Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3972-3976, August 1979
Cell Biology
Stereospecific hexose transport by membrane vesicles from mouse fibroblasts: Membrane vesicles retain increased hexose transport associated with viral transformation (double isotope assay/initial rate of glucose uptake/simian virus 40-transformed cells)
KEN-ICHI INUI, DAVID E. MOLLER, LOYAL G. TILLOTSON, AND KURT J. ISSELBACHER Department of Medicine, Harvard Medical School and Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Contributed by Kurt J. Isselbacher, June 1, 1979
Membrane vesicles isolated from nontransABSTRACT formed BALB/c 3T3 mouse fibroblasts (3T3) and from those cells transformed by simian virus 40 (SV3T3) displayed carrier-mediated and stereospecific uptake of hexose as measured by the difference between D414C]glucose or its analogues and L[3H]glucose uptake. Stereospecific uptake appeared to be linear for 5 sec and reached a maximum at 5-10 min. Stereospecific D-4[C]glucose uptake, osmotically sensitive and temperature dependent, was inhibited by unlabeled D-glucose or its analogues and was stimulated by the countertransport of accumulated unlabeled D-glucose. As with whole cells, the initial rate of stereospecific uptake by SV3T3 membrane vesicles was approximately 2.5-fold greater than that by 3T3 vesicles. Efflux of preloaded D414Clglucose was also faster from SV3T3 than from 3T3 membrane vesicles. The Km value was 5 mM for both the 3T3 and the SV3T3 membrane vesicles, but the Vmax values were 36 and 86 nmol/mg of protein per min, respectively, suggesting an increase in the number or availability of hexose carriers in transformed cell membranes. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicles. The binding of cytochalasin B to the SV3T3 membrane vesicles was significantly greater than that to 3T3 vesicles. Thus, the membrane vesicles retained many of the features of the altered hexose transport observed in whole cells in association with viral transformation.
An increase in the rate of hexose uptake has been reported as one of the early biochemical events in animal cells after their transformation by oncogenic viruses (1). Numerous studies of whole cells have generally concluded that this increased hexose uptake results from specific increases in the number or availability of functional hexose transport sites in the plasma membrane (2-5). However, it has also been proposed that an increased rate of phosphorylation by transformed cells contributes to the enhanced hexose uptake (6). Because the analysis of transport mechanisms in whole cells is complicated by factors such as (i) the simultaneous metabolism of substrates, (ii) the existence of various intracellular compartments, and (iii) the problems of pool size, it seemed desirable to examine hexose transport changes by using an in vitro membrane vesicle system, which allows separation of membrane events from intracellular change. In this and other laboratories, membrane vesicles prepared from transformed and nontransformed cells have been successfully used for the analysis of transport of amino acids (7-12), phosphate (13, 14), and nucleosides (15). The present study was undertaken to evaluate the effect of transformation on hexose transport by plasma membrane vesicles from BALB/c 3T3 mouse fibroblasts (3T3) and simian virus 40-transformed 3T3 (SV3T3) cells. The results revealed that membrane vesicles prepared from SV3T3 reflected the
changes in hexose uptake observed with whole cells and the increased uptake was due to an increase in Vmax for hexose transport. 'MATERIALS AND METHODS Cell Culture. 3T3 and SV3T3 cells were obtained from the Cell Culture Center of the Massachusetts Institute of Technology. Cells were grown in 490-cm2 roller bottles (Corning) at 370C in Dulbecco's modified Eagle's medium, supplemented with 10% (vol/vol) fetal calf serum. Confluent cells were harvested by scraping with a rubber blade at densities of 1-2 X H0P cells per cm2 for 3T3 and 1-4 X 105 cells per cm2 for SV3T3. Preparation of Membrane Vesicles. Mixed membrane vesicles were prepared as reported (7-9), with some modifications. The cells were suspended in 0.25 M sorbitol/I mM Tris-Hepes, pH 7.5/0.5 mM MgCl2 (buffer A) followed by centrifugation. The packed cell pellet was resuspended in 20 vol of buffer A and placed in a nitrogen cavitation bomb. The mixed membrane pellets, obtained by differential centrifugation of homogenates, were suspended in 0.1 M sorbitol/1 mM Tris-Hepes, pH 7.5 (buffer S) and centrifuged again at 100,000 X g for 60 min. The pellets were resuspended in buffer S to give a final protein concentration of 5-10 mg/ml. This fraction was stored at 40C and used as mixed membrane vesicles within 3 days. Membrane vesicles were prepared simultaneously from 3T3 and SV3T3 cells. Compared with the homogenates, the membrane preparations from 3T3 and SV3T3 were enriched approximately 5-fold for 5'-nucleotidase activity. Transport by Whole Cells. Transport studies were carried out as described (16). Corrections for nonspecific trapping of radioactivity were made by subtracting uptake values at the beginning of the assay. Trypan blue exclusion studies were carried out at the end of the experiments; no significant change in cell viability (>80%) was found between 3T3 and SV3T3 cells. Transport by Membrane Vesicles. Membrane vesicles, dispersed in buffer S or specifically conditioned buffer S, were preincubated for 10 min (1.5-3 mg of protein per ml). In most experiments a double isotope medium was used containing D-[14C]Glc (100 MCi/ml) and L-[3H]Glc (200 ,Ci/ml) (1 Ci = 3.7 X 1010 becquerels). In order to obtain initial uptake values, we adopted a droplet technique whereby two 15-gl drops, one containing membrane vesicle suspension and the other containing labeled substrates in buffer S, were placed close to each other at the bottom of a clear plastic test tube (Falcon, 12 X 75 mm). The reaction was initiated by rapid mixing in a Vortex
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Abbreviations: 3T3, BALB/c 3T3 mouse fibroblasts; SV3T3, simian virus 40-transformed 3T3; Glc, glucose; Gal, galactose; 2-dGlc, 2-
deoxy-D-glucose; 3-OMeGlc, 3-0-nmethyl-D-glucose. 3972
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Inui et al. mixer. At the stated time points, 1 ml of ice-cold. stop solution [buffer S containing,0.1 mM phloretin and 0.1% (vol/vol) ethyl
alcohol] was added into the reaction tube. The tube contents immediately poured onto Millipore nitrocellulose filters (0.45 gim, 2.5 cm diameter) and washed with 5 ml of ice-cold stop solution. Filtration and washing were completed within 10 sec. Dried filters were placed in Instagel (Packard) and radioactivity was determined by scintillation counting. In order to obtain reproducible results and also to determine stereospecific transport, corrections for simple diffusion and nonspecific trapping of hexose were made by subtracting the amount of L-Glc associated with each sample. Background was determined by the addition of 15 Mi of isotope mixture to 1 ml of ice-cold stop solution containing 15 ,ul of diluted membrane vesicles. The incubations were carried out at 25 i 10C except where otherwise specified. Assay of a-aminoisobutyric acid uptake, in buffer S, was performed as described (9). Measurement of Intravesicular Volume. The intravesicular volume (pil/mg of protein) available to hexose was calculated from the equilibrium uptake (pmol/mg of protein) of 1 mM D-Glc or 3-O-methyl-D-glucose (3-OMeQlc) obtained at 20 min (14). Estimates of the intravesicular volume made from D-Glc or 3-OMeGlc were similar, but they varied slightly with each preparation [3T3, 2.2 + 0.2 ,ul/mg of protein (SEM); SV3T3, 2.1 1-0.1 pil/mg (SEM)]. Analytical Methods. Protein was determined by the method of Lowry et al. (17).. Purity of mixed membrane vesicles was checked by measuring the activities of marker enzymes-5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (18) for plasma membrane, NADH-cytochrome c reductase [NADH:(acceptor) oxidoreductase, EC 1.6.99.3] (19) for endoplasmic reticulum, and cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) (20) for mitochondria. Materials. Radioactive materials were purchased from New England Nuclear. Cytochalasin B, cytochrome c, and NADH were obtained from Sigma and phloretin was from ICN Pharmaceuticals.
3973
12k
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RESULTS 2-Deoxy-D-glucose (2-dGlc) Transport by Intact Cells. In order to confirm the difference of transport activity by intact 3T3 and SV3T3 cells used in every preparation of membrane vesicles, the uptakes of 2-dGlc and a-anminoisobutyric acid were studied by suspension assay. As shown in Fig. 1, the initial rate of 2-dGlc uptake was 2- to 3-fold greater with intact SV3T3 compared to 3T3 cells. Cytochalasin B specifically inhibited the uptake of 2-dGlc. The initial rate of. Na+-stimulated a-aminoisobutyric acid uptake by intact SV3T3 was similarly increased (data not shown). Modified Assay for Hexose Transport by Membrane Vesicles. The filtration technique, which was effective for measuring ca-aminoisobutyric acid uptake into vesicles (7-9), had to be modified for measurements of hexose uptake because of the greater variability of uptake values and increased nonspecific trapping of hexose on the filter. Thus, in order to obtain more reproducible results, the following modifications were made: (i) stereospecific uptake was measured by the difference between D-[14C]Glc and L-[3H]Glc uptake; (ii) a stop solution containing phloretin was used; (iii) uptake was assayed as early as 5 sec; and (iv) intravesicular volume was also measured. As shown in Fig. 2A, the transport of D-Glc by the membrane vesicles was time dependent and stereospecific and reached equilibrium within 10 min. Stereospecific uptake of D-Glc as measured by the difference between D-['4C]Glc and L-[3H]Glc uptake appeared to be linear for 5 sec, reached a maximum
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-
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FIG. 1. Time course of 2-dGlc uptake by 3T3 and SV3T3 whole cells. Isolated cells were incubated with 1 mM [14C]2-dGlc (1 mCi/ mmol). Each point represents the mean value of four experiments. 3T3: control (A), 20,qM cytochalasin B (-); SV3T3: control (0), 20 guM cytochalasin B (0).
value at 5-10 min, and then gradually declined, concomitant with a gradual increase of L-Glc levels. In order to examine carrier-mediated hexose transport, we focused largely on the initial rate of stereospecific -uptake, Because mammalian cells, except for intestinal and kidney epithelial cells, transport hexose by facilitated diffusion rather than concentrative uptake' it was desirable to determine intravesicular volume. The intravesicular volume available to hexose could be estimated from the equilibrium D-Glc or 3-OMeGlc value obtained at 20 min. Some results for stereospecific D-Glc transport were therefore expressed in terms of MiM concentration [calculated from the amount of uptake (pmol/mg of protein) and intravesicular volume (,ul/mg of protein)] (Fig. 2A). To minimize the loss of D-Glc from vesicles before filtration, a stop solution containing phloretin was used (Fig. 2B). The initial rate of D-Glc uptake was proportional to the amount of vesicle protein in the range used for these studies (data not shown). As evidence that the- vesicular D-Glc represented uptake, rather than nonspecific binding of D-Glc to the membrane surface, the D-Glc accumulated by the vesicles in 20 min was found to be inversely proportional to the sorbitol concentration (data not shown). When these values were extrapolated to infinite medium osmolarity, there was zero uptake (i.e., no binding). Characteristics of Hexose Transport in Membrane Vesicles. To characterize details of the mechanism of hexose transport and its modification by transformation, stereospecific D-Glc transport in 3T3 and SV3T3 membrane vesicles was examined systematically by studying: (i) time course of uptake, (ii) temperature dependence, (iii) competitive inhibition by hexoses, (iv) inhibition of uptake and efflux by cytochalasin B, and (v) countertransport. The initial rates of stereospecific D-Glc (Fig. 3A), 3-OMeGIc (data not shown), and D-Gal (Fig. 3B) uptakes were approximately 2.5-fold greater for SV3T3 vesicles than for 3T3 vesicles, and thus comparable to the increase in 2-dGIc transport observed with intact SV3T3 cells. The rates of uptake for D-Glc and 3-OMeGlc were faster than that for D-Gal. To verify that metabolic changes were not involved in this hexose uptake system, the nonmetabolizable glucose analogue 3-OMeGlc was used. The equilibrium uptake values for D-Glc and 3-OMeGlc were similar.
Cell Biology: Inui et al.
3974
Proc. Natl. Acad. Sci. USA 76 (1979) B
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Time, min Time, min FIG. 2. Time course of Glc uptake (A) by 3T3 membrane vesicles and effect of stop solution (B) on intravesicular Glc retention. (A) Membrane vesicles were preincubated in buffer S for 10 min. The vesicles (15 ,ul, 31 jig of protein) were incubated with isotope mixture (15 ,ul) containing 1)-['4CJGlc (50 mCi/mnmol) and L-[3H]Glc (100 mCi/mmol) (final concentration 1 mM). The uptake reactions were stopped at the stated time points by the addition of ice-cold buffer S containing 0.1 mM phloretin. Initial time points were taken at 3, 5, and 10 sec. D-Glc (0), L-Glc (A), and stereospecific D-Glc uptake (D) were calculated as described in the text. The intravesicular volume, used in calculation of intravesicular concentration, was estimated as 2.2 Al/mg of protein. (B) The vesicles were incubated with isotope mixture for 20 min as described above-and then filtered at the stated time points after the addition of ice-cold buffer S with (-) or without (3) 0.1 mM phloretin. Each point represents the mean value of two experiments.
The initial rates of stereospecific D-Glc uptake by 3T3 and SV3T3 membrane vesicles were temperature dependent. The activation energies for 3T3 and SV3T3 vesicles, calculated from Arrhenius plots, were 5.8' and 8.9 kcal/mol, respectively (data not shown) (1 kcal = 4.184 kj). The nature of the substrate specificity of hexose transport system was examined by competitive inhibition studies (Table 1). D-Glc and 2-dGlc had the strongest inhibitory effect on the stereospecific uptake of D-Glc, followed by 3-OMeGlc and D-Gal. There was no inhibition by L-Glc. Cytochalasin B has been shown to be a potent inhibitor of sugar transport but not phosphorylation in a wide variety of cell types (21, 22). As shown in Fig. 4A, stereospecific D-Glc uptake was strongly inhibited by cytochalasin B, but the extent of the inhibitory effect on the 3T3 membrane vesicles was slightly greater than that for SV3T3 vesicles. The efflux of D-Glc from SV3T3 membrane vesicles was also faster than that from 3T3 (Fig. 4B) and the rate of efflux decreased when the incubation medium contained-cytochalasin B. Vesicles preloaded with high concentration of unlabeled D-Glc showed enhancement of stereospecific D-[l4C]Glc
countertransport, compared with controls (not preloaded) (Fig. 5). The extents of the increases in the initial rates were similar for 3T3 and SV3T3 membrane vesicles. Kinetic Analysis of Stereospecific D-Glc Transport and Inhibition by Cytochalasin B. Lineweaver-Burk plots (Fig. 6A) of the initial rates of stereospecific D-Glc uptake at substrate concentrations between 0.5 and 20 mM revealed that the Km (5 mM) for D-Glc transport was the same for 3T3 and SV3T3 membrane vesicles. However, the Vm, values for stereospecific D-Glc uptake were greater for SV3T3 than for 3T3 vesicles (86 vs. 36 nmol/mg of protein per min, respectively), suggesting an increase in the number or availability of hexose carriers in the transformed cell membranes. The inhibition of stereospecific D-Glc uptake by cytochalasin B showed that this inhibition Table 1. Competitive inhibition by unlabeled hexose of stereospecific l)-Glc uptake by membrane vesicles from 3T3 and SV3T3 cells Uptake Unlabeled hexose
3T3 E
None i-Glc
B
A
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Vesicle
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5 sec
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273 ± 2 336 + 2 475 + 6
control
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stereospecific L)-Glc (A) and D-Gal (B)
uptake by 3T3 (-) and SV,3T3 (0) membrane vesicles. Stereospecific uptake was determined at 1 mM hexose as described for Fig. 2. The intravesicular volumes were 2.4 pl/mg of protein for 3T3 and 2.5 Ail/mg for SV3T3. Each point represents the mean of two experiments.
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100 64 63
55r3 + 9 71 619 + 14 86 753 + 12 D-Gal 105 L,-Glc 922 ± 26 The vesicles were incubated with the isotope mixture (I)-Glc, l-Glc. 1 mM each) containing additional unlabeled hexose at 5 mM. Stereospecific uptake at 5 sec was determined as described for Fig. 2. The intravesicular volumes were 2.4 pl/mg of protein for 3T3 and 2.1 Al/mg for SV:3T3. Each value represents the mean + SEM of three experiments.
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Inui et al.
3975
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FIG. 4. Inhibition of D-Glc uptake (A) and efflux (B) by cytochalasin B in 3T3 and SV3T3 membrane vesicles. (A) The aliquots (15 Ml) of membrane vesicles, dispersed in buffer S with or without 40 gM cytochalasin B, were incubated with isotope mixture (15,ul) at a final concentration of 1 mM as described for Fig. 2. The intravesicular volumes were 1.3 Ml/mg of protein for 3T3 and 2.3 Ml/mg for SV3T3. (B) Vesicles were incubated with 1 mM D-[14C]Glc (100 mCi/mmol) for 20 min. Then the aliquots (15 pl) were diluted into buffer S (750 Ml) with or without cytochalasin B. The intravesicular volumes were 2.4 MI/mg of protein for 3T3 and 2.1 Ml/mg for SV3T3. Each point represents the mean value of two experiments. 3T3: control (A), 20 MM cytochalasin B (-); SV3T3: control (0), 20 MM cytochalasin B (0).
was competitive (Fig. 6B), with apparent dissociation constants
(Ki) for the inhibitor-carrier complex of 0.3 MuM in both 3T3
and SV3T3. Cytochalasin B Binding to the Membrane Vesicles. In order to examine the relationship between cytochalasin B binding and hexose transport with membrane vesicles, cytochalasin B binding studies were performed by the filtration method in a manner similar to hexose transport assays. As shown in Fig. 7, there was a significant increase in [3H]cytochalasin B binding to SV3T3 compared with 3T3 membrane vesicles. DISCUSSION The present results demonstrate that plasma membrane vesicles derived from mouse fibroblasts retain the properties of carrier-mediated hexose transport. When D-Glc, 3-OMeGlc, or D-Gal was used as a substrate, the membrane vesicles from SV3T3 cells were found to have an increase in stereospecific
2 0 1/D-Glc, mM'
FIG. 6. Lineweaver-Burk plots of initial rates of stereospecific D-Glc uptake by 3T3 and SV3T3 membrane vesicles. Stereospecific uptake for 5 sec at concentrations between 0.5 and 20 mM was determined in the absence (A) and the presence (B) of cytochalasin B (0.2 MM) as described for Fig. 2. (A) 3T3 (0); SV3T3 (0). (B) Control (0, 0); cytochalasin B (A). The intravesicular volumes were 2.4 ,l/mg of protein for 3T3 and 2.5 Ml/mg for SV3T3. Each point represents the mean value of three experiments.
hexose uptake compared with 3T3 vesicles, and thus reflected the increase in hexose transport observed with whole SV3T3 cells. Comparable results showing enhancement of amino acid uptake have previously been reported for SV3T3 membrane vesicles (8, 10). Because hexose transport in fibroblasts occurs by facilitated diffusion, it is important to estimate initial rate of uptake with great accuracy. In our studies, this was accomplished by (i) measuring stereospecific uptake, (ii) using double labeling, and (iii) measuring uptake as early as 5 see with a droplet technique. Furthermore, the use of a stop solution containing phloretin resulted in a lower background value and decreased the efflux of D-Glc in the washing buffer, compared with a stop solution without an inhibitor. In order to correct for the small difference in vesicular volume, some uptake results were expressed on the basis of the intravesicular concentration. The results of this study show that the stereospecific D-Glc uptake in the membrane vesicles of fibroblasts satisfies some of the criteria for carrier-mediated process; namely, the process is saturable, temperature dependent, inhibited by D-Glc analogues and inhibitors such as cytochalasin B and phloretin, and undergoes a countertransport effect. Kinetic analysis of stereospecific uptake of D-Glc revealed that the increased uptake
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Time, min FIG. 5. Countertransport effects on stereospecific D-Glc uptake. Vesicles were preincubated with (0, A) or without (0, a) 10 mM unlabeled D-Glc for 10 min, and then the aliquots (15Ml1) (3T3,A, A; SV3T3, 0, 0) were incubated with the isotope mixture (150 Ml) containing 1 mM D-[14C]Glc (10 mCi/mmol) and 1 mM L-[3HIGlc (20 mCi/mmol) during the indicated periods. The intravesicular volumes were 2.3 ,ul/mg of protein for 3T3 and 1.8 Ml/mg for SV3T3. Each point represents the mean value of two experiments.
m
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FIG. 7. Scatchard analysis of cytochalasin B binding to membrane vesicles. [3HlCytochalasin B (15 Ci/mmol) was incubated with 3T3 (-) and SV3T3 (0) membrane vesicles for 30 min over a 3-13,000 nM range of free cytochalasin B. Each point represents the mean of five experiments.
3976
Cell Biology: Inui et al.
was associated with an increase in Vmax, the Km remaining constant. Cytochalasin B competitively inhibited stereospecific uptake, the Ki values being similar in both types of membrane vesicles, suggesting that there was no modification of the transport mechanism due to viral transformation. Recently, equilibrium 13Hjcytochalasin B binding to adipose cell plasma membranes was described and proposed as a means for quantitating the number of functional glucose transport systems in order to elucidate the mechanism of insulin action on glucose transport (23). In the present experiments, [3HJcytochalasin B binding studies showed the binding to SV3T3 membrane vesicles to be significantly greater than to 3T3 vesicles. Colby and Romano (6) reported that enhanced cellular hexose uptake resulting from simian virus 40 transformation was not primarily due to increased activity of glucose carriers but more likely was the result of increased intracellular glucose phosphorylation. The present data, however, suggest that the alteration in hexose transport by transformed cells must also involve changes at the membrane level and that the greater Vmax is due to an increase in the number of transport sites. It is noteworthy that Lever (24, 25) did not observe increased D-Glc uptake in the membrane vesicles from transformed cells; we believe this may be the result of differences in transport assay techniques. Furthermore, in preliminary studies with chicken embryo fibroblasts infected with the temperature-sensitive mutant (TS-68) of Rous sarcoma virus, we observed similar results. The membrane vesicles from TS-68-infected fibroblasts cultured at 370C (transformed) showed a higher rate of stereospecific D-Glc uptake than the vesicles from fibroblasts cultured at 41'C (unpublished data). These findings are consistent with those of Perdue and coworkers with whole cells (4) and their recent report with membrane vesicles (26). In conclusion, we have modified the method for measuring hexose transport by membrane vesicles by using double labeling, early time points, stop solution containing phloretin, and determination of intravesicular concentration. With this assay technique we have been able to demonstrate that membrane vesicles isolated from 3T3 cells and from SV3T3 cells catalyze carrier-mediated transport of hexose and that the enhanced hexose transport activity observed with transformed cells can also be shown in the membrane vesicles derived from them.
Proc. Natl. Acad. Sci. USA 76 (1979) This investigation was supported in part by grants from the National Institutes of Health (AM-01392, AM-03014, and CA-14294). 1. Hatanaka, M. (1974) Biochim. Biophys. Acta 355, 77-104. 2. Isselbacher, K. J. (1972) Proc. Natl. Acad. Sci. USA 69, 585589. 3. Weber, M. J. (1973) J. Biol. Chem. 248,2978--2983. 4. Kletzien, R. R. & Perdue, J. F. (1974) J. Biol. Chem. 249, 3375-3382. 5. Singh, M., Singh, V. N., August, J. T. & Horecker, 13. L. (1978) J. Cell. Physiol. 97, 285--292. 6. Colby, C. & Romano, A. H. (1975) J. Cell. Physiol. 85, 15-24. 7. Quinlan, D. C., Parnes, J. R., Shalom, R., Garvey, T. Q., III, Isselbacher, K. J. & Hochstadt, J. (1976) Proc. Natl. Acad. Sci. USA 73, 1631-1635. 8. Parnes, J. R., Garvey, T. Q., III & Isselbacher, K. J. (1976) J. Cell. Physiol. 89, 789-794. 9. Nishino, H., Schiller, R. M., Parnes, J. R. & Isselbacher, K. J. (1978) Proc. Natl. Acad. Sci. USA 75,2329-2332. 10. Lever, J. E. (1976) Proc. Natl. Acad. Sci. USA 73,2614-2618. 11. Lever, J. E. (1977) J. Biol. Chem. 252, 1990-1997. 12. Hamilton, R. T. & Nilsen-Hamilton, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1907-1911. 13. Lever, J. E. (1978) J. Biol. Chem. 253, 2081-2084. 14. Hamilton, R. T. & Nilsen-Hamilton, M. (1978) J. Biol. Chem. 253,8247-8256. 15. Quinlan, D. C. & Hochstadt, J. (1976) J. Biol. Chem. 251, 344-354. 16. Nishino, H., Christopher, C. W., Schiller, R. M., Gammon, M. T., Ullrey, D. & Isselbacher, K. J. (1978) Proc. Natl. Acad. Sci. USA 75,5048-5051. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. Avruch, J. & Wallach, D. F. H. (1971) Biochim. Biophys. Acta
233,334-347. 19. Mackler, B. (1967) Methods Enzymol. 10, 551-553. 20. Peters, T. J., Muller, M. & de Duve, C. (1972) J. Exp. Med. 136, 1117-1139. 21. Czech, M. P., Lynn, D. G. & Lynn, W. S. (1973) J. Biol. Chem. 22.
248,3636-3641. Kletzien, R. R. & Perdue, J. F. (1973) J. Biol. Chem. 248, 711-
719. 23. Wardzala, L. J., Cushman, S. W. & Salans, L. 13. (1978) J. Biol.
Chem. 253, 8002-8005. 24. Lever, J. E. (1976) J. Cell. Physiol. 89, 779-787. 25. Lever, J. E. (1979) J. Biol. Chem. 254, 2961-2967. 26. Zala, C. A. & Perdue, J. R. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38, 245 (abstr.).
Cell Biology
Stereospecific hexose transport by membrane vesicles from mouse fibroblasts: Membrane vesicles retain increased hexose transport associated with viral transformation (double isotope assay/initial rate of glucose uptake/simian virus 40-transformed cells)
KEN-ICHI INUI, DAVID E. MOLLER, LOYAL G. TILLOTSON, AND KURT J. ISSELBACHER Department of Medicine, Harvard Medical School and Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Contributed by Kurt J. Isselbacher, June 1, 1979
Membrane vesicles isolated from nontransABSTRACT formed BALB/c 3T3 mouse fibroblasts (3T3) and from those cells transformed by simian virus 40 (SV3T3) displayed carrier-mediated and stereospecific uptake of hexose as measured by the difference between D414C]glucose or its analogues and L[3H]glucose uptake. Stereospecific uptake appeared to be linear for 5 sec and reached a maximum at 5-10 min. Stereospecific D-4[C]glucose uptake, osmotically sensitive and temperature dependent, was inhibited by unlabeled D-glucose or its analogues and was stimulated by the countertransport of accumulated unlabeled D-glucose. As with whole cells, the initial rate of stereospecific uptake by SV3T3 membrane vesicles was approximately 2.5-fold greater than that by 3T3 vesicles. Efflux of preloaded D414Clglucose was also faster from SV3T3 than from 3T3 membrane vesicles. The Km value was 5 mM for both the 3T3 and the SV3T3 membrane vesicles, but the Vmax values were 36 and 86 nmol/mg of protein per min, respectively, suggesting an increase in the number or availability of hexose carriers in transformed cell membranes. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicles. The binding of cytochalasin B to the SV3T3 membrane vesicles was significantly greater than that to 3T3 vesicles. Thus, the membrane vesicles retained many of the features of the altered hexose transport observed in whole cells in association with viral transformation.
An increase in the rate of hexose uptake has been reported as one of the early biochemical events in animal cells after their transformation by oncogenic viruses (1). Numerous studies of whole cells have generally concluded that this increased hexose uptake results from specific increases in the number or availability of functional hexose transport sites in the plasma membrane (2-5). However, it has also been proposed that an increased rate of phosphorylation by transformed cells contributes to the enhanced hexose uptake (6). Because the analysis of transport mechanisms in whole cells is complicated by factors such as (i) the simultaneous metabolism of substrates, (ii) the existence of various intracellular compartments, and (iii) the problems of pool size, it seemed desirable to examine hexose transport changes by using an in vitro membrane vesicle system, which allows separation of membrane events from intracellular change. In this and other laboratories, membrane vesicles prepared from transformed and nontransformed cells have been successfully used for the analysis of transport of amino acids (7-12), phosphate (13, 14), and nucleosides (15). The present study was undertaken to evaluate the effect of transformation on hexose transport by plasma membrane vesicles from BALB/c 3T3 mouse fibroblasts (3T3) and simian virus 40-transformed 3T3 (SV3T3) cells. The results revealed that membrane vesicles prepared from SV3T3 reflected the
changes in hexose uptake observed with whole cells and the increased uptake was due to an increase in Vmax for hexose transport. 'MATERIALS AND METHODS Cell Culture. 3T3 and SV3T3 cells were obtained from the Cell Culture Center of the Massachusetts Institute of Technology. Cells were grown in 490-cm2 roller bottles (Corning) at 370C in Dulbecco's modified Eagle's medium, supplemented with 10% (vol/vol) fetal calf serum. Confluent cells were harvested by scraping with a rubber blade at densities of 1-2 X H0P cells per cm2 for 3T3 and 1-4 X 105 cells per cm2 for SV3T3. Preparation of Membrane Vesicles. Mixed membrane vesicles were prepared as reported (7-9), with some modifications. The cells were suspended in 0.25 M sorbitol/I mM Tris-Hepes, pH 7.5/0.5 mM MgCl2 (buffer A) followed by centrifugation. The packed cell pellet was resuspended in 20 vol of buffer A and placed in a nitrogen cavitation bomb. The mixed membrane pellets, obtained by differential centrifugation of homogenates, were suspended in 0.1 M sorbitol/1 mM Tris-Hepes, pH 7.5 (buffer S) and centrifuged again at 100,000 X g for 60 min. The pellets were resuspended in buffer S to give a final protein concentration of 5-10 mg/ml. This fraction was stored at 40C and used as mixed membrane vesicles within 3 days. Membrane vesicles were prepared simultaneously from 3T3 and SV3T3 cells. Compared with the homogenates, the membrane preparations from 3T3 and SV3T3 were enriched approximately 5-fold for 5'-nucleotidase activity. Transport by Whole Cells. Transport studies were carried out as described (16). Corrections for nonspecific trapping of radioactivity were made by subtracting uptake values at the beginning of the assay. Trypan blue exclusion studies were carried out at the end of the experiments; no significant change in cell viability (>80%) was found between 3T3 and SV3T3 cells. Transport by Membrane Vesicles. Membrane vesicles, dispersed in buffer S or specifically conditioned buffer S, were preincubated for 10 min (1.5-3 mg of protein per ml). In most experiments a double isotope medium was used containing D-[14C]Glc (100 MCi/ml) and L-[3H]Glc (200 ,Ci/ml) (1 Ci = 3.7 X 1010 becquerels). In order to obtain initial uptake values, we adopted a droplet technique whereby two 15-gl drops, one containing membrane vesicle suspension and the other containing labeled substrates in buffer S, were placed close to each other at the bottom of a clear plastic test tube (Falcon, 12 X 75 mm). The reaction was initiated by rapid mixing in a Vortex
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Abbreviations: 3T3, BALB/c 3T3 mouse fibroblasts; SV3T3, simian virus 40-transformed 3T3; Glc, glucose; Gal, galactose; 2-dGlc, 2-
deoxy-D-glucose; 3-OMeGlc, 3-0-nmethyl-D-glucose. 3972
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Inui et al. mixer. At the stated time points, 1 ml of ice-cold. stop solution [buffer S containing,0.1 mM phloretin and 0.1% (vol/vol) ethyl
alcohol] was added into the reaction tube. The tube contents immediately poured onto Millipore nitrocellulose filters (0.45 gim, 2.5 cm diameter) and washed with 5 ml of ice-cold stop solution. Filtration and washing were completed within 10 sec. Dried filters were placed in Instagel (Packard) and radioactivity was determined by scintillation counting. In order to obtain reproducible results and also to determine stereospecific transport, corrections for simple diffusion and nonspecific trapping of hexose were made by subtracting the amount of L-Glc associated with each sample. Background was determined by the addition of 15 Mi of isotope mixture to 1 ml of ice-cold stop solution containing 15 ,ul of diluted membrane vesicles. The incubations were carried out at 25 i 10C except where otherwise specified. Assay of a-aminoisobutyric acid uptake, in buffer S, was performed as described (9). Measurement of Intravesicular Volume. The intravesicular volume (pil/mg of protein) available to hexose was calculated from the equilibrium uptake (pmol/mg of protein) of 1 mM D-Glc or 3-O-methyl-D-glucose (3-OMeQlc) obtained at 20 min (14). Estimates of the intravesicular volume made from D-Glc or 3-OMeGlc were similar, but they varied slightly with each preparation [3T3, 2.2 + 0.2 ,ul/mg of protein (SEM); SV3T3, 2.1 1-0.1 pil/mg (SEM)]. Analytical Methods. Protein was determined by the method of Lowry et al. (17).. Purity of mixed membrane vesicles was checked by measuring the activities of marker enzymes-5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (18) for plasma membrane, NADH-cytochrome c reductase [NADH:(acceptor) oxidoreductase, EC 1.6.99.3] (19) for endoplasmic reticulum, and cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) (20) for mitochondria. Materials. Radioactive materials were purchased from New England Nuclear. Cytochalasin B, cytochrome c, and NADH were obtained from Sigma and phloretin was from ICN Pharmaceuticals.
3973
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RESULTS 2-Deoxy-D-glucose (2-dGlc) Transport by Intact Cells. In order to confirm the difference of transport activity by intact 3T3 and SV3T3 cells used in every preparation of membrane vesicles, the uptakes of 2-dGlc and a-anminoisobutyric acid were studied by suspension assay. As shown in Fig. 1, the initial rate of 2-dGlc uptake was 2- to 3-fold greater with intact SV3T3 compared to 3T3 cells. Cytochalasin B specifically inhibited the uptake of 2-dGlc. The initial rate of. Na+-stimulated a-aminoisobutyric acid uptake by intact SV3T3 was similarly increased (data not shown). Modified Assay for Hexose Transport by Membrane Vesicles. The filtration technique, which was effective for measuring ca-aminoisobutyric acid uptake into vesicles (7-9), had to be modified for measurements of hexose uptake because of the greater variability of uptake values and increased nonspecific trapping of hexose on the filter. Thus, in order to obtain more reproducible results, the following modifications were made: (i) stereospecific uptake was measured by the difference between D-[14C]Glc and L-[3H]Glc uptake; (ii) a stop solution containing phloretin was used; (iii) uptake was assayed as early as 5 sec; and (iv) intravesicular volume was also measured. As shown in Fig. 2A, the transport of D-Glc by the membrane vesicles was time dependent and stereospecific and reached equilibrium within 10 min. Stereospecific uptake of D-Glc as measured by the difference between D-['4C]Glc and L-[3H]Glc uptake appeared to be linear for 5 sec, reached a maximum
10 *; .W 0
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-
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FIG. 1. Time course of 2-dGlc uptake by 3T3 and SV3T3 whole cells. Isolated cells were incubated with 1 mM [14C]2-dGlc (1 mCi/ mmol). Each point represents the mean value of four experiments. 3T3: control (A), 20,qM cytochalasin B (-); SV3T3: control (0), 20 guM cytochalasin B (0).
value at 5-10 min, and then gradually declined, concomitant with a gradual increase of L-Glc levels. In order to examine carrier-mediated hexose transport, we focused largely on the initial rate of stereospecific -uptake, Because mammalian cells, except for intestinal and kidney epithelial cells, transport hexose by facilitated diffusion rather than concentrative uptake' it was desirable to determine intravesicular volume. The intravesicular volume available to hexose could be estimated from the equilibrium D-Glc or 3-OMeGlc value obtained at 20 min. Some results for stereospecific D-Glc transport were therefore expressed in terms of MiM concentration [calculated from the amount of uptake (pmol/mg of protein) and intravesicular volume (,ul/mg of protein)] (Fig. 2A). To minimize the loss of D-Glc from vesicles before filtration, a stop solution containing phloretin was used (Fig. 2B). The initial rate of D-Glc uptake was proportional to the amount of vesicle protein in the range used for these studies (data not shown). As evidence that the- vesicular D-Glc represented uptake, rather than nonspecific binding of D-Glc to the membrane surface, the D-Glc accumulated by the vesicles in 20 min was found to be inversely proportional to the sorbitol concentration (data not shown). When these values were extrapolated to infinite medium osmolarity, there was zero uptake (i.e., no binding). Characteristics of Hexose Transport in Membrane Vesicles. To characterize details of the mechanism of hexose transport and its modification by transformation, stereospecific D-Glc transport in 3T3 and SV3T3 membrane vesicles was examined systematically by studying: (i) time course of uptake, (ii) temperature dependence, (iii) competitive inhibition by hexoses, (iv) inhibition of uptake and efflux by cytochalasin B, and (v) countertransport. The initial rates of stereospecific D-Glc (Fig. 3A), 3-OMeGIc (data not shown), and D-Gal (Fig. 3B) uptakes were approximately 2.5-fold greater for SV3T3 vesicles than for 3T3 vesicles, and thus comparable to the increase in 2-dGIc transport observed with intact SV3T3 cells. The rates of uptake for D-Glc and 3-OMeGlc were faster than that for D-Gal. To verify that metabolic changes were not involved in this hexose uptake system, the nonmetabolizable glucose analogue 3-OMeGlc was used. The equilibrium uptake values for D-Glc and 3-OMeGlc were similar.
Cell Biology: Inui et al.
3974
Proc. Natl. Acad. Sci. USA 76 (1979) B
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Time, min Time, min FIG. 2. Time course of Glc uptake (A) by 3T3 membrane vesicles and effect of stop solution (B) on intravesicular Glc retention. (A) Membrane vesicles were preincubated in buffer S for 10 min. The vesicles (15 ,ul, 31 jig of protein) were incubated with isotope mixture (15 ,ul) containing 1)-['4CJGlc (50 mCi/mnmol) and L-[3H]Glc (100 mCi/mmol) (final concentration 1 mM). The uptake reactions were stopped at the stated time points by the addition of ice-cold buffer S containing 0.1 mM phloretin. Initial time points were taken at 3, 5, and 10 sec. D-Glc (0), L-Glc (A), and stereospecific D-Glc uptake (D) were calculated as described in the text. The intravesicular volume, used in calculation of intravesicular concentration, was estimated as 2.2 Al/mg of protein. (B) The vesicles were incubated with isotope mixture for 20 min as described above-and then filtered at the stated time points after the addition of ice-cold buffer S with (-) or without (3) 0.1 mM phloretin. Each point represents the mean value of two experiments.
The initial rates of stereospecific D-Glc uptake by 3T3 and SV3T3 membrane vesicles were temperature dependent. The activation energies for 3T3 and SV3T3 vesicles, calculated from Arrhenius plots, were 5.8' and 8.9 kcal/mol, respectively (data not shown) (1 kcal = 4.184 kj). The nature of the substrate specificity of hexose transport system was examined by competitive inhibition studies (Table 1). D-Glc and 2-dGlc had the strongest inhibitory effect on the stereospecific uptake of D-Glc, followed by 3-OMeGlc and D-Gal. There was no inhibition by L-Glc. Cytochalasin B has been shown to be a potent inhibitor of sugar transport but not phosphorylation in a wide variety of cell types (21, 22). As shown in Fig. 4A, stereospecific D-Glc uptake was strongly inhibited by cytochalasin B, but the extent of the inhibitory effect on the 3T3 membrane vesicles was slightly greater than that for SV3T3 vesicles. The efflux of D-Glc from SV3T3 membrane vesicles was also faster than that from 3T3 (Fig. 4B) and the rate of efflux decreased when the incubation medium contained-cytochalasin B. Vesicles preloaded with high concentration of unlabeled D-Glc showed enhancement of stereospecific D-[l4C]Glc
countertransport, compared with controls (not preloaded) (Fig. 5). The extents of the increases in the initial rates were similar for 3T3 and SV3T3 membrane vesicles. Kinetic Analysis of Stereospecific D-Glc Transport and Inhibition by Cytochalasin B. Lineweaver-Burk plots (Fig. 6A) of the initial rates of stereospecific D-Glc uptake at substrate concentrations between 0.5 and 20 mM revealed that the Km (5 mM) for D-Glc transport was the same for 3T3 and SV3T3 membrane vesicles. However, the Vm, values for stereospecific D-Glc uptake were greater for SV3T3 than for 3T3 vesicles (86 vs. 36 nmol/mg of protein per min, respectively), suggesting an increase in the number or availability of hexose carriers in the transformed cell membranes. The inhibition of stereospecific D-Glc uptake by cytochalasin B showed that this inhibition Table 1. Competitive inhibition by unlabeled hexose of stereospecific l)-Glc uptake by membrane vesicles from 3T3 and SV3T3 cells Uptake Unlabeled hexose
3T3 E
None i-Glc
B
A
added
Vesicle
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-Glc
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5 sec
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273 ± 2 336 + 2 475 + 6
control
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stereospecific L)-Glc (A) and D-Gal (B)
uptake by 3T3 (-) and SV,3T3 (0) membrane vesicles. Stereospecific uptake was determined at 1 mM hexose as described for Fig. 2. The intravesicular volumes were 2.4 pl/mg of protein for 3T3 and 2.5 Ail/mg for SV3T3. Each point represents the mean of two experiments.
875 557
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100 64 63
55r3 + 9 71 619 + 14 86 753 + 12 D-Gal 105 L,-Glc 922 ± 26 The vesicles were incubated with the isotope mixture (I)-Glc, l-Glc. 1 mM each) containing additional unlabeled hexose at 5 mM. Stereospecific uptake at 5 sec was determined as described for Fig. 2. The intravesicular volumes were 2.4 pl/mg of protein for 3T3 and 2.1 Al/mg for SV:3T3. Each value represents the mean + SEM of three experiments.
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Inui et al.
3975
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FIG. 4. Inhibition of D-Glc uptake (A) and efflux (B) by cytochalasin B in 3T3 and SV3T3 membrane vesicles. (A) The aliquots (15 Ml) of membrane vesicles, dispersed in buffer S with or without 40 gM cytochalasin B, were incubated with isotope mixture (15,ul) at a final concentration of 1 mM as described for Fig. 2. The intravesicular volumes were 1.3 Ml/mg of protein for 3T3 and 2.3 Ml/mg for SV3T3. (B) Vesicles were incubated with 1 mM D-[14C]Glc (100 mCi/mmol) for 20 min. Then the aliquots (15 pl) were diluted into buffer S (750 Ml) with or without cytochalasin B. The intravesicular volumes were 2.4 MI/mg of protein for 3T3 and 2.1 Ml/mg for SV3T3. Each point represents the mean value of two experiments. 3T3: control (A), 20 MM cytochalasin B (-); SV3T3: control (0), 20 MM cytochalasin B (0).
was competitive (Fig. 6B), with apparent dissociation constants
(Ki) for the inhibitor-carrier complex of 0.3 MuM in both 3T3
and SV3T3. Cytochalasin B Binding to the Membrane Vesicles. In order to examine the relationship between cytochalasin B binding and hexose transport with membrane vesicles, cytochalasin B binding studies were performed by the filtration method in a manner similar to hexose transport assays. As shown in Fig. 7, there was a significant increase in [3H]cytochalasin B binding to SV3T3 compared with 3T3 membrane vesicles. DISCUSSION The present results demonstrate that plasma membrane vesicles derived from mouse fibroblasts retain the properties of carrier-mediated hexose transport. When D-Glc, 3-OMeGlc, or D-Gal was used as a substrate, the membrane vesicles from SV3T3 cells were found to have an increase in stereospecific
2 0 1/D-Glc, mM'
FIG. 6. Lineweaver-Burk plots of initial rates of stereospecific D-Glc uptake by 3T3 and SV3T3 membrane vesicles. Stereospecific uptake for 5 sec at concentrations between 0.5 and 20 mM was determined in the absence (A) and the presence (B) of cytochalasin B (0.2 MM) as described for Fig. 2. (A) 3T3 (0); SV3T3 (0). (B) Control (0, 0); cytochalasin B (A). The intravesicular volumes were 2.4 ,l/mg of protein for 3T3 and 2.5 Ml/mg for SV3T3. Each point represents the mean value of three experiments.
hexose uptake compared with 3T3 vesicles, and thus reflected the increase in hexose transport observed with whole SV3T3 cells. Comparable results showing enhancement of amino acid uptake have previously been reported for SV3T3 membrane vesicles (8, 10). Because hexose transport in fibroblasts occurs by facilitated diffusion, it is important to estimate initial rate of uptake with great accuracy. In our studies, this was accomplished by (i) measuring stereospecific uptake, (ii) using double labeling, and (iii) measuring uptake as early as 5 see with a droplet technique. Furthermore, the use of a stop solution containing phloretin resulted in a lower background value and decreased the efflux of D-Glc in the washing buffer, compared with a stop solution without an inhibitor. In order to correct for the small difference in vesicular volume, some uptake results were expressed on the basis of the intravesicular concentration. The results of this study show that the stereospecific D-Glc uptake in the membrane vesicles of fibroblasts satisfies some of the criteria for carrier-mediated process; namely, the process is saturable, temperature dependent, inhibited by D-Glc analogues and inhibitors such as cytochalasin B and phloretin, and undergoes a countertransport effect. Kinetic analysis of stereospecific uptake of D-Glc revealed that the increased uptake
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Time, min FIG. 5. Countertransport effects on stereospecific D-Glc uptake. Vesicles were preincubated with (0, A) or without (0, a) 10 mM unlabeled D-Glc for 10 min, and then the aliquots (15Ml1) (3T3,A, A; SV3T3, 0, 0) were incubated with the isotope mixture (150 Ml) containing 1 mM D-[14C]Glc (10 mCi/mmol) and 1 mM L-[3HIGlc (20 mCi/mmol) during the indicated periods. The intravesicular volumes were 2.3 ,ul/mg of protein for 3T3 and 1.8 Ml/mg for SV3T3. Each point represents the mean value of two experiments.
m
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FIG. 7. Scatchard analysis of cytochalasin B binding to membrane vesicles. [3HlCytochalasin B (15 Ci/mmol) was incubated with 3T3 (-) and SV3T3 (0) membrane vesicles for 30 min over a 3-13,000 nM range of free cytochalasin B. Each point represents the mean of five experiments.
3976
Cell Biology: Inui et al.
was associated with an increase in Vmax, the Km remaining constant. Cytochalasin B competitively inhibited stereospecific uptake, the Ki values being similar in both types of membrane vesicles, suggesting that there was no modification of the transport mechanism due to viral transformation. Recently, equilibrium 13Hjcytochalasin B binding to adipose cell plasma membranes was described and proposed as a means for quantitating the number of functional glucose transport systems in order to elucidate the mechanism of insulin action on glucose transport (23). In the present experiments, [3HJcytochalasin B binding studies showed the binding to SV3T3 membrane vesicles to be significantly greater than to 3T3 vesicles. Colby and Romano (6) reported that enhanced cellular hexose uptake resulting from simian virus 40 transformation was not primarily due to increased activity of glucose carriers but more likely was the result of increased intracellular glucose phosphorylation. The present data, however, suggest that the alteration in hexose transport by transformed cells must also involve changes at the membrane level and that the greater Vmax is due to an increase in the number of transport sites. It is noteworthy that Lever (24, 25) did not observe increased D-Glc uptake in the membrane vesicles from transformed cells; we believe this may be the result of differences in transport assay techniques. Furthermore, in preliminary studies with chicken embryo fibroblasts infected with the temperature-sensitive mutant (TS-68) of Rous sarcoma virus, we observed similar results. The membrane vesicles from TS-68-infected fibroblasts cultured at 370C (transformed) showed a higher rate of stereospecific D-Glc uptake than the vesicles from fibroblasts cultured at 41'C (unpublished data). These findings are consistent with those of Perdue and coworkers with whole cells (4) and their recent report with membrane vesicles (26). In conclusion, we have modified the method for measuring hexose transport by membrane vesicles by using double labeling, early time points, stop solution containing phloretin, and determination of intravesicular concentration. With this assay technique we have been able to demonstrate that membrane vesicles isolated from 3T3 cells and from SV3T3 cells catalyze carrier-mediated transport of hexose and that the enhanced hexose transport activity observed with transformed cells can also be shown in the membrane vesicles derived from them.
Proc. Natl. Acad. Sci. USA 76 (1979) This investigation was supported in part by grants from the National Institutes of Health (AM-01392, AM-03014, and CA-14294). 1. Hatanaka, M. (1974) Biochim. Biophys. Acta 355, 77-104. 2. Isselbacher, K. J. (1972) Proc. Natl. Acad. Sci. USA 69, 585589. 3. Weber, M. J. (1973) J. Biol. Chem. 248,2978--2983. 4. Kletzien, R. R. & Perdue, J. F. (1974) J. Biol. Chem. 249, 3375-3382. 5. Singh, M., Singh, V. N., August, J. T. & Horecker, 13. L. (1978) J. Cell. Physiol. 97, 285--292. 6. Colby, C. & Romano, A. H. (1975) J. Cell. Physiol. 85, 15-24. 7. Quinlan, D. C., Parnes, J. R., Shalom, R., Garvey, T. Q., III, Isselbacher, K. J. & Hochstadt, J. (1976) Proc. Natl. Acad. Sci. USA 73, 1631-1635. 8. Parnes, J. R., Garvey, T. Q., III & Isselbacher, K. J. (1976) J. Cell. Physiol. 89, 789-794. 9. Nishino, H., Schiller, R. M., Parnes, J. R. & Isselbacher, K. J. (1978) Proc. Natl. Acad. Sci. USA 75,2329-2332. 10. Lever, J. E. (1976) Proc. Natl. Acad. Sci. USA 73,2614-2618. 11. Lever, J. E. (1977) J. Biol. Chem. 252, 1990-1997. 12. Hamilton, R. T. & Nilsen-Hamilton, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1907-1911. 13. Lever, J. E. (1978) J. Biol. Chem. 253, 2081-2084. 14. Hamilton, R. T. & Nilsen-Hamilton, M. (1978) J. Biol. Chem. 253,8247-8256. 15. Quinlan, D. C. & Hochstadt, J. (1976) J. Biol. Chem. 251, 344-354. 16. Nishino, H., Christopher, C. W., Schiller, R. M., Gammon, M. T., Ullrey, D. & Isselbacher, K. J. (1978) Proc. Natl. Acad. Sci. USA 75,5048-5051. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. Avruch, J. & Wallach, D. F. H. (1971) Biochim. Biophys. Acta
233,334-347. 19. Mackler, B. (1967) Methods Enzymol. 10, 551-553. 20. Peters, T. J., Muller, M. & de Duve, C. (1972) J. Exp. Med. 136, 1117-1139. 21. Czech, M. P., Lynn, D. G. & Lynn, W. S. (1973) J. Biol. Chem. 22.
248,3636-3641. Kletzien, R. R. & Perdue, J. F. (1973) J. Biol. Chem. 248, 711-
719. 23. Wardzala, L. J., Cushman, S. W. & Salans, L. 13. (1978) J. Biol.
Chem. 253, 8002-8005. 24. Lever, J. E. (1976) J. Cell. Physiol. 89, 779-787. 25. Lever, J. E. (1979) J. Biol. Chem. 254, 2961-2967. 26. Zala, C. A. & Perdue, J. R. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38, 245 (abstr.).
