"
Microsomes--
Disrupted microsomes were subjected to different
concentrations of NEM and then assayed for residual Glc-6-Pase activity
with different concentrations of Glc-6-P. The activities were
normalized to the value obtained for time 0 and plotted against time
(Fig. 7A). The figure shows
that there was no effect (within the experimental error) of Glc-6-P
concentration on the inhibition rate constant for a given NEM
concentration. Moreover, the NEM inhibition is a pseudo-first order
mechanism with one modified site. The second-order rate constant was
calculated plotting the first order rate constant against NEM
concentration; its value is 1.1 min1
M
1 (see Fig. 7A, inset). A
protection experiment with a broad range of substrates and products of
Glc-6-Pase catalytic subunit from NEM inhibition is presented in Fig.
7B. All of the compounds tested had a protection effect. We
must note that glucose and mannose can both protect, but only if they
are used at a high concentration (>200-250 mM), which is
in agreement with the dissociation constant of these two sugars with
the Glc-6-Pase catalytic subunit (40).
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DISCUSSION |
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NEM is a relatively specific sulfhydryl reagent commonly used to assess the structure of proteins (35, 41, 42). We have to note that reactions with amino groups can occur (43, 44) but need high pH conditions (>8.5-9.0). NEM can cross phospholipid membranes. Thus, studying the topology of a membrane protein using only this reagent is difficult. However in our case, the high reactivity of this compound toward cysteine residues makes it a good tool to examine the structure of the liver microsomal Glc-6-Pase system.
Relatively low concentrations of NEM (<1-2 mM) inhibited
the activity of the Glc-6-Pase system when microsomes were incubated in
nondisrupting conditions. However, when the membrane was subsequently treated with histones, the activity measured was identical to those of
the NEM-unmodified controls (Fig. 2C), showing that the catalytic subunit activity is not affected by an alkylation by 1-2
mM NEM of one or more of its cysteine residues. When an
inhibition time course is made in nondisrupted microsomes (Fig. 2,
A and B), a two site (two different thiol
groups), first order kinetic inhibition is demonstrated; the first site
was modified after only 2 min, and the second site needed more time to
be alkylated by NEM. The modification rate of the two sites depends on
NEM concentration (Fig. 2A). The rate constant for the
second modified site was unchanged, whatever the Glc-6-P concentration
used to assay the residual activity (Figs. 2B and
3B), but surprisingly, the rate constant of the first
modified site depends on the substrate concentration used to determine
residual activity (Figs. 2B and 3A). Indeed, the
inhibition was released when the concentration of Glc-6-P used was
higher than 20 mM (Figs. 2B and 3A).
Here in the first phase, the rate constants (k1) are
apparent constants. An extrapolation of the
k1app to 0 mM Glc-6-P has been done
to calculate the second order constant of the reaction. The value of
the second order rate constant kI for the first (quickly)
modified thiol is 2360 min1 M
1
(Fig. 3C), whereas the calculated value for the second site
is 33 min
1 M
1.
A light scattering technique (36) has demonstrated that the Glc-6-P translocase was the only transporter inhibited by NEM alkylation (Figs. 5 and 6) and that the Glc-6-P transport inhibition was closely correlated to the activity loss. Therefore, the two thiol groups, modified with different velocities, are in the T1 protein.
The extent of inhibition due to the NEM alkylation of the T1 first reactive site depends on the Glc-6-P concentration used to assay the residual activity (Fig. 2B). This dependence is a saturation process (Fig. 4). We explain the effect of Glc-6-P on the first inhibition phase by the binding of this compound to T1, which causes a conformational change releasing the effects of NEM modification. Whether this Glc-6-P molecule is regulatory and/or transported is still unclear. In 1991, Nordlie and co-workers reported that T1 could be regulated by intramicrosomal levels of Glc-6-P (45). The conformational change observed in our conditions could be the reflection of such a regulation.
Protection experiments, performed with nondisrupted vesicles in the presence of Glc-6-P or vanadate, have shown that vanadate has no effect on the inhibition process (Table I). However, a high concentration of Glc-6-P, the transported compound, partially prevents the second site from NEM modification and increases the reactivity of the first site, by improving its accessibility to NEM. From these observations, it seems that two areas or domains, each containing a reactive cysteine residue, can be found in T1. A description of their particular features is attempted in the working model proposed in Fig. 8.
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Recently, a human membrane protein that exhibits sequence similarities with bacterial phosphoester transporters, such as UhpT (the sugar phosphate carrier of Escherichia coli), and that is mutated in patients suffering from glycogen storage disease 1b (no Glc-6-P transport), has been cloned (46). In bacteria, two UhpT cysteines can be modified by sulfhydryl reagents leading to an inactive protein (47, 48). The behavior of the mammalian T1 reported in this paper is rather close to that observed with UhpT. Hence, it seems possible that the putative Glc-6-P translocase recently cloned is T1.
Experiments performed with histone-disrupted microsomes allow us to look directly at the effects of NEM on the Glc-6-Pase catalytic subunit activity without the rate limitations imposed by the translocases. The catalytic subunit loses its activity at NEM concentration higher than those used to inhibit the whole system in nondisrupting conditions (IC50 = 7-8 mM instead of 0.5 mM). The incubation of the histones with dithionitrobenzoate (Ellman's reagent) did not result in a yellow coloration mark of titrable cysteines. Thus, the higher IC50 cannot be attributed to the presence of cysteines in the histones. We therefore have to assume that a thiol group of the Glc-6-Pase catalytic subunit can be modified but needs rather high NEM concentrations.
The NEM inhibition of the Glc-6-Pase catalytic subunit (Fig.
7A) is a second order mechanism (k = 1.1 min1 M
1).The presence of
substrates in the NEM incubation assay fully protects the enzyme from
the inhibition, which suggests that the modified cysteine is located in
the protein active site or that substrate binding hides the thiol group
via a conformational change in the Glc-6-Pase structure. The
identification of the residue alkylated by NEM would be helpful for
completing the recently proposed Glc-6-Pase topology (49, 50) and
improving our knowledge of the Glc-6-Pase active site.
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ACKNOWLEDGEMENT |
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We thank Dr. Peter E. Ross for access to his Hitachi spectrofluorometer.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council and the Royal Society (to A. B.).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.:
44-1382-632445; Fax.: 44-1382-633847; E-mail:
aburchell{at}ninewells.dundee.ac.uk.
1 The abbreviations used are: Glc-6-P, glucose-6-phosphate; Glc-6-Pase, glucose-6-phosphatase; NEM, N-ethylmaleimide; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).
2 Glc-6-Pase sequence from fish: S. Nagl, W. E. Mayer, and J. Klein, GenBankTM accession number AF008945.
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REFERENCES |
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