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Specificity of the rat hepatocyte monosaccharide transporter
hr. J. Biochem. Vol. 16, No. 12, pp. 1251-1253, 1984 Prmted in Great Britain. All rights reserved
Copyright 0
0020-71 IX/84 $3.00 + 0.00 1984 Pergamon Press Ltd
SPECIFICITY OF THE RAT HEPATOCYTE MONOSACCHARIDE TRANSPORTER Department
KEITH R. F. ELLIOTT, ALISON J. BATE and JAMES D. CRAIK* of Biochemistry, The Medical School, University of Manchester, Manchester [Tel. 061-273-82411
Ml3 9PT, U.K.
(Received 29 May 1984) Abstract-l. The effect of a number of hexoses and pentoses on 3-O-methyl-D-glucose transport into isolated hepatocytes is reported. 2. The hexoses tested inhibited transport in a competitive manner, with K, values ranging from 80 to 190 mM. 3. No significant inhibition was seen with either D-ribose or D-arabinose.
INTRODUCTION
After addition of phloretin to stop transport the suspension was centrifuged at 2000g in IO-ml conical tubes through a I-ml layer of a mixture of I-bromodedecane and ndodecane [14: 1 (v/v) giving a specific gravity of 1.021 g/ml] into a solution of 4% (v/v) perchloric acid in 10% (w/v) sucrose. After removal of the supernatant and I-bromododecane/n-dodecane the cell pellet was dispersed and recentrifuged before the radioactivity was counted in PCS using a Beckman LS9800 scintillation counter with automatic quench correction.
Monosaccharide transport across the hepatocyte plasma membrane has been shown to be via a rapid, high K,, stereospecific facilitated diffusion system (Williams et al., 1968; Baur and Heldt, 1977; Craik and Elliott, 1979, 1980; Elliott and Craik, 1982). Most of the published data have concentrated on the major physiologically important hexoses, i.e. D-glucose, D-fructose and D-galactose (Williams et al., 1968; Sestoft and Fleron, 1974; Baur and Heldt, 1977; Craik and Elliott, 1980), although we have also produced data on 2-deoxy-D-glucose (Elliott and Craik, 1982) and a detailed kinetic analysis of 3-O-methyl-D-glucose transport (Craik and Elliott, 1979). There is a considerable amount of information on the specificity of other monosaccharide transport systems, notably that of the human erythrocyte (Barnett et al., 1973, 1975). This has led to hypotheses on the nature of the binding interactions between substrates and transporter. In this paper we report the effects of some hexoses and pentoses on the transport of 3-O-methyl-~-glucose into isolated hepatocytes which indicate that there are differences between this system and that of the human erythrocyte.
RESULTS AND DISCUSSION Hexose
Pentose
sugars
Neither D-ribose nor D-arabinose inhibited 3-O-methyl-D-glucose transport to any significant extent up to 100mM (Fig. 2). The experimental
MATERIALS AND METHODS Sugars, 1-bromododecane and n-dodecane were obtained from Sigma (London) Chemical Co., Poole, Dorset, U.K. 3-O-Methyl-u-[l-iH]glucose (specific radioactivity 6.5 Cij mmol) was obtained from the Radiochemical Centre, Amersham, Bucks, U.K. Other materials were as previously described (Craik and Elliott, 1979, 1980). Cell preparation and incubations were as previously described (Craik and Elliott, 1979, 1980) with the following modification to the transport methodology to simplify the technique. 3-O-methyl-o-glucose transport was measured at 2O’C in the presence or absence of competing sugar as the amount of 3-O-methyl-D-glucose taken up in 5 sec. This leads to an underestimate of the true initial rate of zero-tram entry of between 10 and 20”, at the concentrations used. *Present address: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
sugars
All the hexose sugars tested inhibited 3-O -methyl-D-glucose transport in a competitive manner. Figure 1 illustrates the Dixon plots obtained with D-glucose and D-mannose as inhibitors. The K, values are given in Table 1.
especially at scatter on the data, low 3-O -methyl-D-glucose and high competing sugar concentrations is such that it is not possible to draw a definite conclusion that there is no inhibition. However, if D-ribose or D-arabinose do compete then the K, values will be high, in excess of 300 mM. Comparison
with other
transporters
Table 1 details the results for inhibition reported here along with previously published data from this laboratory on K,,, values for zero-trans entry. Where direct comparison is possible, i.e. for D-glucose, D-fructose and D-galactose the K,,, value and K, value are very similar. These data, along with those on the mutual inhibition between D-frucose and D-galactose (Craik and Elliott, 1980) and inhibition of D-glucose transport by 2-deoxy-D-glucose (Baur and Heldt, 1977), are consistent with the hypothesis that all the hexoses studied are transported via the same system. It would appear from the data that the 2, 3 and 4 hydroxyl groups are all important in glucose binding to the carrier. Alteration at the 2 position either by
1251
KEITH R. F ELLIOTT et ai.
1252
(Al
(A)
&
I
I 100
0 Glucose (mM
7; 0h 0
i
”
IJ)
12
;: ” 0, --3
10
z r
Monnose (mM
100 1
Fig. 1. Dixon plots of inhibition of 3-O-methyl-D-glucose zero-tram entry by (A) o-glucose and (B) D-mannose. Points are +S.E.M. of (A) 3 and (B) 6 separate hepdtocyte preparations. 3-O-Methyl-n-glucose concentrations were (A) 0.5 mM, (A) 1.O mM, (II) 2.0 mM and (0) 30 mM.
epimerization (i.e. D-mannuse) or loss of the hydroxyl (i.e. 2-deoxy-D-glucose) results in an increase in Kmor K,, as does epimerization at the 4 position (i.e. n-galactose). On the other hand, methylation of the 3-hydroxyl decreases the K,,,. Obviously, much more investigation is necessary to provide a better description of the structural features necessary for binding. However, it is clear that these differ from those of the human erythrocyte (Barnett et al., 1973), where similar effects are seen with D-mannose and D-galactose, but 3-0-methyl-n-glucose has a high K, and that for 2-deoxy-D-glucose is low. The apparent lack of inhibition by the pentose sugars, D-ribose and D-arabinose, is similar to the situation found with human erythrocytes where both Table I. Kinetic constants for monosaccharide port into isolated hepatocytes 3.O-Methyl-o-glucose D-GlUCOSe
o-Fructose o-Galactose 2.Deoxy-u-glucose
u-ManMlse o-Ribose o-Arabinose
IC,(mM) 20+3 66& 14 212 + 32 174+32 167245
trans-
K,(mM) 80 130 140
191) > 300 >300
K,, values are taken from Elliott and Craik (1982) and arc for zero-wuns entry calculated by the method of Wilkinson (1961) ( i. S.E.M. for between 21 and 63 initial rate measurements). Ic, values are obtained from Dixon plots for zero-lrans entfv of 3-O-methyl-o-glucose assuming competmve inhibltion.
RI bose
r
‘Or
0
50
::
100
( mM )
(91
i
TA-
08
I-
O
50 Arablnose
f mM 1
100
Fig. 2. Dixon plots of the effects of (A) n-ribose and (B) n-arabinose on 3-O-methyl-D-ghrcose zero-lruns entry. Points are +S.E.M. of (A) 3 and (B) 4 separate hepatocyte preparations. 3-O-Methyl-o-glucose concentrations were (A) 0.5mM, (A) l.OmM, (II) 2.0mM and (U) 30mM.
show a high X;, for transport (see Stein, 1967). In the human erythrocyte, and other systems, L-arabinose is a better substrate [see Stein (1967)] being in effect an analogue of D-galactose. On the basis of the data presented here it is not possible to determine whether these pentoses interact weakly with the hexose transporter or not at ah. Further investigation may clarify this situation. Ackno~iedgement-This grant from the Medical
work was supported Research Council.
in part by a
REFERENCES Barnett J. E. G., Holman G. D., Chalkley R. A. and Munday K. A. (1975) Evidence for two asymmetric conformational states in the human erythrocyte sugartransport system. Biochem. J. 145, 417419. Barnett J. E. G., Hohan G. D. and Munday K. A. (1973) Structural requirements for binding to the sugartransport system of the human erythrocyte. Biochern. /. 131, 211-221. Baur H. and Heldt H. W. (1977) Transport of hexoses across the liver-cell membrane. Eur. f. Bio~hem. 74,
397-403. Craik J. D. and Elliott K. R. F. (1979) Kinetics of 3-Omethyl-o-glucose transport in isolated rat hepatocytes. Biochem. J. 182, 503-508. Craik J. D. and Elliott K. R. F. (1980) Transport of o-fructose and n-galactose into isolated rat hepatocytes. ~~oc~ern..i. 192, 373-375. Elliott K. R. F. and Craik J. D. (1982) Sugar transport across the hepatocyte plasma membrane. T~LIILY. hiochem. sot. 10. 12-13.
Sugar transport in hepatocytes Sestoft L. and Fleron P. (1974) Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver. Biochim. biophys. Actn 345, 27-38. Stein W. D. (1967) The Movement of Molecules Across Cell Membranes. Academic Press, New York.
1253
Wilkinson G. N. (1961) Statistical estimations in enzyme kinetics, Biochem. J. 80, 324-332. Williams T. F., Exton J. H., Park C. R. and Regen D. M. (1968) Stereospecific transport of glucose in the perfused rat liver. Am. J. Physiol. 215, 120&1209.
Copyright 0
0020-71 IX/84 $3.00 + 0.00 1984 Pergamon Press Ltd
SPECIFICITY OF THE RAT HEPATOCYTE MONOSACCHARIDE TRANSPORTER Department
KEITH R. F. ELLIOTT, ALISON J. BATE and JAMES D. CRAIK* of Biochemistry, The Medical School, University of Manchester, Manchester [Tel. 061-273-82411
Ml3 9PT, U.K.
(Received 29 May 1984) Abstract-l. The effect of a number of hexoses and pentoses on 3-O-methyl-D-glucose transport into isolated hepatocytes is reported. 2. The hexoses tested inhibited transport in a competitive manner, with K, values ranging from 80 to 190 mM. 3. No significant inhibition was seen with either D-ribose or D-arabinose.
INTRODUCTION
After addition of phloretin to stop transport the suspension was centrifuged at 2000g in IO-ml conical tubes through a I-ml layer of a mixture of I-bromodedecane and ndodecane [14: 1 (v/v) giving a specific gravity of 1.021 g/ml] into a solution of 4% (v/v) perchloric acid in 10% (w/v) sucrose. After removal of the supernatant and I-bromododecane/n-dodecane the cell pellet was dispersed and recentrifuged before the radioactivity was counted in PCS using a Beckman LS9800 scintillation counter with automatic quench correction.
Monosaccharide transport across the hepatocyte plasma membrane has been shown to be via a rapid, high K,, stereospecific facilitated diffusion system (Williams et al., 1968; Baur and Heldt, 1977; Craik and Elliott, 1979, 1980; Elliott and Craik, 1982). Most of the published data have concentrated on the major physiologically important hexoses, i.e. D-glucose, D-fructose and D-galactose (Williams et al., 1968; Sestoft and Fleron, 1974; Baur and Heldt, 1977; Craik and Elliott, 1980), although we have also produced data on 2-deoxy-D-glucose (Elliott and Craik, 1982) and a detailed kinetic analysis of 3-O-methyl-D-glucose transport (Craik and Elliott, 1979). There is a considerable amount of information on the specificity of other monosaccharide transport systems, notably that of the human erythrocyte (Barnett et al., 1973, 1975). This has led to hypotheses on the nature of the binding interactions between substrates and transporter. In this paper we report the effects of some hexoses and pentoses on the transport of 3-O-methyl-~-glucose into isolated hepatocytes which indicate that there are differences between this system and that of the human erythrocyte.
RESULTS AND DISCUSSION Hexose
Pentose
sugars
Neither D-ribose nor D-arabinose inhibited 3-O-methyl-D-glucose transport to any significant extent up to 100mM (Fig. 2). The experimental
MATERIALS AND METHODS Sugars, 1-bromododecane and n-dodecane were obtained from Sigma (London) Chemical Co., Poole, Dorset, U.K. 3-O-Methyl-u-[l-iH]glucose (specific radioactivity 6.5 Cij mmol) was obtained from the Radiochemical Centre, Amersham, Bucks, U.K. Other materials were as previously described (Craik and Elliott, 1979, 1980). Cell preparation and incubations were as previously described (Craik and Elliott, 1979, 1980) with the following modification to the transport methodology to simplify the technique. 3-O-methyl-o-glucose transport was measured at 2O’C in the presence or absence of competing sugar as the amount of 3-O-methyl-D-glucose taken up in 5 sec. This leads to an underestimate of the true initial rate of zero-tram entry of between 10 and 20”, at the concentrations used. *Present address: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
sugars
All the hexose sugars tested inhibited 3-O -methyl-D-glucose transport in a competitive manner. Figure 1 illustrates the Dixon plots obtained with D-glucose and D-mannose as inhibitors. The K, values are given in Table 1.
especially at scatter on the data, low 3-O -methyl-D-glucose and high competing sugar concentrations is such that it is not possible to draw a definite conclusion that there is no inhibition. However, if D-ribose or D-arabinose do compete then the K, values will be high, in excess of 300 mM. Comparison
with other
transporters
Table 1 details the results for inhibition reported here along with previously published data from this laboratory on K,,, values for zero-trans entry. Where direct comparison is possible, i.e. for D-glucose, D-fructose and D-galactose the K,,, value and K, value are very similar. These data, along with those on the mutual inhibition between D-frucose and D-galactose (Craik and Elliott, 1980) and inhibition of D-glucose transport by 2-deoxy-D-glucose (Baur and Heldt, 1977), are consistent with the hypothesis that all the hexoses studied are transported via the same system. It would appear from the data that the 2, 3 and 4 hydroxyl groups are all important in glucose binding to the carrier. Alteration at the 2 position either by
1251
KEITH R. F ELLIOTT et ai.
1252
(Al
(A)
&
I
I 100
0 Glucose (mM
7; 0h 0
i
”
IJ)
12
;: ” 0, --3
10
z r
Monnose (mM
100 1
Fig. 1. Dixon plots of inhibition of 3-O-methyl-D-glucose zero-tram entry by (A) o-glucose and (B) D-mannose. Points are +S.E.M. of (A) 3 and (B) 6 separate hepdtocyte preparations. 3-O-Methyl-n-glucose concentrations were (A) 0.5 mM, (A) 1.O mM, (II) 2.0 mM and (0) 30 mM.
epimerization (i.e. D-mannuse) or loss of the hydroxyl (i.e. 2-deoxy-D-glucose) results in an increase in Kmor K,, as does epimerization at the 4 position (i.e. n-galactose). On the other hand, methylation of the 3-hydroxyl decreases the K,,,. Obviously, much more investigation is necessary to provide a better description of the structural features necessary for binding. However, it is clear that these differ from those of the human erythrocyte (Barnett et al., 1973), where similar effects are seen with D-mannose and D-galactose, but 3-0-methyl-n-glucose has a high K, and that for 2-deoxy-D-glucose is low. The apparent lack of inhibition by the pentose sugars, D-ribose and D-arabinose, is similar to the situation found with human erythrocytes where both Table I. Kinetic constants for monosaccharide port into isolated hepatocytes 3.O-Methyl-o-glucose D-GlUCOSe
o-Fructose o-Galactose 2.Deoxy-u-glucose
u-ManMlse o-Ribose o-Arabinose
IC,(mM) 20+3 66& 14 212 + 32 174+32 167245
trans-
K,(mM) 80 130 140
191) > 300 >300
K,, values are taken from Elliott and Craik (1982) and arc for zero-wuns entry calculated by the method of Wilkinson (1961) ( i. S.E.M. for between 21 and 63 initial rate measurements). Ic, values are obtained from Dixon plots for zero-lrans entfv of 3-O-methyl-o-glucose assuming competmve inhibltion.
RI bose
r
‘Or
0
50
::
100
( mM )
(91
i
TA-
08
I-
O
50 Arablnose
f mM 1
100
Fig. 2. Dixon plots of the effects of (A) n-ribose and (B) n-arabinose on 3-O-methyl-D-ghrcose zero-lruns entry. Points are +S.E.M. of (A) 3 and (B) 4 separate hepatocyte preparations. 3-O-Methyl-o-glucose concentrations were (A) 0.5mM, (A) l.OmM, (II) 2.0mM and (U) 30mM.
show a high X;, for transport (see Stein, 1967). In the human erythrocyte, and other systems, L-arabinose is a better substrate [see Stein (1967)] being in effect an analogue of D-galactose. On the basis of the data presented here it is not possible to determine whether these pentoses interact weakly with the hexose transporter or not at ah. Further investigation may clarify this situation. Ackno~iedgement-This grant from the Medical
work was supported Research Council.
in part by a
REFERENCES Barnett J. E. G., Holman G. D., Chalkley R. A. and Munday K. A. (1975) Evidence for two asymmetric conformational states in the human erythrocyte sugartransport system. Biochem. J. 145, 417419. Barnett J. E. G., Hohan G. D. and Munday K. A. (1973) Structural requirements for binding to the sugartransport system of the human erythrocyte. Biochern. /. 131, 211-221. Baur H. and Heldt H. W. (1977) Transport of hexoses across the liver-cell membrane. Eur. f. Bio~hem. 74,
397-403. Craik J. D. and Elliott K. R. F. (1979) Kinetics of 3-Omethyl-o-glucose transport in isolated rat hepatocytes. Biochem. J. 182, 503-508. Craik J. D. and Elliott K. R. F. (1980) Transport of o-fructose and n-galactose into isolated rat hepatocytes. ~~oc~ern..i. 192, 373-375. Elliott K. R. F. and Craik J. D. (1982) Sugar transport across the hepatocyte plasma membrane. T~LIILY. hiochem. sot. 10. 12-13.
Sugar transport in hepatocytes Sestoft L. and Fleron P. (1974) Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver. Biochim. biophys. Actn 345, 27-38. Stein W. D. (1967) The Movement of Molecules Across Cell Membranes. Academic Press, New York.
1253
Wilkinson G. N. (1961) Statistical estimations in enzyme kinetics, Biochem. J. 80, 324-332. Williams T. F., Exton J. H., Park C. R. and Regen D. M. (1968) Stereospecific transport of glucose in the perfused rat liver. Am. J. Physiol. 215, 120&1209.