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INTRODUCTION |
Hepatic glucose-6-phosphatase system catalyzing the last step of
both gluconeogenesis and glycogenolysis plays a pivotal role in the
maintenance and regulation of blood glucose levels (1, 2). Accordingly,
a huge increase in the activity of the enzyme can be observed after
birth (3, 4). This developmental change forms the liver to be a
gluconeogenic organ and serves the adaptation of the organism to the
feeding-starvation cycles in the postnatal life. The proper timing of
this event is of vital importance; in several cases very low
glucose-6-phosphatase activities could be detected in the liver of
newborns died in sudden infant death syndrome (5); also, the mean
activity of the enzyme was also lower in preterm children (6).
Several factors can contribute to the fetal-to-neonatal transition of
the glucose-6-phosphatase system. The postnatal hypoglycemia and the
consequently high glucagon/insulin ratio may have a primary role (7).
Indeed, it has been reported that dibutyryl cyclic AMP stimulated the
transcription of the glucose-6-phosphatase gene in cultured fetal
hepatocytes (8). Long chain fatty acids contributed to the induction by
the stabilization of the transcript (8). However, the rise in the
amount of mRNA and enzyme protein is lower than the dramatic
elevation of the activity (9), indicating that other,
post-transcriptional factors can also affect the protein(s) of the
glucose-6-phosphatase system around the birth.
In adult liver, the catalytic unit of the system is an integral protein
of the endoplasmic reticulum membrane. On the basis of its proposed
structure (10, 11) and the existence of an intravesicular substrate
pool (12), it seems very likely that the loop containing amino acids
that contribute to the catalytic site is orientated toward the lumen of
the endoplasmic reticulum, in accordance with the compartmentational or
substrate transport model (2, 13-16). The phenomenon of latency (the
enzyme activity is lower in intact microsomal vesicles than in
disrupted ones) and the strict substrate specificity of the
phosphohydrolase in intact microsomal vesicles (both determined by the
activity/specificity of the substrate transporter T1) can be explained
by this topological situation. However, several observations indicate
that the compartmentational model is not sufficient in the case of
nongluconeogenic organs and tissues, including fetal liver. Diminished
latency, altered substrate specificity, different kinetic properties,
or unusual lability of the enzyme could be observed in nongluconeogenic
organs expressing glucose-6-phosphatase (17), esophagus (18, 19), adult
adrenal (20), pancreatic
-cells (21), skeletal muscle (22),
astrocytes (23), etc. Moreover, even the enzyme from the nuclear
envelope of adult hepatocytes (24) or the microsomal enzyme prepared
from glucocorticoid-treated animals (3, 25) exhibited moderate latency.
A recent study indicated that the intravesicular glucose accumulation
upon glucose-6-phosphate hydrolysis, a characteristic feature of adult
liver microsomes, was absent in microsomes from newborn mice (25).
These observations altogether clearly show that the arrangement of the
glucose-6-phosphatase system as it can be seen in the endoplasmic
reticulum of the adult liver is a special situation supported by
developmental and local factors. Therefore, experiments were undertaken
to investigate the presence and the characteristics of the
phosphohydrolase and the related transport activities in fetal rat
liver microsomes and their change around the birth.
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EXPERIMENTAL PROCEDURES |
Preparation of Rat Liver Microsomes--
Fetal rat liver
microsomes from 18-day-old fetuses and adult rat liver microsomes from
male Sprague-Dawley rats (180-230 g) were prepared according to Ref.
26. Microsomal fractions were resuspended in a buffer (buffer A)
containing: 100 mM KCl, 20 mM NaCl, 1 mM MgCl2, 20 mM
Mops,1 pH 7.2. The
suspensions were rapidly frozen and maintained under liquid nitrogen
until used. Intactness of microsomal vesicles was checked by
investigating the osmotically induced changes in microsomal vesicle
size upon the addition of the nonpermeant compound sucrose by the light
scattering technique. Microsomes used in this study showed a sustained
shrinking upon sucrose addition (see Fig. 1). Microsomal protein
concentrations were determined by biuret reaction using bovine serum
albumin as standard.
Uptake Measurements--
Liver microsomes (1 mg protein/ml) were
incubated in buffer A containing 0.2-30 mM
glucose-6-phosphate plus [14C]glucose-6-phosphate (8-10
µCi/ml) or 0.2 mM glucose-1-phosphate plus
[14C]glucose-1-phosphate (8-10 µCi/ml) or 1 mM glucose plus [3H]glucose (9 mCi/ml) or 1 mM sucrose plus [14C]sucrose (1 µCi/ml) at
22 °C. In a series of incubations, the pore-forming antibiotic
alamethicin (0.05 mg/ml) was added to distinguish the intravesicular
and the bound radioactivity (27, 28). The alamethicin-releasable
portion of radioactivity was regarded as intravesicular (12). At the
indicated time intervals, samples (0.1 ml) were rapidly filtered
through cellulose acetate/nitrate filter membranes (pore size, 0.22 µm) and were washed with 4 ml of Hepes (20 mM) buffer, pH
7.2, containing 250 mM sucrose and 0.5 mM
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. During the
measurement of glucose-6-phosphate or glucose-1-phosphate uptake
parallel filters were treated after washing with
ZnSO4-Ba(OH)2 to separate glucose from
glucose-6-phosphate or glucose-1-phosphate according to Ref. 12. The
radioactivity associated with microsomes retained by filters was
measured by liquid scintillation counting.
Assay of Glucose-6-phosphatase--
Glucose-6-phosphatase
activity was measured after 5 min of incubation in buffer A at 22 °C
on the basis of [14C]glucose production from
[14C]glucose-6-phosphate according to Ref. 12.
Permeabilized microsomes were treated with 0.05 mg alamethicin/mg
protein. Alternatively, the phosphohydrolase activity toward various
phosphoesters was assayed in native and permeabilized microsomal
vesicles on the basis of phosphate measurement according to Ref. 29.
Glucose-6-phosphate content of the incubates was measured enzymatically
with glucose-6-phosphate dehydrogenase as described in detail earlier
(12).
Light Scattering Measurements--
Osmotically induced changes
in microsomal vesicle size and shape (30) were monitored at 400 nm at
right angles to the incoming light beam using a fluorimeter
(Perkin-Elmer model 650-10S) equipped with a temperature-controlled
cuvette holder (22 °C) and magnetic stirrer as described elsewhere
(31, 32). The mV output signals were acquired at 0.25-s intervals,
using a MacLabTM hardware (AD Instruments), equipped with a
Chart v3.2.5. software.
Antibodies against Various Sections of the Catalytic Subunit of
Glucose-6-phosphatase--
Antibodies specific to 14 amino acid
peptides in the glucose-6-phosphatase catalytic subunit were raised in
Cheviot sheep. The peptides were synthesized at Severn Biotech Ltd. and
received as a lyophilized powder. Sequences of the peptides from the
human glucose-6-phosphatase protein against which specific antisera were raised are shown in Table I. Each
peptide had an N-terminal cysteine residue added to allow for thiol
coupling to the carrier protein, keyhole limpet hemocyanin (Sigma
H2133). 20 mg of keyhole limpet hemocyanin was dissolved in 2 ml of 50 mM sodium phosphate (4:1 dibasic to monobasic). The
solution was dialyzed overnight against the same buffer at 4 °C. The
dialyzed keyhole limpet hemocyanin was treated with 17 µl of 300 mM N-ethylmaleimide (5 µmol). After 30 min 6.2 mg of
m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (Pierce 22312) was added, and after a further 30 min the pH was
adjusted to 6.0 with 1 N HCl. This mixture was dialyzed overnight at 4 °C against 20 mM
NaH2PO4/135 mM NaCl, pH 5.6. An equal volume of 20 mM NaH2PO4/150
mM NaCl, pH 8.0, was added to the dialysate. The pH was
adjusted quickly to 6.7. 9 mg of peptide was then added and dissolved
by vigorous vortexing. The pH was determined and brought back to 6.7 with 1 N NaOH if necessary. The reaction was over in 2 h, after which the coupled peptide was stored at
20 °C. 185 µg
of coupled peptide was injected subcutaneously into Cheviot sheep with
Freund's adjuvant on two separate occasions. A third injection of 370 µg was given for peptide A, and two further injections of 370 µg
each were given for peptides B, C, and D. Serum from the sheep was
stored at
70 °C until use. The four anti-peptide antisera
recognize the rat glucose-6-phosphatase catalytic subunit as shown by
Western blot analysis.
For the determination of the effect of the antibodies on
glucose-6-phosphatase activity, native fetal or adult rat microsomes were preincubated in buffer A in the presence of each of the four anti-peptide antibodies for 50 min at room temperature, under continuous agitation. The microsomal protein concentration was 0.35 mg/ml, the antibodies were 100 times diluted in the preincubation mixture. The incubation was started with the addition 10 mM
glucose-6-phosphate. The phosphohydrolase activity was detected by
measuring glucose or phosphate production (see above).
Western Blot Analysis--
Proteins of the microsomal membranes
were separated by electrophoresis on SDS-polyacrylamide gels (15%) and
electrophoretically transferred to nitrocellulose (22). Western blots
were immunoreacted with a sheep IgG previously shown to be monospecific
for the glucose-6-phosphatase catalytic subunit (33). The
immonoreactive band was revealed by a biotin-streptavidin horseradish
peroxidase-linked detection system with 4-chloro-1-naphthol as the substrate.
Materials--
Glucose-6-phosphate (dipotassium salt),
mannose-6-phosphate (dipotassium salt), alamethicin, glucose kit
(Trinder method), D-[1-3H(N)]glucose (0.9 mCi/ml; 15.5 Ci/mmol), and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were obtained from Sigma.
D-[14C(U)]glucose-6-phosphate (0.1 mCi/ml; 300 mCi/mmol) was from American Radiolabeled Chemicals Inc.
(St. Louis, MO). [U-14C]sucrose 0.2 mCi/ml; 612 mCi/mmol)
and D-[14C(U)]glucose-1-phosphate (0.1 mCi/ml; 285 mCi/mmol) were obtained from Amersham Pharmacia Biotech.
Cellulose acetate/nitrate filter membranes (pore size, 0.22 µm) were
from Millipore. All other chemicals were of analytical grade.
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RESULTS |
Transport of Glucose-6-phosphate, Glucose, and Phosphate in Fetal
Rat Liver Microsomal Vesicles--
The intactness of the microsomal
vesicles and their permeability toward the substrates and products of
the glucose-6-phosphatase system were investigated by the light
scattering technique. Both fetal and adult microsomal vesicles were
practically impermeable toward sucrose, excluding the possibility of
the nonspecifically increased permeability. Glucose-6-phosphate was
able to enter both types of microsomal vesicles, but the rate of entry
was somewhat lower in the fetal microsomes. No major differences could
be observed in the permeation of glucose and phosphate (Fig.
1). Other hexose phosphates, which are
the substrates of the catalytic subunit, did not permeate the
microsomal membrane, their addition caused a prolonged shrinking both
in adult and fetal microsomes with the exception of
glucose-1-phosphate, which was slowly permeable only in fetal vesicles
(Table II).

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Fig. 1.
Influx of glucose-6-phosphate, glucose, and
phosphate into liver microsomal vesicles evaluated by a light
scattering technique. Light scattering measurements were performed
as described under "Experimental Procedures." Light scattering
increase was assumed to reflect shrinkage of microsomal vesicles (30,
31). Osmotically induced changes in microsomal vesicle size and shape
were initiated by adding 0.1 ml (black arrowhead) of
concentrate solutions of sucrose (Sucr), glucose-6-phosphate
(G6P), potassium phosphate (Pi, pH 7.0),
or glucose (Glc) to 1.5 ml of the microsomal suspensions (in
a hypoosmotic buffer at pH 7.0; 70 or 35 µg protein/ml, for adult and
fetal microsomes, respectively), giving the final concentration 75 mM (sucrose), 50 mM (glucose), or 30 mM (glucose-6-phosphate and phosphate). Alamethicin (10 µg/ml; open arrowheads) was then added to fully
permeabilize microsomal vesicles (28). The addition of the poorly
permeable sucrose resulted in a sustained shrinkage of vesicles
indicating intactness of microsomal membrane. The recovery of initial
signal (swelling phase) after glucose-6-phosphate, glucose, or
potassium phosphate addition was assumed to reflect their entry into
vesicles. The shrinking phase (dotted lines) has been
graphically reconstructed by taking into account the loss of the light
scattering intensity due to dilution of microsomal suspensions after
solute additions. Traces are representative of two to five separate
measurements.
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Table II
Latency of the phosphohydrolase activity towards various sugar
phosphoesters in fetal and adult rat liver microsomes
Microsomal vesicles (1 mg protein/ml) were incubated in buffer A in the
presence of various phosphoesters (10 mM) for 30 min
(fetal) or 5 min (adult) at room temperature. Phosphate production was
measured. Permeabilized microsomes were treated with 50 µg/ml
alamethicin. Data are the means ± S.D. of three to six
experiments. The influx rates of the phosphoesters (25 mM)
into microsomal vesicles expressed as half-time of the light scattering
signal are also shown (n = 2-5).
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Transport activities were also assessed using radiolabeled compounds
and a rapid filtration method. Fetal rat liver microsomes incubated in
the presence of various concentrations of glucose-6-phosphate (plus
[14C]glucose-6-phosphate as a tracer) rapidly accumulated
glucose-6-phosphate + glucose reaching a steady-state level in 2 min.
More than 90% of the radioactivity associated to the microsomes was
releasable by the pore-forming alamethicin, indicating the
intravesicular accumulation. In contrast to adult microsomes, fetal
vesicles did not accumulate the isotope over the equilibrium. Separate measurement of glucose-6-phosphate and glucose revealed that both compounds were present in the intraluminal space of vesicles; however,
the amount of the intravesicular glucose was very low compared with
that of adult microsomes and hardly reached the detection limit (Table
III). Upon glucose-1-phosphate addition (which is a poor substrate of the phosphohydrolase, see Table II), the
difference in the intravesicular isotope accumulation was much
less expressive between fetal and adult microsomes, due to the very low
glucose production (Table III).
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Table III
Uptake and hydrolysis of glucose-6-phosphate in fetal rat liver
microsomal vesicles
Liver microsomes (1 mg/ml) were incubated in buffer A containing 0.2 glucose-6-phosphate (in some experiments glucose-1-phosphate) plus
[14C]glucose-6-phosphate ([14C]glucose-1-phosphate)
(8-10 µCi/ml). In a series of incubations the pore-forming
antibiotic alamethicin (0.05 mg/ml) was added to distinguish the
intravesicular and the bound radioactivity. The alamethicin releasable
(i.e. intravesicular) portion of radioactivity is shown. At
the steady-state level of uptake (5-10 min), samples (0.1 ml) were
rapidly filtered and washed; the intravesicularly accumulated glucose
deriving from glucose-6-phosphate (glucose-1-phosphate) hydrolysis
during the measurement of glucose-6-phosphate uptake was measured
parallelly as described under "Experimental Procedures." For the
measurement of intravesicular glucose and sucrose spaces, 1 mM glucose plus [3H]glucose (9 mCi/ml) or 1 mM sucrose plus [14C]sucrose (1 µCi/ml) were
incubated with microsomal vesicles for 2 h at 22 °C. At the end
of the incubation the intravesicular radioactivity was assessed as
described for glucose-6-phosphate. Apparent intravesicular isotope
spaces were calculated according to the formula: apparent
intravesicular space (µl/mg protein) = intravesicular accumulation
(nmol/mg protein)/concentration of the added compound (nmol/µl). The
same measurements were also executed on adult rat liver microsomal
vesicles. The abbreviations used in the table are: G6P,
glucose-6-phosphate; G1P, glucose-1-phosphate; NM, not measurable.
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The steady-state level of intravesicular glucose-6-phosphate increased
linearly by increasing the extravesicular concentration of
glucose-6-phosphate. Consequently, the intravesicular
glucose-6-phosphate accessible space (calculated by dividing the amount
of accumulated glucose-6-phosphate by the extravesicular
glucose-6-phosphate concentration) was independent of the
glucose-6-phosphate concentration in the medium (Fig.
2).

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Fig. 2.
The intravesicular glucose-6-phosphate space
in fetal rat liver microsomal vesicles. Microsomes were incubated
in the presence of 0.2-30 mM glucose-6-phosphate plus
[14C]glucose-6-phosphate as tracer for 5 min. The
glucose-6-phosphatase activities and the intravesicular
glucose-6-phosphate contents were measured (for details see the legend
to Table III). The glucose-6-phosphate-accessible intravesicular space
of microsomes was calculated using the following formula:
intravesicular glucose-6-phosphate space (µl/mg protein) = intravesicular glucose-6-phosphate content (nmol/mg
protein)/extravesicular glucose-6-phosphate concentration (nmol/µl).
Glucose-6-phosphate space is shown as a function of both extravesicular
glucose-6-phosphate concentration (open symbols) and
microsomal glucose-6-phosphatase activity (filled symbols).
The values are the means ± S.D. of three to six
experiments.
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Fetal rat liver microsomal vesicles incubated in the presence of 1 mM glucose took up the radioactive tracer until a
steady-state level reached over a 5-min period of incubation (Fig.
3). The uptake was attributable to
intravesicular accumulation, because the majority of radioactivity
(>90%) could be released by permeabilizing the vesicles with the
pore-forming alamethicin. The steady-state level of intravesicular
glucose was stable for at least 2 h. In the steady-state phase of
glucose uptake, the apparent intravesicular glucose space, which was
calculated by dividing the intravesicular glucose content (nmol/mg
protein) with the extravesicular glucose concentration (nmol/µl), was
0.23 ± 0.04 (µl/mg protein) at 1 mM glucose
concentration. This apparent space was about 50% of that observed in
adult rat liver microsomes by using the same method (Table III).

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Fig. 3.
Glucose uptake by fetal rat liver microsomal
vesicles. Microsomes were incubated in the presence of 1 mM glucose plus [3H]glucose as tracer and
intravesicular glucose contents were measured as detailed under
"Experimental Procedures." The data are the means ± S.D. of
four experiments.
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Because the intravesicular glucose-6-phosphate, glucose-1-phosphate,
and glucose accessible spaces of fetal liver microsomes were lower than
the corresponding maximal values obtained in adult microsomes, we have
estimated the total intravesicular space measuring the sucrose
accessible space of microsomes. Sucrose, a poorly permeant compound,
reached a steady-state level of uptake at 2 h of incubation. The
value of the intravesicular sucrose space was also 2-fold higher in
adult rat liver microsomes (Table III).
Glucose-6-phosphatase Activity in Fetal Rat Liver Microsomal
Vesicles--
The total phosphohydrolase activity in fetal microsomes
was less than 5% of the adult value (Tables II and
IV). According to Western blot analysis,
the amount of the enzyme protein was less than in adult microsomes, yet
it was clearly detectable (Fig. 4). The
latency of the enzyme was almost absent; permeabilization of the
vesicles elevated the activity slightly, whereas in adult microsomes
more than 50% of the activity was latent (Table II). The difference in
glucose-6-phosphatase activities obtained by the addition of various
concentrations of glucose-6-phosphate did not affect the extent of the
intravesicular glucose-6-phosphate accessible space (Fig. 2.).
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Table IV
Effect of antibodies against various sections of glucose-6-phosphatase
on its catalytic activity in fetal and adult rat liver microsomes
Native microsomes (0.35 mg protein/ml) were preincubated buffer A in
the presence of each of the four anti-peptide antibodies (1% of the
incubation volume) for 50 min at room temperature under continuous
agitation. The incubations were started with the addition 10 mM glucose-6-phosphate. The phosphohydrolase activity was
detected by measuring glucose or phosphate production. Data are the
means ± S.D. of 4-9 measurements.
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Fig. 4.
Immunoblot analysis of fetal and adult rat
liver microsomes. 5 and 10 µg (adult; lanes 1 and
2) or 10 µg (fetal; lane 3) of microsomal
protein was loaded. The arrow indicates a 38-kDa molecular
mass.
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Hydrolysis of other phosphoesters (10 mM) by fetal
microsomes showed diminished latency compared with adult microsomes
(Table II). Very moderate latency was observed in the case of
glucose-1-phosphate and fructose-6-phosphate, whereas the
phosphohydrolase activity toward mannose-6-phosphate,
deoxyglucose-6-phosphate, and glucosamine-6-phosphate remained latent
in two-thirds. The eventual conversion of the hexose phosphates to
glucose-6-phosphate during the incubation (which has been reported in
brain microsomes, for example; Ref. 23) was checked by measuring the
glucose-6-phosphate content at 0 and 30 min of incubation. We have
found low glucose-6-phosphate contents, presumably due to the impurity
of chemicals. The highest value, in the case of fructose-6-phosphate,
was 0.04 mM at the start of incubation (0.4% of the added
fructose-6-phosphate) and 0.07 mM at 30 min. Other
incubates contained less than 0.01 mM glucose-6-phosphate,
a value that did not increase during the incubation (data not shown).
Inhibition of the Phosphohydrolase Activity by Antibodies Against
Different Sections of Glucose-6-phosphatase--
Native fetal and
adult microsomal vesicles were preincubated in the presence of
antibodies raised against different sections of glucose-6-phosphatase:
the N- and C-terminal parts, an external loop, and an internal loop
containing the His residue that belongs to the active site of the
enzyme. After 50 min of preincubation 10 mM
glucose-6-phosphate was added, and the glucose-6-phosphatase activities
were measured. In adult microsomes neither of the antibodies affected
the phosphohydrolase activity. On the contrary, in fetal microsomes
antibodies A and D, raised against two loops supposed to be orientated
toward the cytosol and the lumen in adult liver, strongly inhibited the
enzyme (Table IV).
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DISCUSSION |
The results of the present study demonstrate that the components
of the hepatic glucose-6-phosphatase system are already present in the
fetal liver, although at a lower extent. In accordance with earlier
studies the catalytic subunit of the enzyme can be demonstrated by
Western blotting, although the microsomal phosphohydrolase activity in
fetal liver is less than the 5% of the adult one. The transporters
belonging to the system (T1, T2, and T3) are also present, because
microsomal transport of glucose-6-phosphate, glucose, and phosphate can
also be detected by uptake measurements. However, the intraluminal
glucose-6-phosphate and glucose spaces are 40-50% of the values
observed in adult rat liver microsomal vesicles. These alterations
might suggest that the corresponding transporters are present in a
lower percentage of fetal microsomal vesicles. More probably, they can
reflect the differences in the protein content and/or vesicle size
between fetal and adult microsomal preparations. This latter
possibility is supported by the facts that (i) the light scattering
behavior of fetal and adult microsomes is similar in the presence of
glucose or glucose-6-phosphate and (ii) the intravesicular sucrose
space, which is nearly as extensive as the intravesicular water space
(32), is proportionally lower in fetal microsomes.
Beyond the quantitative differences, fetal liver microsomes also differ
qualitatively. Permeabilization of the microsomal vesicles by
alamethicin slightly increases the phosphohydrolase activity,
i.e. the latency of the enzyme is minimal. Using other substrates than glucose-6-phosphate, we have also observed reduced latency that is accompanied with a reduced substrate specificity. The
intravesicular accumulation of glucose due to glucose-6-phosphate hydrolysis is hardly measurable, in accordance with the observation of
Annabi and van de Werve (25). The intravesicular glucose-6-phosphate space, which is in inverse ratio to the extravesicular
glucose-6-phosphate concentration and the consequent
glucose-6-phosphatase activity in adult rat liver microsomes (12), is
stable independently of extravesicular glucose-6-phosphate
concentrations. It suggests that in fetal rat liver microsomes
glucose-6-phosphatase does not utilize an intravesicular substrate pool.
The differences between fetal and adult glucose-6-phosphatase system
can be explained in several ways. The nonspecific increase of membrane
permeability in fetal microsomal vesicles is out of the question on the
basis of normal sucrose response in light scattering and the presence
of intravesicular pools. The altered (diminished) specificity of
glucose-6-phosphate transporter and/or the presence of other
transporter(s) responsible for the permeation of other phosphoesters
may also be supposed. However, other hexose-6-phosphates are not
permeable on the basis of light scattering experiments. Furthermore,
the altered balance of transport and hydrolysis, i.e. the
very low phosphohydrolase activity together with the quasi normal
transport processes, may cease the rate-limiting property of
glucose-6-phosphate transport, which is characteristic of adult
microsomes. This explanation can account for the diminished latency of
the system in the presence of glucose-6-phosphate and the low
intravesicular glucose accumulation upon glucose-6-phosphate hydrolysis. In itself it is not enough to explain the decreased latency
of phosphohydrolase toward other phosphoesters. On the basis of the
results we suppose that the changed orientation of the proposed active
site of glucose-6-phosphatase can be responsible for the altered
features of glucose-6-phosphatase system in fetal rat liver microsomes.
This feature of the enzyme is not without precedents: multiple
topological orientations linked with different functions has been
reported in the case of other membrane proteins (34-36). The premise
of the presence of a phosphohydrolase orientated with its catalytic
site toward the cytosolic surface of the endoplasmic reticulum membrane
is compatible with the reduced latency, the relative substrate
specificity in native microsomes, the phosphohydrolase-independent intravesicular glucose-6-phosphate pool, and the small intravesicular glucose accumulation. The inhibitory effect of an antibody against the
loop containing the proposed active site of the enzyme, which was
present only in the fetal microsomal vesicles, strongly supports this
hypothesis. Because glucose-6-phosphate transport is present in fetal
microsomes, it means that the transport and hydrolysis are at least
partially uncoupled. Due to the altered orientation, the enzyme can act
as a rather nonspecific phosphohydrolase or, alternatively, can
catalyze the inverse reaction (37), i.e. it may replace the
glucokinase activity missing in the neonatal period (38).
It has been observed previously that the latency of the
glucose-6-phosphatase system (and other microsomal enzymes with
different time course; Ref. 39) increases rapidly around the birth. It may be caused by the change of orientation of the enzyme or by the
replacement of the fetal isozyme with an adult form. In the first case
it can be supposed that the folding of the protein converts it into a
more active, more specific, intralumenally orientated form. The
activation of preformed proteins by folding together with the increased
transcription of glucose-6-phosphatase gene may serve the rapid
accommodation to the new gluconeogenic/glycogenolytic role of the liver
after the birth.