(Received for publication, February 14, 1995; and in revised form, June 15, 1995)
From the
Brief (1-2 h) exposure of Clone 9 cells to inhibitors of
oxidative phosphorylation such as azide is known to markedly increase
glucose uptake. Clone 9 cells express GLUT1 but not GLUT2, -3, and -4,
and the azide effect was not accompanied by any increase in cellular or
plasma membrane GLUT1 level. To identify the molecular event underlying
this apparent increase in GLUT1 intrinsic activity, we studied the
acute effects of azide on the substrate binding activity of GLUT1 in
Clone 9 cells by measuring glucose-sensitive cytochalasin B binding.
The glucose-displaceable, cytochalasin B binding activity was barely
detectable in membranes isolated from Clone 9 cells under control
conditions but was readily detectable after a 60-min incubation of
cells in the presence of 5 mM azide showing a 3-fold increase
in binding capacity with no change in binding affinity. Furthermore,
the cytochalasin B binding activity of purified human erythrocyte GLUT1
reconstituted in liposomes was significantly reduced in the presence of
cytosol derived from azide-treated Clone 9 cells but not in the
presence of cytosol from control cells; this effect was heat-labile and
abolished by the presence of the peptide corresponding to the GLUT1
COOH-terminal sequence. These results suggest that a cytosolic protein
in Clone 9 cells binds to GLUT1 at its COOH-terminal domain and
inhibits its substrate binding and that azide-induced metabolic
alteration releases GLUT1 from this inhibitory interaction. Studying
the binding of cytosolic proteins derived from S-labeled
Clone 9 cells to glutathione S-transferase fusion protein
containing glucose transporter COOH-terminal sequences, we identified
28- and 70-kDa proteins that bind specifically to the cytoplasmic
domain of GLUT1 and GLUT4 in vitro. We also found a
P-labeled, 85-kDa protein that binds to GLUT4 but not to
GLUT1 and only in cytosol derived from azide-treated cells. The roles,
if any, of these glucose transporter-binding proteins in the
azide-sensitive modulation of GLUT1 substrate binding activity in Clone
9 cells are yet to be determined.
A family of six distinct intrinsic membrane proteins (facilitative glucose transporters) catalyze the glucose uptake and release in animal cells(1, 2) . GLUT1, the member (isoform) of this family that is abundant in human erythrocytes and transformed cell lines, catalyzes glucose uptake in Clone 9 cells(2, 3, 4) , a ``nontransformed'' rat liver cell line(4) . As in nucleated avian erythrocytes(5) , mammalian muscle, and adipose cells(6) , the glucose utilization in Clone 9 cells is rate-limited and metabolically regulated at the transport step(3) . A brief (1-2 h) exposure to cyanide or azide, inhibitors of oxidation phosphorylation, stimulates glucose uptake in Clone 9 cells 6-8-fold (3, 7) . This acute effect of azide occurs without any increase in cellular GLUT1 content(3) , indicating that it is due to a post-translational modulation. A longer (up to 24 h) incubation with azide causes an additional 2-fold stimulation of glucose uptake in these cells(7) . In contrast to the acute effect, however, this later effect of azide is accompanied by 2- and 8-fold increases in GLUT1 and GLUT1 mRNA cellular content, respectively(8) , indicating that it is largely due to a pretranslational modulation.
The two well
known post-translational mechanisms for glucose transport regulation in
animal cells include changes in transporter intrinsic activity (2) and transporter redistribution from a microsomal storage
pool to the cell surface (recruitment)(9, 10) . A 2-h
incubation with azide only slightly increases plasma membrane GLUT1
levels in Clone 9 cells(3) , indicating that the acute effect
of azide on glucose transport is due to an increased GLUT1 intrinsic
activity rather than GLUT1 recruitment. Furthermore, kinetic studies
have shown that the acute glucose transport stimulation by azide is due
to an increased V with no change in K
for glucose(4) , suggesting
that azide most likely increases GLUT1 catalytic turnover.
Alternatively, however, it is also possible that azide converts GLUT1
from an inactive state to an active state in terms of substrate
binding; GLUT1 in Clone 9 cells may be largely kept in an inactive
state where it cannot bind substrate, and an azide-induced metabolic
alteration may convert this inactive GLUT1 to an active (unmasked)
state where it can now bind glucose with a given affinity and is hence
functional.
In the present study, we tested the alternative
possibility discussed above. We studied the substrate binding capacity
of GLUT1 in Clone 9 cells by measuring D-glucose-sensitive CB ()binding and examined if it is increased after azide
treatment in association with the transport stimulation. CB, a well
known inhibitor of facilitative glucose transporters, binds to GLUT1,
and this binding is readily and specifically displaced by D-glucose but not by L-glucose(11) . This D-glucose-induced cytochalasin B displacement has been used
extensively as an assay for substrate binding activity of
GLUT1(12) . We demonstrate here that the glucose-displaceable
cytochalasin B binding activity is barely detectable in membranes
isolated from control Clone 9 cells, but it is increased greatly after
a 1-h incubation with azide. We further show that the
glucose-displaceable cytochalasin B binding capacity of GLUT1 purified
from human erythrocytes and reconstituted as proteoliposomes is
significantly reduced in the presence of cytosol isolated from
azide-treated Clone 9 cells but not from untreated Clone 9 cells. We
also describe three distinct cytosolic proteins that bind to the
glutathione S-transferase (GST) fusion proteins containing the
COOH-terminal cytoplasmic domain of GLUT1 and GLUT4 but not to
irrelevant fusion proteins. Possible roles of these putative glucose
transporter-binding proteins in the regulation of glucose transporter
function in response to metabolic alteration are discussed.
For P
labeling, four 100-mm confluent plates of cells were rinsed four times
with sodium phosphate-free DMEM and incubated for 2 h with 2.5 ml of
sodium phosphate-free DMEM containing 3.5 µl of 5 M NaCl
and 1.25 mCi of
P (9,000 Ci/mmol). After 2 h, cells were
treated with diluent or azide (dissolved in sodium phosphate-free DMEM)
for 1 additional hour prior to harvest. All subsequent steps were as
described above except that 1 ml of lysis buffer was used per two
plates of cells.
Figure 1:
Equilibrium cytochalasin B binding to
basal and azide-treated Clone 9 cell total membranes. Clone 9 cells
were incubated without (A) or with (B) 5 mM sodium azide for 60 min, and total membranes were isolated from
them. Membranes (100 µg protein) were incubated with a varying
concentration (0-10 µM) of CB and a tracer amount
(0.02 µCi) of [H]CB in the absence (
)
or in the presence of 500 mMD-glucose (
), 100
µM cytochalasin E (
), or 500 mMD-glucose plus 100 µM cytochalasin E (
) for
20 min at room temperature and then separated from the medium by
centrifugation. Both membrane-bound and free
[
H]CB were measured, from which amounts of bound
ligand were calculated as the percentages of the total (bound plus
free) and plotted as a function of the CB concentration used for the
binding experiment.
The CE-sensitive and CE-insensitive CB binding components mentioned
above were analyzed (not illustrated) according to
Scatchard(20) . The CE-sensitive CB binding was estimated by
measuring the difference in ligand binding observed in the presence and
in the absence of 10M CE. The
CE-insensitive binding component was estimated by measuring CB binding
in the presence of 10
M CE and substracting
linear binding component. The CE-sensitive binding in Scatchard plot
revealed a single binding component with apparent K
and B
(total binding capacity) of
approximately 2 µM and 80 pmol/mg protein, respectively,
for both control and azide-treated cells. The CE-insensitive CB binding
also revealed an apparent linearity in Scatchard plot, yielding an
apparent K
of 0.2 µM for
both control and azide-treated cells. The plot, however, gave B
values of approximately 15 and 20 pmol/mg
protein for control and azide-treated cells, respectively, indicating
that azide treatment selectively increases the high affinity,
CE-insensitive CB binding in Clone 9 cell membranes. It should be noted
that the K
value for this apparently
azide-sensitive, high affinity binding component is similar to the K
value known for cytochalasin B binding
to GLUT1 (11) .
We next quantitated the glucose-sensitive
cytochalasin B binding to Clone 9 cell membranes that is
stereospecifically displaced by D-glucose, the physiological
substrate of GLUT1, by quantitating difference in cytochalasin B
binding observed between the presence of 500 mMD-
and L-glucose (Fig. 2). For control cells, CB bound
less in the presence of D-glucose than in the presence of L-glucose, but the difference was so slight that exact
quantitation was rather difficult (Fig. 2). However, when we
repeated these measurements with membranes derived from azide-treated
cells, cytochalasin B binding was clearly less in the presence of D-glucose compared with that in the presence of L-glucose (Fig. 2), demonstrating that a significant
portion of the ligand bound to membranes derived from azide-treated
cells is readily displaceable by D-glucose but not by L-glucose. This D-glucose-sensitive CB binding was a
saturable function of CB concentrations for both control and
azide-treated cells (Fig. 2). Scatchard analysis of the D-glucose-sensitive, CB binding data (not illustrated)
revealed a single CB binding component with an apparent K and B
value of
0.26 ± 0.03 µM and 1.8 ± 1.6 pmol/mg protein
for control cells (n = 3) and 0.25 ± 0.03
µM and 6.7 ± 1.2 pmol/mg protein for azide-treated
cells (n = 4), respectively. These K
values are similar to the K
values of the CE-insensitive, CB binding component, but an order
of magnitude smaller than that of the CE-sensitive, CB binding
component discussed above. Data of the D-glucose-displaceable
CB binding to membranes of azide-treated Clone 9 cells measured in the
presence of 10
M CE (not illustrated) gave K
and B
of 0.31
µM and 6.1 pmol/mg protein, respectively, indicating that
the glucose effect was not affected in the presence of an excess of CE.
Figure 2:
Effects of acute azide treatment (5
mM) on D-glucose-sensitive CB binding in Clone 9
cells. Clone 9 cell total membranes (100 µg of protein) were
incubated with 500 mMD-glucose or L-glucose
for 20 min, and the equilibrium binding of cytochalasin B was measured
in the presence of 500 mMD-glucose and L-glucose, respectively, using 0.02 µCi of
[H]CB as tracer in the presence of a varying CB
concentration (0-10 µM). The D-glucose-sensitive CB binding was calculated as the
difference in CB binding observed in the presence of L- and D-glucose for Clone 9 cells before (
) and after (
)
sodium azide (5 mM) treatment for 60 min and plotted as a
function of CB concentration. Each data point represents an average of
data obtained in three independent measurements with S.D. shown as vertical bars.
We next examined expression of glucose transporter isoforms in Clone 9 cells. GLUT1 is the only isoform detectable in Northern blot of Clone 9 cells(7) . Our Western blot analysis shows that Clone 9 cells express GLUT1 protein in abundance without any detectable amounts of GLUT2, GLUT4 (Fig. 3), or GLUT3 (not illustrated). Again, the acute treatment (2 h) with azide did not affect Clone 9 cell GLUT1 content to any significant degree (Fig. 3).
Figure 3: Western blot analysis of glucose transporters in Clone 9 cells. Total membranes (40 µg of proteins) of Clone 9 cells before (Con) or after (Azide) azide treatment (5 mM for 60 min) were applied for each lane and immunoblotted using antipeptide antibodies specific to GLUT1, 2, and 4. Also included are purified human erythrocyte GLUT1 (HEGT, 1 µg of protein), rat liver cell total membranes (Hep, 40 µg of protein) and rat epididymal adipocyte microsomes (Adip, 40 µg protein) as controls to confirm isoform specificities of the antibodies. These were reproduced in two other experiments, whereas the positive blot with GLUT4 IgG of an apparent molecular mass of 50 kDa seen in hepatocyte membranes was not reproducible.
These findings strongly suggest that the D-glucose-sensitive portion of the saturable CB binding is due to GLUT1 and that the acute azide treatment increases the GLUT1 CB binding capacity without GLUT1 content in Clone 9 cells. The exact nature of this azide-induced apparent increase in GLUT1 CB binding, particularly its relevance to GLUT1 substrate (glucose) binding function, can only be speculated at this time.
To test this
possibility, we examined whether the cytochalasin B binding activity of
purified GLUT1 and/or its sensitivity to D-glucose are
affected by cytosol derived from basal and azide-treated Clone 9 cells (Fig. 4). Human erythrocyte GLUT1 was purified and reconstituted
in liposomes(18) . Equilibrium binding of cytochalasin B to
this preparation was measured as a function of CB concentrations and
analyzed in Scatchard plot. When suspended in Tris buffer, purified
GLUT1 typically bound CB with K and B
of 0.2-0.4 µM and 14-16
nmol/mg protein, respectively (Fig. 4A). The incubation
of purified GLUT1 in Tris buffer containing 5 mM azide for up
to 60 min prior to measurement did not affect its CB binding activity
(not illustrated). When suspended in cytosol of control Clone 9 cells,
GLUT1 bound CB equally well, showing apparent K
and B
values that were practically
identical to those measured in buffer alone (Fig. 4A, inset). In the presence of the cytosol derived from
azide-treated cells, however, CB binding was significantly reduced
compared with those in buffer or in the presence of the cytosol derived
from control cells (Fig. 4A), and this reduction was
due to a decrease in B
without any significant
change in K
(Fig. 4, A (inset) and C). Furthermore, this reduction in B
by azide-treated cytosol was largely (50% or
more) abolished in the presence of the peptide corresponding to the
cytoplasmic COOH-terminal domain of GLUT1 (Fig. 4C).
These findings suggest the presence of a factor in azide-treated cell
cytosol that interacts with purified GLUT1 at its COOH-terminal domain
and inhibits its CB binding activity and that this factor is virtually
missing in cytosol obtained from control cells. There was no reduction
in CB binding when this experiment was repeated using azide-treated
cytosol preheated for 15 min at 70 °C (Fig. 4B),
indicating that the factor is heat-labile, thus most likely a protein.
Figure 4:
Effects of cytosol derived from basal and
azide-treated Clone 9 cells on the CB binding activity of purified,
human erythrocyte GLUT1 reconstituted in liposomes. A,
liposomes containing GLUT1 (5 µg of protein) were incubated with
Tris (pH 7.4) buffer or Clone 9 cell cytosol (150-200 µg
protein) for 30 min, and then equilibrium CB binding to GLUT1-liposomes
was measured in a manner similar to those of Fig. 1and Fig. 2. Data were expressed in the percentage bound as a
function of CB concentrations and also analyzed (insets in A and B) according to Scatchard (20) in Tris
buffer (), in control cytosol (
), and in azide-treated
cytosol (
). B, equilibrium binding of CB to
GLUT1-containing liposomes suspended in control cytosol (
) and in
azide-treated cytosol preheated for 15 min at 70 °C (
). The
GLUT1 preparation used here shows a slightly higher K value
for CB binding than that used in A. Experiments were otherwise
similar to that in A. These results were reproduced in three
independent sets of experiments. C, CB binding to purified
GLUT1 (5 µg) suspended in 500 µl of control and azide-treated
Clone 9 cytosols were measured in the presence or absence of the
synthetic peptide (10 µg) corresponding to GLUT1 COOH-terminal
sequence (residues 451-492) using a CB concentration of
10
M. Values for bound/free are means of
three measurements with S.D. shown as vertical
bars.
Figure 5: SDS-gel electrophoretic separation of GST fusion proteins. Purified GST fusion proteins were separated using 12% polyacrylamide gel and stained with Coomassie Blue (see ``Experimental Procedures''). Lane 1, high molecular mass markers; lane 2, GST; lane 3, GST-G1C; lane 4, GST-G4CR; lane 5, GST-G4C; lane 6, low molecular weight markers. Sizes (in kDa) of the molecular mass markers used are specified in the margins.
The Clone 9 cells were metabolically labeled with S, and labeled cytosolic proteins were separated free of
membrane proteins by centrifugation at 185,000
g for
60 min. The cytosol were then incubated with purified GST fusion
proteins coupled to glutathione-agarose beads. The bound cytosolic
proteins were separated from unbound ones by centrifugation. To prevent
nonspecific binding, the GST fusion protein beads were washed three
times with PBS before use. Bound proteins were eluted from the beads by
adding 5 mM glutathione. The eluted proteins were then
separated on 10% SDS-PAGE and subjected to autoradiography.
A typical result of such an experiment revealed two cytosolic proteins with apparent molecular masses of 70 and 28 kDa that bind to GLUT1 and GLUT4 GST fusion proteins but not to GST (Fig. 6) or to the GST fusion protein of ``incorrectly orientated GLUT4 COOH terminus'' (not illustrated). The 70-kDa protein binding was much more intense for GLUT4 fusion protein than for GLUT1 fusion protein (Fig. 6). Azide treatment (5 mM for 1 h) did not reproducibly affect either the 28- or the 70-kDa protein binding appreciably in these experiments. It is interesting to note that there is a 70-kDa protein in rat adipocyte cytosol that binds to the GST fusion proteins of GLUT1 and 4, although the relationship if any of this adipocyte protein to the 70-kDa Clone 9 cell protein is yet to be determined. The 28-kDa protein binding, on the other hand, appeared unique to Clone 9 cells; no 28-kDa protein binding to GLUT1 or GLUT4 fusion proteins was detectable when rat adipocyte or hepatocyte cytosols were employed in these experiments (not illustrated).
Figure 6:
Binding of the 28- and 70-kDa Clone 9
cell cytosolic proteins to GLUT1 and GLUT4 COOH-terminal domains. 75
µg of each GST fusion protein and 150 µg of S-labeled control or azide-treated Clone 9 cell cytosol
proteins were used for each binding assay. Proteins bound to GST fusion
proteins were subsequently eluted with 150 µl of 10 mM
glutathione for 30 min and then 150 µl of solubilizer for 10 min.
The eluates were applied on a 150-mm 10% SDS-PAGE slab gel and
separated as described. The gel was incubated in enhancer for 1 h and
in tap water for 0.5 h. It was then dried and autoradiograghed for
2-7 days at -70 °C. GST, GST alone; G1C, GST fusion protein of GLUT1 COOH terminus; G4C,
GST fusion protein of GLUT4 COOH terminus for both control and
azide-treated cytosols. Three molecular mass marker (in kDa) positions
are shown in the right margin. 70- and 28-kDa glucose
transporter-binding proteins are indicated by arrowheads.
Similar results were reproduced in three other
experiments.
To
test if either the 70- or the 28-kDa GLUT1-binding protein is a
phosphoprotein, we repeated these experiments using P-labeled Clone 9 cell cytosol (Fig. 7). Neither
70- nor 28-kDa protein binding was detected with
P-labeled
cytosols for GLUT1 and GLUT4 fusion proteins, indicating that the 70-
and 28-kDa GLUT1-binding protein are not phosphoproteins. Instead,
there was a
P-labeled, 85-kDa protein that bound to GLUT4
fusion protein but not to GLUT1 fusion protein. Furthermore, this
85-kDa phosphoprotein binding was seen only in cytosol of azide-treated
cells and not of control cells (Fig. 7). The 85-kDa binding was
not always (two in four independent experiments) reproducible. The
failure in detection of this protein by
S-labeled cytosol
would suggest that its metabolic turnover is rather slow.
Figure 7:
Binding of P-labeled, 85-kDa
Clone 9 cell cytosolic protein to GLUT4 COOH-terminal domain GST fusion
protein. Experiments are similar to these of Fig. 6above except
that
P-labeled cytosol was used. Both glutathione eluates (GSH) and 1% SDS eluates (SDS) were analyzed by
SDS-PAGE. Lanes 1, GST alone; lanes 2, GST fusion
protein of GLUT1 COOH terminus; lanes 3, GST fusion protein of
GLUT4 COOH terminus; CONT, for control cytosol; AZIDE, azide-treated cytosol. An arrowhead indicates
the 85-kDa protein position. Molecular mass markers are also shown in
kDa.
It is known that anaerobic glycolysis in many animal cells is physiologically suppressed under normal aerobic conditions and that this suppression is released when oxidative metabolism is inhibited (21) . Reversible activation of phosphofructokinase, a major rate-limiting enzyme in the glycolytic pathway, has been shown to be important in this regulation(22) . However, an intrinsic facilitation of glycolysis itself would provide little protection against a reduced oxidative pathway unless sufficient intracellular glucose is available to support the increased need of glucose consumption by glycolysis. Thus, for many mammalian cells where glucose entry is rate-limited under basal condition and glycogen storage is minimal(23) , the adaptive response to a reduction in oxidative metabolism must include a substantial enhancement of glucose transport(4) . Such an adaptive response has been described not only in the Clone 9 cells studied here, but also in rat myocardial cells(24) , muscle cells(25) , avian erythrocytes(3, 26) , and yeast (27) as well.
Our results in the present study revealed that cytochalasin B, a
potent inhibitor of GLUT1 function, binds to Clone 9 cell membranes at
three distinct classes of saturable binding sites, namely, a
cytochalasin E-sensitive site, a D-glucose-sensitive site, and
a site insensitive to both D-glucose and cytochalasin E. Our
data also show that the D-glucose-sensitive site is a glucose
transporter; the K for CB binding to this
site is practically identical to the K
for CB binding to GLUT1 and also to the K
value for CB as an inhibitor of GLUT1 transport activity.
These findings are reminiscent of the characteristics of the CB binding
sites in human erythrocyte membranes(19) , where a D-glucose-sensitive site (site I), a CE-sensitive site (site
II), and a site (site III) insensitive to both D-glucose and
CE exist and where site I is GLUT1 and site II is cytoskeletal.
We have shown in the present study that the amount of the CE-insensitive, D-glucose-sensitive CB binding site, presumably GLUT1 (GLUT2, 3, and 4 were not detectable in Western blots), is very small in Clone 9 cells, amounting to a low percentage of the total CB binding sites in membranes and thus barely detectable. This corresponds to 1-2 pmol/mg membrane protein. The GLUT1 content in human erythrocyte membranes has been estimated to be 300 pmol/mg protein(19) . The binding capacity of this site, however, increased in Clone 9 cells after acute treatment with azide to 5-7 pmol/mg membrane protein. This increase is large enough to account for most of the acute increase in glucose transport by azide(5) . Incidentally, the azide treatment also increases CE-insensitive CB binding capacity, and this increase is almost stoichiometric to the increase in glucose-sensitive CB binding capacity. It is interesting in this regard to recall that in human erythrocytes CB binding to site III becomes glucose-sensitive when site II is saturated or selectively removed by extraction with EDTA(18) , suggesting that site III is an altered form of GLUT1 inactivated by molecular association with a cytoskeletal component.
Most revealing and intriguing in the present study is the finding that the CB binding activity of purified GLUT1 is inhibited by cytosol of azide-treated Clone 9 cells. This effect is specific to the cytosol isolated from azide-treated cells and was absent in cytosol from control cells. This effect was largely abolished by the peptide corresponding to the COOH-terminal cytoplasmic domain of GLUT1. This effect was lost after heat-treatment, suggesting that it is due to protein. One simple interpretation of these observations is that there is a cytosolic protein, X, in Clone 9 cells that interacts with the COOH-terminal cytoplasmic domain of GLUT1 to form a reversible X-GLUT1 complex where GLUT1 is incompetent to bind CB and glucose. It is further possible that the total amount of X in Clone 9 cells is rather limited and is available in copy number similar to that of GLUT1, such that practically all of X are in complex with GLUT1 in control cells and that azide treatment effectively dissociates X-GLUT1 complex releasing X almost quantitatively to the cytosol.
The biochemical or physicochemical basis of the ``masking'' and ``unmasking'' of GLUT1 by X proposed above is unknown. X could be an adaptor protein whose physical interaction with GLUT1 makes GLUT1 sterically unable to bind substrate. Alternatively, X could be an enzyme that modulates GLUT1 activity via a covalent change such as phosphorylation and dephosphorylation(28, 29) . The chemical identity of X is unknown at present. It could be an intermediate of glucose metabolism that responds to the metabolic alteration secondary to a decrease in oxidative phosphorylation.
We
have identified three proteins with apparent molecular masses of 28,
70, and 85 kDa in Clone 9 cell cytosol that bind to the COOH-terminal
cytoplasmic domains of glucose transporters expressed as GST fusion
proteins. They did not bind to irrelevant fusion proteins, indicating
that the binding is specific to glucose transporters. The 28- and
70-kDa proteins were labeled by S but not by
P, suggesting they are not phosphoproteins. The 28-kDa
protein bound more to GLUT1 than GLUT4, whereas the 70-kDa protein
binding was more intense to GLUT4. Azide treatment did not affect these
protein bindings significantly. The 85-kDa protein was labeled by
P, indicating that it is a phosphoprotein. The 85-kDa
protein bound only to GLUT4 fusion protein but not to GLUT1 fusion
protein. One cannot rule out the possibility, however, that this
protein may bind to the GLUT1 fusion protein under more appropriate
experimental conditions or that it may bind to intact GLUT1. The
P-labeling of this 85-kDa protein is seen only in
azide-treated cytosol but not in control cytosol, strongly suggesting
that the binding is sensitive to metabolic alteration induced by azide
treatment. This is particularly interesting because the putative
inhibitory factor that inactivates purified GLUT1 glucose binding is
seen only in azide-treated cytosol. More experiments are needed to
establish the functional significance, if any, of these glucose
transporter-binding proteins. These would include purification, partial
sequencing, and cloning of this protein. Once purified protein is
available in a large quantity, its role in GLUT1 modulation would be
directly studied by injecting the protein into Clone 9 cells.