(Received for publication, April 4, 1995; and in revised form, July 12, 1995)
From the
We have investigated the kinetics of tracer uptake into rat
liver microsomes in relation to [C]glucose
6-phosphate (Glu-6-P) hydrolysis by glucose 6-phosphatase (Glu-6-Pase).
1) The steady-state levels of intravesicular tracer accumulated during
the rapid (AMP
) and slow (AMP
) phases of uptake
both demonstrate Michaelis-Menten kinetics relative to outside Glu-6-P
concentrations with K
values similar to
those observed for the initial burst (V
)
and steady-state (V
) rates of Glu-6-P hydrolysis.
2) The AMP
/AMP
ratio is constant (mean value
= 0.105 ± 0.018) over the whole range of outside Glu-6-P
concentrations and is equal to the AMP
/AMP
ratio (0.109 ± 0.032). 3) Linear relationships are
observed between the initial rates of glucose transport during the slow
uptake phase (V
) and
[AMP
], and between
[V
] and [AMP
]. 4)
The value of V
exceeds by more than
10-fold that of V
. 5) It
is concluded that the substrate transport model is incompatible with
those results and that AMP
represents a membrane
exchangeable glucose pool. 6) We propose a new version of the
conformational model in which the catalytic site lies deep within a
hydrophilic pocket of an intrinsic membrane protein and communicates
with the extra- and intravesicular spaces through channels with
different glucose permeabilities.
The localization of glucose 6-phosphatase (Glu-6-Pase) ()(EC 3.1.3.9) within the membrane of the endoplasmic
reticulum (ER) has led to consider that glucose 6-phosphate (Glu-6-P)
hydrolysis in vivo should be associated with transport
functions allowing both substrate and products to cross the ER
membrane. In agreement with that idea, Glu-6-Pase activity was found to
be increased in disrupted rat liver microsomes as compared with intact
vesicles, and the enzyme activity has therefore been described as
latent(1, 2) . The basis of Glu-6-Pase latency has led
to much controversy in the literature, and two theories have emerged
during the last 20 years. In the substrate transport model (1, 3, 4, 5, 6, 7) ,
it is postulated that the enzyme is part of a multicomponent system in
which the catalytic site is oriented toward the lumen of the ER and
associated with specific transport proteins for Glu-6-P (T1), phosphate
(T2), and glucose (T3)(4) . Accordingly, Glu-6-Pase latency has
been interpreted as evidence for a rate-limiting step in Glu-6-P
transport through T1. In contrast, the conformational
model(8, 9, 10) proposes that Glu-6-Pase is
a single entity sequestered within the ER membrane. The catalytic site
is thus freely accessible from the cytoplasm, and Glu-6-Pase latency
would result from changes in the membrane environment that modify the
interactions of the enzyme with its substrates and products.
In agreement with the conformational model, our recent kinetic studies using a fast-sampling, rapid-filtration apparatus (FSRFA, U. S. patent 07/697,769) have brought evidence for a tight coupling between Glu-6-P transport and Glu-6-Pase activity (11, 12, 13) and failed to demonstrate any significant transport of D-glucose into rat liver microsomes under zero-trans uptake conditions(13, 14) . Those studies have resolved, however, neither the nature of the labeled species that equilibrate during the rapid phase of intramicrosomal uptake nor the kinetic relationships between fast tracer uptake and Glu-6-P hydrolysis and between rapid and slow tracer uptakes. Since all of those issues appear critical to the validation of the substrate transport(1, 2, 3, 4, 5, 6, 7) or conformational (9, 10, 11, 12) models, they have been addressed in the present studies. Our results demonstrate that the steady-state rate of Glu-6-P hydrolysis by intact microsomes exceeds by more than 10-fold the steady-state rate of glucose efflux from microsomes, thus ruling out the substrate transport model(1, 2, 3, 4, 5, 6, 7) . It is further shown that the tracer taken up during the rapid phase of intravesicular accumulation is not the precursor for hydrolysis during the slow uptake phase but represents instead an exchangeable glucose pool. Accordingly, we propose a modified version of the combined flexibility-substrate transport model (9, 10) in which glucose released by Glu-6-P hydrolysis accumulates within a hydrophilic pocket and has access to either of the extra- or intramicrosomal compartments through outer and inner channels with different intrinsic permeabilities to glucose.
Figure 1:
Correlation
between glucose transport rates and steady-state tracer concentrations
achieved during the rapid (A) and slow (B) phases of
uptake into microsomes. [AMP] and
[AMP
] values are the theoretical ones shown in Table 1. V
values were
calculated as described in the text, while efflux rates correspond to
either V
(
) or the efflux data of Fig.
5B (
) in (13) . Lines shown correspond
to the linear regression analysis of the data points ± S.E.R.
values. When not shown, errorbars were smaller than
symbol sizes.
According to the substrate transport
model(1, 2, 3, 4, 5, 6, 7) derived in
a previous paper(11) , which is similar in its concept and
predictions to the tandem model of transport and phosphorylation
analyzed by Wohlhueter and Plagemann(15) , the biexponential
uptake time courses of tracer uptake into microsomes (11, 13) would attribute the rapid and slow phases of
uptake to Glu-6-P and glucose equilibration inside of the vesicles,
respectively. Compiled in Table 1are the experimental and
theoretical tracer concentrations corresponding to AMP and
AMP
at each of the Glu-6-P concentrations used in our
studies. Interestingly, there is a marked difference between
[AMP
] and [AMP
] in that
tracer accumulation against a concentration gradient is apparent with
[AMP
] only (values under brackets in Table 1), in agreement with the substrate transport model
provided that glucose exit from microsomes is slower than both Glu-6-P
transport and hydrolysis, and in agreement with our former conclusion (11, 13) that [
C]glucose is the
major species to be found at the steady-state level of tracer uptake
when using subsaturating Glu-6-P concentrations. Moreover, the apparent
decreasing accumulation ratio observed with AMP
at
increasing Glu-6-P concentrations is compatible with a situation where
the V
for Glu-6-P hydrolysis would be higher
than the V
for transport while the K
of both processes would be
similar(15) , in agreement with the kinetic parameters shown in Table 2for V
and V
. We thus have investigated directly
the possibility that AMP
might represent intramicrosomal
steady-state Glu-6-P concentrations by plotting the initial rates of
uptake during the slow phase of tracer accumulation (V
) against the theoretical
values of [AMP
] shown in Table 1. Indeed,
the expectation here was to find a Michaelis-Menten relationship with
kinetic parameters similar to those characterizing V
. As shown in Fig. 1A, however,
a linear relationship is observed over the accessible range of
concentrations, thus ruling out that the tracer taken up during the
rapid phase is the precursor for hydrolysis during the slow phase of
uptake. It should be noted that the slope value of 113.5 ± 5.3
pmol/s
mg of protein
mM is equivalent in microsomes
to a first order rate constant of 0.142 ± 0.024 s
(t = 4.9 ± 0.8 s).
An important
observation to be made in Table 1is that the
AMP/AMP
ratio is quite constant over the whole
range of Glu-6-P concentrations with mean values of 0.105 ±
0.018 and 0.101 ± 0.004 for experimental and theoretical values,
respectively. Accordingly, we were led to consider the hypothesis that
[AMP
] might represent glucose concentrations
within a third compartment located in between the extra- and
intravesicular spaces in which the sugar could accumulate rapidly and
reach a steady-state concentration before being able to access the
intramicrosomal volume to any significant extent. Compatible with that
hypothesis are the results of Table 2showing that: 1) both
AMP
and V
follow
Michaelis-Menten kinetics relative to outside Glu-6-P concentrations
with K
similar to that of Glu-6-P
hydrolysis, and 2) the AMP
/AMP
ratio of
0.109 ± 0.032 is similar to the AMP
/AMP
ratio determined from Table 1. Quite importantly too, the
results of Table 2demonstrate that the V
value represents 7.4%
only of that for V
, thus showing that the
amount of glucose produced from Glu-6-P hydrolysis and liberated into
the intramicrosomal compartment represents a minor fraction only of
glucose directly released into the incubation medium. Alternative
explanations relating to the consideration that a major fraction of our
membrane vesicles might be either leaky or ``inside-out''
oriented can be safely ruled out since the latency of glucose
dehydrogenase activity is routinely 95-100% in our vesicle
preparations(13) , in agreement with the former demonstration
that mannose 6-phosphate is not hydrolyzed to any measurable extent
during the first minute of incubation and that both uptake into
microsomes and total glucose production by intact vesicles are fully
inhibited by 5 mM phlorizin(11) . Moreover, as
discussed in the companion paper(13) , such results cannot be
the consequence of damage to a major fraction of the vesicles when
using the FSRFA. Finally, the observation that the bulk of glucose
accumulation into microsomes occurs during the steady-state phase of
Glu-6-P hydrolysis (13) cannot be the result of a slow vesicle
equilibration with glucose released into the incubation medium. Indeed,
by taking into account both the experimental conditions (13) and the kinetic parameters for Glu-6-P hydrolysis (Table 2), it can be evaluated that the glucose concentration in
the extravesicular medium never exceeds 10-16 µM at
0.2 mM outside Glu-6-P over a 2-min incubation period. In
fact, then, glucose can be accumulated under such conditions up to
15-20- or 160-270-fold relative to outside Glu-6-P or
glucose concentrations, respectively.
A final test of the substrate
transport model (1, 7) can be proposed on the
rationale that, when a steady-state level of tracer accumulation into
microsomes has been reached, the rate of tracer influx into microsomes
(Glu-6-P transport and hydrolysis) is equal to the rate of tracer
efflux from microsomes (glucose efflux). Accordingly, V should be equal to the initial rate of efflux from microsomes
measured at the steady-state of intravesicular glucose concentration.
That this prediction of the substrate transport model fails is shown in Fig. 1B where both V
(opensymbols) and the initial steady-state rate of glucose
efflux (closedsymbols) corresponding to the efflux
data of Fig. 5B in (13) have been plotted against the
theoretical values of [AMP
] shown in Table 1. No saturation could be observed over the accessible
range of concentrations, and the slope values of 154 ± 4 for V
and 11.2 ± 0.6 pmol/s
mg of
protein. mM for efflux are equivalent in microsomes to first
order rate constant values of 0.193 ± 0.029 s
(t = 3.59 ± 0.54 s) and 0.0140 ±
0.0025 s
(t = 49.5 ± 8.8 s)
that closely match those found in the companion paper (13) for
the rapid and slow phases of tracer uptake, respectively. Moreover, the
slope ratio of 0.073 ± 0.006 between the steady-state glucose
efflux and total glucose production rates confirms directly the
conclusion above that glucose produced from Glu-6-P hydrolysis is
mostly released into the incubation medium.
The demonstration that most of glucose produced from Glu-6-P hydrolysis is released directly into the extramicrosomal space strongly argues against the current version of the substrate transport model(1, 7) . All of the kinetic evidence presented herein and in the companion paper (13) point out, however, to intimate relationships between glucose accumulation into microsomes and Glu-6-Pase activity, and between the rapid and slow phases of glucose uptake. Moreover, our results appear quite compatible with the hypothesis of an exchangeable glucose pool located in between the extra- and intravesicular spaces. Such a view fits nicely within the concept of the combined conformational flexibility-substrate transport model of Schulze et al.(10) , an updated version of which is shown in Fig. 2and justified below.
Figure 2:
Modified version of the combined
conformational flexibility-substrate transport model. Microsomal
Glu-6-Pase is depicted as a transmembrane protein with the catalytic
site lying in a hydrophilic pocket deep inside of the protein where
Glu-6-P hydrolysis occurs at rate V = V
under conditions described
in the text. Exchanges between the extra- and intravesicular spaces are
made possible through outer and inner channels with different intrinsic
permeabilities to glucose (k
, k
) and Glu-6-P (k
). (Glu-6-P) and (G) represent Glu-6-P and
glucose concentrations, while indices o, m, and i refer to the incubation medium, the hydrophilic pocket, and the
intramicrosomal space, respectively. More details are given in the
text.
According to Fig. 2, Glu-6-Pase is an intrinsic membrane protein that would span the entire microsomal membrane, in agreement with the cloned sequence of the enzyme catalytic subunit that predicts up to six membrane-spanning segments in the secondary structure of the protein (16, 17) . The catalytic site would be embedded to some extent within the protein interior and would form a hydrophilic pocket in an otherwise hydrophobic environment. That pocket would be accessible directly from the external medium via a hydrophilic outer channel that is not, however, freely accessible to just any molecule. In that sense, there should be some gating mechanism that might account for the unidirectionality of glucose transport and allow for both substrate and inhibitor specificities (T1-like function). Glu-6-P would be hydrolyzed within the hydrophilic pocket, and glucose released at that site could be either exported to the external medium through the outer channel or transported into the intravesicular space via the inner channel (T3-like activities).
The difference in the time
scales needed to reach a steady-state rate of Glu-6-P hydrolysis and a
steady-state level of glucose accumulation into microsomes can be
explained in a three-compartment system like the one defined by the
enzyme structure postulated in Fig. 2by assuming that Glu-6-P
transport through the outer channel, Glu-6-P hydrolysis in the
hydrophilic pocket, and glucose permeation through the outer channel (Fig. 2, upperpart of the cycle) are
all faster than glucose transport through the inner channel. If,
additionally, glucose efflux through the outer channel is slower than
both Glu-6-P transport into and Glu-6-P hydrolysis within the pocket,
then free glucose would accumulate into that compartment until a steady
state is reached. At that point, the rate of glucose exit would be
equal to the slower activity of either Glu-6-P transport or Glu-6-P
hydrolysis. Since similar K for
hydrolysis are observed in deoxycholate-treated membranes and, in
native membranes, during the burst and the steady-state phases of
glucose production (Table 2), we would favor at this point the
hypothesis that hydrolysis is slower than Glu-6-P transport.
Alternatively, Glu-6-P access to the catalytic site and hydrolysis
might be tightly coupled events(10, 11) . Under these
conditions, apply where V
and K
refer
to the kinetic parameters of Glu-6-P hydrolysis during the steady-state
phase of total glucose production (Table 2) while
(G
), (G
), and k
are defined in the
legend to Fig. 2.
Since a steady-state rate of Glu-6-P hydrolysis insures constant G within the hydrophilic pocket, the
glucose pool can slowly equilibrate with the intravesicular space
through the inner channel. Under these conditions, and apply where (G
) and k
are defined in the legend to Fig. 2.
It should be stressed that the hypotheses that were made in
deriving the above equations are indeed compatible with 1) the t values in the range of 2.5-5.0 s observed for the first
exponential term in the glucose accumulation curves (13) that
would characterize the filling time of the hydrophilic pocket (rate
constant k in ) and 2)
the t values in the range of 30-50 s (13) observed for the slow phase of glucose uptake into
microsomes that would characterize glucose equilibration between the
hydrophilic pocket and the intramicrosomal space (rate constant k
in and ).
An
important feature of the model depicted in Fig. 2is illustrated
by and showing that
[AMP] and [AMP
] both
represent [G
]. Accordingly, the
[AMP
]/[AMP
] ratio in Table 1, where the [AMP
] and
[AMP
] values have been calculated relative to the
intramicrosomal volume, would represent in fact the apparent fractional
volume of the hydrophilic pocket relative to the intramicrosomal space,
hence its constant value and independence on Glu-6-P concentrations. As
shown in Table 2, however, both AMP
and AMP
should follow Michaelis-Menten kinetics with K
typical of Glu-6-P hydrolysis. Indeed, the condition
[AMP
] = [AMP
] calls
for a reassessment of the plot shown in Fig. 1A, which
should depict, according to with
[G
] = 0, the dependence
of V
on
[G
]. Accordingly, the t value determined in that figure (4.9 ± 0.8 s), which should
characterize k
, is underestimated by a factor of
10. That correction thus agrees with the determination of k
from 1) the uptake time courses according to , which is equivalent to in (13) (t in the range of 30-50 s), and 2) the
efflux data shown in Fig. 1B (closedsymbols, t = 49.5 ± 8.8 s), where
glucose permeation through the inner channel represents the overall
rate-limiting step of the efflux process.
The simple scheme depicted
in Fig. 2is also compatible with the following results
presented herein and in the companion paper(13) : 1) the
linearity of V relative to (G
) with slope k
( and Fig. 1B, opensymbols), and its michaelian dependence on
(Glu-6-P
) ( and and Table 2); 2) the independence of the t of glucose
equilibration into microsomes from the presence of varying
concentrations of Glu-6-P or vanadate(13) ; 3) the similar
steady-state levels of intramicrosomal glucose achieved under
symmetrical influx and efflux conditions of Glu-6-P and vanadate
concentrations(13) ; and 4) the direct correlation between the
steady-state levels of intramicrosomal
[
C]glucose accumulation and V
measured at increasing cold Glu-6-P or vanadate
concentrations(13) . In that context, our recent studies with
histone IIA-treated microsomes (18) also agree with the model
of Fig. 2in that higher uptake values were associated with
higher Glu-6-Pase activities in treated as compared with normal
microsomes. Moreover, the failure of histone IIA-treated microsomes to
take up [
C]mannose from
[
C]mannose 6-phosphate (18) would
suggest that the inner channel is quite specific for glucose and that
histones mostly affect the gating mechanism of the upper channel.
While the molecular arrangement within the transverse plane of the microsomal membrane proposed in Fig. 2for Glu-6-Pase does not exclude that the catalytic subunit be associated with one or several proteins (subunits), it is quite clear too that our studies ( (11, 12, 13) and 19 and this paper) failed to demonstrate the existence of both T1 and T3 (GLUT7) as entities fully separated from the catalytic process. Accordingly, the scheme of Fig. 2should be viewed as the minimum mechanistic model that is needed to explain the steady-state kinetics of Glu-6-P hydrolysis and glucose exchange through the ER membrane. That scheme, in its general principle at least, thus represents by now the only alternative to the more classic substrate transport hypothesis. The recent demonstration that the Glu-6-Pase gene of glycogen storage disease type 1b and 1c patients is normal (20) clearly indicates, however, that a major component of the Glu-6-Pase system is presently missing in the model of Fig. 2. It is thus reasonable to propose that an extra protein component(s) might be needed to account for other characteristics of the complete system, an obvious candidate(s) being a soluble or a membrane protein(s) that would modulate the transport functions through the outer and/or inner channels. Indeed, the characterization of such a component(s) should prove of paramount importance to the understanding of a number of questions that are not readily answered by the scheme of Fig. 2like the apparent unidirectionality of glucose transport (13) and the molecular mechanism responsible for the hystereric transition from a ``detergent-like'' to a ``steady-state'' form of the enzyme(11) . Independent of the complexity of the Glu-6-Pase system, however, the model of Fig. 2strongly suggests that the release of phosphate inside of the hydrophilic pocket might contribute to the hysteretic transition by a mechanism that has yet to be determined.