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Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells
Vo1.160, No. 3,1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1409-1414
May 15,1989
Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells James S. Clegg and Susan A. Jackson University of California Bodega Marine Laboratory Bodega Bay, California 94923 Received April 7, 1989
ABSTRACT. L-929 cells permeabilized by dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Unlabeled 3-phospho~lycerate(PGA) added to DSP cells reduces the specific activity of lactate coming from [' C]glncose but the extent of this reduction can not be accounted for on the basis of free diffusion of PGA coming from [14C]glucose. Studies on other glycolytic intermediates, although preliminary, yield similar results. PGA also inhibits the production of lactate from glucose; however, this effect, like that of the reduction of lactate specific activity, becomes apparent only at concentrations of PGA well in excess of those considered to be physiological. We conclude that channelling of PGA, and probably other intermediates, occurs but is of the "leaky" type. ~ 19s9AcademicP..... ~o.
Abundant evidence has been obtained for the interaction in vitro of various glycolytic enzymes with each other (for example, 1-4), with ribosomes (5), mitochondria (6) and elements of the cytoskeleton (7-9). Excellent reviews are available (10-13). However, we know little about the details of these relationships in intact cells, and even less about their physiological significance.
Srivastava and Bernhard (4) and Keleti and
Ovadi (10) have summarized evidence for chalmelling of intermediates between several glycolytic enzymes. Because these data have been obtained chiefly from isolated enzymes some doubt exists concerning the existence of channelling in vivo. Testing that possibility in intact cells is extremely difficult. We recently found that L - 9 2 9 cells permeabilized with dextran sulfate allow molecules of very high molecular weight to enter and leave, yet carry out vigorous glycolysis when supplemented with glucose, ATP and NAD + (14). Consequently, these cells provide a means by which the possibility of intermediate channelling might be examined further. MATERIALS AND METHODS Cell Culture and Handling. Mouse L929 cells (L cells) from American Type Tissue Culture Collection (Rockville, Maryland) were grown to confluency in sealed flasks at 37°C with Medium 199 plus 10% fetal calf serum (Flow Laboratories, Inc.) and were harvested by treatment with tryypsin-EDTA. For permeabilization cells were washed 0006-291X/89,~ $1.50 1409
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
once in Buffer A (93raM NaC1, 5raM KC1, 5mM MgClz, 35raM Hepes, 0.1raM EGTA, pH 7.4) and resuspended in Buffer A. Permeabilization. The procedure follows, in general, that of Kucera and Paulus (15). Equal volumes of dextran sulfate, (Sigma Chemical Corporation, St. Louis, Missouri) of 5xl0SDa (lmg/ml in Buffer A), and cell suspension in buffer A, were combined in test tubes and incubated at 4°C for two consecutive 12.5 minute periods, swirling vigorously at halftime. Cells were then centrifuged at 4°C and 650g, washed once in Buffer A and resuspended in Buffer K (150raM sorbitol, 70mM potassium gluconate, 5mM MgClz, 5raM NaHzPO4, 35mM Hepes, 0.1mM EGTA, pH 7.55). Cells were counted in a hemocytometer after exposure to 0.1% trypan blue (TB) to determine cell numbers and the percentage of cells stained with TB. Populations of at least 85% TB + cells were used. Glyeolyzing conditions. The incubation medium for dextran sulfate-permeabilized (DSP) cells consisted of Buffer K plus 5mM glucose, 2mM ATP and lmM NAD +. These conditions allow maximal rates of glycolysis, linear for at least 30 minutes (14). 0.5ml of cell suspension were incubated in tilted 12x75mm capped test tubes at 37°C and 50 rpm in a reciprocating water bath shaker for 30 minutes. Activity was stopped by addition of perchloric acid (PCA) to a final concentration of 6%. Lactate was measured in PCA supernatants using Sigma procedure no. 826-UV (Sigma Chemical Company, St. Louis, Missouri). Complete details have been published (14). Effects of 3-phosphoglycerate (PGA) and other glycolytie intermediates. Glycolytic intermediates (Sigma Chemical Corporation ) were added to the medium of DSP cells incubated with uniformly labeled [1 C]glucose (Amersham Corp., Arlington Heights, Illinois) under the usual glycolyzing conditions. Lactate was isolated from charcoltreated 6% PCA supernatants using high pressure liquid chromatography (column HPX87H, from Bio-Rad, Richmond, California). Fractions from the lactate peak were assayed as usual, and for radioactivity by adding aliquots to 15 ml of scintillation fluid (ACS, Amersham Corporation) and counting to 1% error in a scintillation spectrometer. RESULTS Table I summarizes experiments on the effects of adding PGA to the medium of DSP cells incubated with radioactive glucose. Note first that the predicted specific activity of lactate from [14C] glucose in the absense of PGA was very close to that actually observed. However, in the presence of unlabelled PGA the specific activity of lactate was reduced. Although the extent of this reduction was generally related to the concentration of added PGA, some variation occurred when different preparations of cells were used. It soon became apparent that PGA also inhibited lactate production (Figure 1) at PGA concentrations above 0.1mM. The inset in Figure 1 shows the ratio of lactate produced in the presence of added PGA compared to its absence. This presentation of the data minimizes the variability observed from day to day. The main body of Figure 2 describes an experiment in which both the dilution of lactate specific activity and the inhibition of lactate production were measured: both show similar PGA concentration dependence. The inset indicates the relative reduction in specific activity of lactate from labeled glucose due to added unlabeled PGA for these data (open circles). The continuous lines are calculations of the concentration of [14C]PGA from [14C]glucose that would be required to account for the measured 1410
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Effect of unlabeled 3-phosphoglycerate (PGA) on the specific activity of lactate derived from [14C]glucoseZ in dextran sulfate permeabilized cells mM in Medium [14C]glucose [12C]PGA
Specific Activity (clam/mole) Glucose Lactate
O/P 2
1.7 1.7
0 1.7
3956 3956
1828 1363
0.92 0,69
2.5 2.5 5.0
0 2.5 5.0
582 582 582
255 163 88
0.88 0.56 0.30
5.0 5.0
0 1.0
828 828
402 118
0.97 0.29
5.0 5.0 5.0 5.0
0 0.1 0.5 1.0
974 974 974 974
509 482 420 378
1.05 0.99 0.86 0.78
1. [14C]glucose was uniformly labeled. 2. O/P refers to the ratio of the observed (measured) lactate specific activity compared to that predicted from the known specific activity of added glucose.
reduction in lactate specific activity, assuming that PGA from glucose is well-mixed with exogenous unlabeled PGA, and that both are present throughout the medium: The fractional reduction in lactate specific activity = [12C]PGA added/[12C]PGA added + [14C]PGA coming from [14C]glucose, all in mM concentrations. The calculation suggests that the concentration of [14C]PGA from [14C]glucose would have to be between 2 and 4mM from the onset of incubation to account for the reduction in lactate specific activity that we observe. Table II describes the results obtained when other glycolytic intermediates are added to DSP cells undergoing glycolysis. Unlike PGA, these intermediates do not appear to inhibit total lactate production, although pyruvate might be slightly inhibitory. However, all intermediates do decrease lactate specific activity to varying degrees. DISCUSSION We interpret these data as evidence that PGA is channelled in DSP cells but that this channelling is somewhat leaky. Exactly "how leaky" is difficult to say but we note that high concentrations of unlabeled PGA are required to demonstrate this effect, and that similar results were obtained by Mowbray and Moses (3) in their study of a glycolytic complex isolated from bacteria. The amounts of labeled PGA that are 1411
Vol. 160, No. 3, 1989
i
=
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
. . . .
i
,
,
,
,
,
, , , , ,, , , , , 1i 0
ovv[
~ ~
0.5 m M P G A
2
i.
~
0
e
0.5 ~.
1.0
Q)
T 0
--I 0
P
r
~
O
20(: I
i
0.2
I
I
0.4
l
/'z'x
o
'
'o'.s
. . . .
1:o
mM [12C] PGA
mM PGA
Figure 1. Production of lactate (nmoles/106 cells/30 min) by dextran sulfate permeabilized cells as a function of added 3-phosphoglycerate (PGA). Three separate studies are shown in the main body. The inset shows the ratio of lactate produced in the presence of PGA to that produced in its absence. Standard deviations are for n=4. Figure 2. Lactate production (nmoles/106 cells/30 min) and specific activity (cpm/nmole) from [lnC]glucose as a function of unlabeled 3-phosphoglycerate (PGA) added to DSP cells. The continuous lines in the inset are calculated values for the mM concentrations of [14C]PGA required to produce relative specific activity values as a function of added [lZC]PGA (see text for calculation). produced from [14C]glucose in our experiments are not known; however, we have evaluated the concentrations that would be needed to account for the observed reductions in lactate specific activity (inset, Figure 2) given two assumptions: (1) that all [14C]PGA is no___jtchannelled but mixes freely with added unlabeled P G A and (2) that TABLE II Effect of unlabeled glycolytic intermediates on the production and specific activity of lactate from [14C]glucose in dextran sulfate permeabilized cells Intermediate added (lmM) none G6P FDP DHAP PGA PYR
Specific Activity (cpm/nmole)
O/P
Production (nmole/106cells)
+I/-I
402 170 258 324 178 218
0.97 0.48 0.62 0.78 0.42 0.53
221 206 238 223 99 183
1 0.93 1.08 1.01 0.45 0.83
Glucose was present at 5mM and 828 cpm/nmole. O/P is defined in Table I, and I refers to the added intermediate, present (+) or absent (-). Abbreviations: G6P (glucose 6-phosphate); FDP (fructose 1,6-bisphosphate); DHAP (dihydroxyacetone phosphate); PGA (3-phosphoglycerate); PYR (pyruvate). 1412
t
n~l~ [~2CIPG~, J
O 40(]
i
-
F~ " ~ ~~ ° ~ t ~"- 0.2"t
~-
I-I-
0~mMi [14C]PGA
,4
!
600
1.0
.
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
both species of PGA are found throughout the volume of the incubation medium, as well as within the DSP cell. The data in Figure 2 indicate that PGA coming from glucose, and distributed as above, would have to be about 3mM; thus, this intermediate alone would consume 1.5mM of the total glucose available. Continuing to operate on the assumption of no glycolytic channelling (free diffusion of intermediates) comparable levels of other glycolytic intermediates would also have to be produced. The calculation under consideration assumes that the [14C]PGA concentrations are present from the onset of incubation (obviously an unrealistic condition, but one we include because it does not favor the channelling possibility). On the other hand, we note that the outcome is not always so clear cut and that larger dilutions of lactate specific activity are sometimes observed (Tables I and II). Part of this variability may result from the variable degrees of integrity observed for different DSP cell preparations (14). Even in these cases, however, the lactate specific activity reductions are not so large as to be easily accounted for without channelling. Consider the data in Table II. Using the same calculation we have estimated the global concentrations of the five intermediates that would be required to explain the observed reductions in lactate specific activity. In equivalents of glucose used, these mM estimates are 0.9(G6P), 1.6(FDP), 1.8(DHAP), 0.4(PGA), and 0.6(PA). Thus, more glucose would be required for these intermediates alone than is present in the entire incubation medium (5mM). Finally, we note that the rate of glycolysis in DSP cells is vigorous and linear (14), conditions that do not favor the possibility of a global build up of intermediates. We were surprised to find that PGA inhibited lactate production in addition to its effect on reducing its specific activity. Both effects might involve a common mechanism in view of their similar concentration dependence (Figure 2). Be that as it may, both effects became appreciable only when added PGA was in excess of 0. lmM, concentrations which are higher than those in intact non-muscle cells (16). These studies were done on cells whose plasma membranes have been perturbed by dextran sulfate (14). Thus, interpretation must be tempered by the realization that the cell interior has been disturbed. While these conditions might somehow generate an organization of enzymes that results in an artifactual channelling of intermediates that seems unlikely to us. When considering, in addition, the evidence from numerous other laboratories that favors the existence of channelling of glycolytic intermediates (1-4, 713) we believe our results on DSP cells are, more likely than not, a reflection of events that occur in intact cells. ACKNOWLEDGMENTS
We gratefully acknowledge the skilled experience of Diane Cosgrove in manuscript preparation, and support from NSF grant 88-20347. 1413
Vot. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Tompa, P., Bar, J., and Batke, J. (1986) Eur. J. Biochem. 159, 117-124. Friedrich, P., and Hajdee, J. (1987) Biochem. Soc. Trans. 15, 973-977. Mowbray, J., and Moses, V. (1976) Eur. J. Biochem. 66, 25-36. Srivastava, D.K., and Bernhard, S.A. (1987) Science 234, 1081-1086. Ryazanov, A.G., Askmarina, L.I., and Muronitz, V.I. (1988) Eur. J. Biochem. 171, 301-305. Wilson, J.E. (1980) Curr. Top. Cell. Regul. 16, 1-54. Westrin, H., and Backman, L. (1983) Eur. J. Biochem. 136, 407-411. Walsh, J.L., and Knull, H.R. (1988) Biochim. Biophys. Acta 952, 83-91. Durrieu, C., Bernier-Valentin, F., and Rousset, B. (1987) Molec. Cell. Biochem. 74, 55-65. Keleti, T., and Ovadi, J. (1988) Curr. Top. Cell Regul. 29, 1-33. Srere, P. (1988) Ann. Rev. Biochem. 56, 21-62. Clarke, F.M., Morton, D.J., Stephan, P., and Wiedemann, J. (1985) In Cell Motility: Mechanisms and Regulation (H. Ishikawa, Ed.), pp. 235-250, University of Tokyo Press. Masters, C.J., and Reid, S. (1987) Curt. Top. Biol. Med. Res. (Isozymers) 14, 45-58. Clegg, J.S., and Jackson, S.A. (1988) Biochem. J. 225, 335-344. Kucera, R., and Paulus, H. (1982) Arch. Biochem. Biophys. 214, 102-113. Ottaway, J.H., and Mowbray, J. (1977) Curr. Top. Cell. Regul. 12, 108-209.
t414
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1409-1414
May 15,1989
Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells James S. Clegg and Susan A. Jackson University of California Bodega Marine Laboratory Bodega Bay, California 94923 Received April 7, 1989
ABSTRACT. L-929 cells permeabilized by dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Unlabeled 3-phospho~lycerate(PGA) added to DSP cells reduces the specific activity of lactate coming from [' C]glncose but the extent of this reduction can not be accounted for on the basis of free diffusion of PGA coming from [14C]glucose. Studies on other glycolytic intermediates, although preliminary, yield similar results. PGA also inhibits the production of lactate from glucose; however, this effect, like that of the reduction of lactate specific activity, becomes apparent only at concentrations of PGA well in excess of those considered to be physiological. We conclude that channelling of PGA, and probably other intermediates, occurs but is of the "leaky" type. ~ 19s9AcademicP..... ~o.
Abundant evidence has been obtained for the interaction in vitro of various glycolytic enzymes with each other (for example, 1-4), with ribosomes (5), mitochondria (6) and elements of the cytoskeleton (7-9). Excellent reviews are available (10-13). However, we know little about the details of these relationships in intact cells, and even less about their physiological significance.
Srivastava and Bernhard (4) and Keleti and
Ovadi (10) have summarized evidence for chalmelling of intermediates between several glycolytic enzymes. Because these data have been obtained chiefly from isolated enzymes some doubt exists concerning the existence of channelling in vivo. Testing that possibility in intact cells is extremely difficult. We recently found that L - 9 2 9 cells permeabilized with dextran sulfate allow molecules of very high molecular weight to enter and leave, yet carry out vigorous glycolysis when supplemented with glucose, ATP and NAD + (14). Consequently, these cells provide a means by which the possibility of intermediate channelling might be examined further. MATERIALS AND METHODS Cell Culture and Handling. Mouse L929 cells (L cells) from American Type Tissue Culture Collection (Rockville, Maryland) were grown to confluency in sealed flasks at 37°C with Medium 199 plus 10% fetal calf serum (Flow Laboratories, Inc.) and were harvested by treatment with tryypsin-EDTA. For permeabilization cells were washed 0006-291X/89,~ $1.50 1409
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
once in Buffer A (93raM NaC1, 5raM KC1, 5mM MgClz, 35raM Hepes, 0.1raM EGTA, pH 7.4) and resuspended in Buffer A. Permeabilization. The procedure follows, in general, that of Kucera and Paulus (15). Equal volumes of dextran sulfate, (Sigma Chemical Corporation, St. Louis, Missouri) of 5xl0SDa (lmg/ml in Buffer A), and cell suspension in buffer A, were combined in test tubes and incubated at 4°C for two consecutive 12.5 minute periods, swirling vigorously at halftime. Cells were then centrifuged at 4°C and 650g, washed once in Buffer A and resuspended in Buffer K (150raM sorbitol, 70mM potassium gluconate, 5mM MgClz, 5raM NaHzPO4, 35mM Hepes, 0.1mM EGTA, pH 7.55). Cells were counted in a hemocytometer after exposure to 0.1% trypan blue (TB) to determine cell numbers and the percentage of cells stained with TB. Populations of at least 85% TB + cells were used. Glyeolyzing conditions. The incubation medium for dextran sulfate-permeabilized (DSP) cells consisted of Buffer K plus 5mM glucose, 2mM ATP and lmM NAD +. These conditions allow maximal rates of glycolysis, linear for at least 30 minutes (14). 0.5ml of cell suspension were incubated in tilted 12x75mm capped test tubes at 37°C and 50 rpm in a reciprocating water bath shaker for 30 minutes. Activity was stopped by addition of perchloric acid (PCA) to a final concentration of 6%. Lactate was measured in PCA supernatants using Sigma procedure no. 826-UV (Sigma Chemical Company, St. Louis, Missouri). Complete details have been published (14). Effects of 3-phosphoglycerate (PGA) and other glycolytie intermediates. Glycolytic intermediates (Sigma Chemical Corporation ) were added to the medium of DSP cells incubated with uniformly labeled [1 C]glucose (Amersham Corp., Arlington Heights, Illinois) under the usual glycolyzing conditions. Lactate was isolated from charcoltreated 6% PCA supernatants using high pressure liquid chromatography (column HPX87H, from Bio-Rad, Richmond, California). Fractions from the lactate peak were assayed as usual, and for radioactivity by adding aliquots to 15 ml of scintillation fluid (ACS, Amersham Corporation) and counting to 1% error in a scintillation spectrometer. RESULTS Table I summarizes experiments on the effects of adding PGA to the medium of DSP cells incubated with radioactive glucose. Note first that the predicted specific activity of lactate from [14C] glucose in the absense of PGA was very close to that actually observed. However, in the presence of unlabelled PGA the specific activity of lactate was reduced. Although the extent of this reduction was generally related to the concentration of added PGA, some variation occurred when different preparations of cells were used. It soon became apparent that PGA also inhibited lactate production (Figure 1) at PGA concentrations above 0.1mM. The inset in Figure 1 shows the ratio of lactate produced in the presence of added PGA compared to its absence. This presentation of the data minimizes the variability observed from day to day. The main body of Figure 2 describes an experiment in which both the dilution of lactate specific activity and the inhibition of lactate production were measured: both show similar PGA concentration dependence. The inset indicates the relative reduction in specific activity of lactate from labeled glucose due to added unlabeled PGA for these data (open circles). The continuous lines are calculations of the concentration of [14C]PGA from [14C]glucose that would be required to account for the measured 1410
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Effect of unlabeled 3-phosphoglycerate (PGA) on the specific activity of lactate derived from [14C]glucoseZ in dextran sulfate permeabilized cells mM in Medium [14C]glucose [12C]PGA
Specific Activity (clam/mole) Glucose Lactate
O/P 2
1.7 1.7
0 1.7
3956 3956
1828 1363
0.92 0,69
2.5 2.5 5.0
0 2.5 5.0
582 582 582
255 163 88
0.88 0.56 0.30
5.0 5.0
0 1.0
828 828
402 118
0.97 0.29
5.0 5.0 5.0 5.0
0 0.1 0.5 1.0
974 974 974 974
509 482 420 378
1.05 0.99 0.86 0.78
1. [14C]glucose was uniformly labeled. 2. O/P refers to the ratio of the observed (measured) lactate specific activity compared to that predicted from the known specific activity of added glucose.
reduction in lactate specific activity, assuming that PGA from glucose is well-mixed with exogenous unlabeled PGA, and that both are present throughout the medium: The fractional reduction in lactate specific activity = [12C]PGA added/[12C]PGA added + [14C]PGA coming from [14C]glucose, all in mM concentrations. The calculation suggests that the concentration of [14C]PGA from [14C]glucose would have to be between 2 and 4mM from the onset of incubation to account for the reduction in lactate specific activity that we observe. Table II describes the results obtained when other glycolytic intermediates are added to DSP cells undergoing glycolysis. Unlike PGA, these intermediates do not appear to inhibit total lactate production, although pyruvate might be slightly inhibitory. However, all intermediates do decrease lactate specific activity to varying degrees. DISCUSSION We interpret these data as evidence that PGA is channelled in DSP cells but that this channelling is somewhat leaky. Exactly "how leaky" is difficult to say but we note that high concentrations of unlabeled PGA are required to demonstrate this effect, and that similar results were obtained by Mowbray and Moses (3) in their study of a glycolytic complex isolated from bacteria. The amounts of labeled PGA that are 1411
Vol. 160, No. 3, 1989
i
=
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
. . . .
i
,
,
,
,
,
, , , , ,, , , , , 1i 0
ovv[
~ ~
0.5 m M P G A
2
i.
~
0
e
0.5 ~.
1.0
Q)
T 0
--I 0
P
r
~
O
20(: I
i
0.2
I
I
0.4
l
/'z'x
o
'
'o'.s
. . . .
1:o
mM [12C] PGA
mM PGA
Figure 1. Production of lactate (nmoles/106 cells/30 min) by dextran sulfate permeabilized cells as a function of added 3-phosphoglycerate (PGA). Three separate studies are shown in the main body. The inset shows the ratio of lactate produced in the presence of PGA to that produced in its absence. Standard deviations are for n=4. Figure 2. Lactate production (nmoles/106 cells/30 min) and specific activity (cpm/nmole) from [lnC]glucose as a function of unlabeled 3-phosphoglycerate (PGA) added to DSP cells. The continuous lines in the inset are calculated values for the mM concentrations of [14C]PGA required to produce relative specific activity values as a function of added [lZC]PGA (see text for calculation). produced from [14C]glucose in our experiments are not known; however, we have evaluated the concentrations that would be needed to account for the observed reductions in lactate specific activity (inset, Figure 2) given two assumptions: (1) that all [14C]PGA is no___jtchannelled but mixes freely with added unlabeled P G A and (2) that TABLE II Effect of unlabeled glycolytic intermediates on the production and specific activity of lactate from [14C]glucose in dextran sulfate permeabilized cells Intermediate added (lmM) none G6P FDP DHAP PGA PYR
Specific Activity (cpm/nmole)
O/P
Production (nmole/106cells)
+I/-I
402 170 258 324 178 218
0.97 0.48 0.62 0.78 0.42 0.53
221 206 238 223 99 183
1 0.93 1.08 1.01 0.45 0.83
Glucose was present at 5mM and 828 cpm/nmole. O/P is defined in Table I, and I refers to the added intermediate, present (+) or absent (-). Abbreviations: G6P (glucose 6-phosphate); FDP (fructose 1,6-bisphosphate); DHAP (dihydroxyacetone phosphate); PGA (3-phosphoglycerate); PYR (pyruvate). 1412
t
n~l~ [~2CIPG~, J
O 40(]
i
-
F~ " ~ ~~ ° ~ t ~"- 0.2"t
~-
I-I-
0~mMi [14C]PGA
,4
!
600
1.0
.
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
both species of PGA are found throughout the volume of the incubation medium, as well as within the DSP cell. The data in Figure 2 indicate that PGA coming from glucose, and distributed as above, would have to be about 3mM; thus, this intermediate alone would consume 1.5mM of the total glucose available. Continuing to operate on the assumption of no glycolytic channelling (free diffusion of intermediates) comparable levels of other glycolytic intermediates would also have to be produced. The calculation under consideration assumes that the [14C]PGA concentrations are present from the onset of incubation (obviously an unrealistic condition, but one we include because it does not favor the channelling possibility). On the other hand, we note that the outcome is not always so clear cut and that larger dilutions of lactate specific activity are sometimes observed (Tables I and II). Part of this variability may result from the variable degrees of integrity observed for different DSP cell preparations (14). Even in these cases, however, the lactate specific activity reductions are not so large as to be easily accounted for without channelling. Consider the data in Table II. Using the same calculation we have estimated the global concentrations of the five intermediates that would be required to explain the observed reductions in lactate specific activity. In equivalents of glucose used, these mM estimates are 0.9(G6P), 1.6(FDP), 1.8(DHAP), 0.4(PGA), and 0.6(PA). Thus, more glucose would be required for these intermediates alone than is present in the entire incubation medium (5mM). Finally, we note that the rate of glycolysis in DSP cells is vigorous and linear (14), conditions that do not favor the possibility of a global build up of intermediates. We were surprised to find that PGA inhibited lactate production in addition to its effect on reducing its specific activity. Both effects might involve a common mechanism in view of their similar concentration dependence (Figure 2). Be that as it may, both effects became appreciable only when added PGA was in excess of 0. lmM, concentrations which are higher than those in intact non-muscle cells (16). These studies were done on cells whose plasma membranes have been perturbed by dextran sulfate (14). Thus, interpretation must be tempered by the realization that the cell interior has been disturbed. While these conditions might somehow generate an organization of enzymes that results in an artifactual channelling of intermediates that seems unlikely to us. When considering, in addition, the evidence from numerous other laboratories that favors the existence of channelling of glycolytic intermediates (1-4, 713) we believe our results on DSP cells are, more likely than not, a reflection of events that occur in intact cells. ACKNOWLEDGMENTS
We gratefully acknowledge the skilled experience of Diane Cosgrove in manuscript preparation, and support from NSF grant 88-20347. 1413
Vot. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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