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Activation of phosphofructokinase from rat tissues by 6-phosphogluconate and fructose 2,6-bisphosphate
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 232, No. 2, August 1, pp. 579-584, 1984
Activation of Phosphofructokinase from Rat Tissues by 6-Phosphogluconate and Fructose 2,6-Bisphosphate’ JAMES SOMMERCORN,2 TINA STEWARD, Department
AND
RICHARD A. FREEDLAND
of Physiol&d Sciences, S&ml of Veterinary Medicine, 95616 University of California, Davis, Cal&rnio
Received February
2, 1934, and in revised form April
2, 1934
6-Phosphogluconate activates phosphofructokinase from liver, adipose tissue, kidney, and skeletal muscle by decreasing the apparent S0.5for fructose 6-phosphate without affecting the maximum velocity. The response of phosphofructokinase to 6-phosphogluconate is hyperbolic, with apparent activation constants similar to concentrations of 6-phosphogluconate in tissues. Phosphofructokinase from these tissues is also activated by fructose 2,6-bisphosphate, but the apparent activation constants are much less than the concentrations of fructose 2,6-bisphosphate in tissues. Under most conditions, the effects of 6-phosphogluconate and fructose 2,6-bisphosphate are additive. However, with low concentrations of fructose 6-phosphate there is synergism between the effecters. Whereas fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP, 6-phosphogluconate does not. Thus, the effecters probably act at different sites on the enzyme. The relative effect of 6-phosphogluconate is much greater on phosphofructokinase from the lipogenic tissues, adipose, and liver, than it is on the enzyme from kidney or skeletal muscle. Thus, the influence of 6-phosphogluconate on phosphofructokinase, which could coordinate the disposition of glucose 6phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis, may be important for lipogenesis. 6-Phosphogluconate, an intermediate of the oxidative branch of the hexosemonophosphate pathway, activates both hepatic phosphofructokinase (ATP:D-fructoseSphosphate-1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis (1,2) and pyruvate kinase, the second regulated glycolytic enzyme (3,4). The influence of 6-phosphogluconate on these two enzymes may provide a means of coordinating fluxes through two pathways which contribute substrates for the syni The work was supported by U. S. Public Health Service Grant AM23319. a Present address: Howard Hughes Medical Institute, SL-15 University of Washington, Seattle, Wash. 98195. a To whom correspondence should be addressed.
thesis of fatty acids. To assess the importance to lipogenesis of the influence of 6phosphogluconate on phosphofructokinase, we have studied the effect on the enzyme from adipose, another lipogenic tissue, and on the enzyme from kidney and skeletal muscle, tissues which do not synthesize fatty acids. MATERIALS
AND
METHODS
Male Sprague-Dawley rats were either purchased from Hilltop Lab Animals, Inc. (Scottsdale, Pa.) or they were obtained from a colony which is kept by the Department of Animal Science on campus. The animals ranged in weight from 150 to 540 g, and they were fed a commercial diet (Rat Chow, Ralston Purina Co., St. Louis, MO.) ad Zibitum Sodium pentobarbital was injected intraperitoneally into the rats to induce general anesthesia. They were then decapitated, and
579
0003-9861/34 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
580
SOMMERCORN,
STEWARD,
the liver, skeletal muscle (latissimus dorsi), kidneys, and abdominal adipose tissue were removed and placed in cold saline on ice. After they had cooled, the tissues were placed in 4 vol of buffer which comprised 50 mM triethanolamine/HCl (pH 7.5), 250 mrd sucrose, 100 mM NaF, and 1.0 mM EDTA, and they were disrupted, at 4’C, with a Potter-Elvehjem tissue homogenizer. The homogenates were sedimented at 27,OOOgfor 30 min at 4”C, and the resultant supernates were assayed for phosphofructokinase activity as described previously (1, 5). Extracts from the various tissues were diluted with homogenizing buffer to give similar rates of oxidation of NADH. Protein was assayed by the method of Lowry et al. (6) using bovine serum albumin as a standard. Metabolites and coupling enzymes were obtained from Sigma Chemical Company (St. Louis, MO.).
RESULTS
AND
DISCUSSION
6-Phosphogluconate activated phosphofructokinase in extracts of all the tissues we tested. The effect was achieved by decreasing the S0.5for fructose 6-phosphate without affecting the maximum velocity. 6-Phosphogluconate also made the response of phosphofructokinase to fructose 6-phosphate more hyperbolic. These results, which agree with those obtained with phosphofructokinase from liver (1, 2), are shown in Fig. 1 with the enzyme from kidney.4 The activity of phosphofructokinase from liver is a hyperbolic function of the concentration of 6-phosphogluconate (1). This is also true of phosphofructokinase from adipose, kidney, and skeletal muscle. Apparent activation constants are listed in Table I. The ability of 6-phosphogluconate to activate phosphofructokinase is influenced by adenine nucleotides. Increasing concentrations of ATP progressively decrease the magnitude of the activation of phosphofructokinase by 6-phosphogluconate (Fig. 2). In the presence of 2.5 mM ATP, which is a physiological concentration (l), 6phosphogluconate had very little influence on enzyme activity. Although 6-phosphogluconate does not completely reverse the ’ 6-Phosphogluconate also activates phosphofructokinase from brain and heart muscle. We have not examined further the influence of 6-phosphogluconate on phosphofructokinase from these two tissues.
AND
FREEDLAND
4o c KIDNEY ’
PFK ’
’
0.2 [Fru-6-P],mM
’
;-I
0.3
2.0
FIG. 1. Activation of phosphofructokinase by 6phosphogluconate and fructose 2,6-bisphosphate. Phosphofructokinase in an extract of kidney was assayed in the presence of 2.3 mM ATP, 1.3 rnM ADP and 0.33 mM AMP. The units of specific activity are nanomoles minute-’ milligram protein-‘. The symbols refer to assays with 0, no effecters; 0,30 PM 6-phosphogluconate; A, 30 nM fructose 2,6-bisphosphate; and X, both effecters.
inhibition of the enzyme by high concentrations of ATP, it does activate phosphofructokinase assayed in the presence of a mixture of adenine nucleotides which contains a high concentration of ATP (Table I and Ref. (1)). It is likely that this results from the fact that AMP and ADP overcome the inhibition of phosphofructokinase by ATP (7, 8). In Table II, the activation by 6-phosphogluconate of phosphofructokinase from several tissues is shown. The relative effect of 6-phosphogluconate, expressed as the ratio of enzyme activity in the presence of 200 I.LM 6-phosphogluconate divided by the activity without 6-phosphogluconate, was greater on phosphofructokinase from liver and adipose tissue than on the enzyme from kidney or muscle. Whereas 200 PM 6-phosphogluconate, a concentration which provides the maximum activation of phosphofructokinase from liver (1) as well as from the other tissues (data not shown), increased the activity of phosphofructokinase from kidney and muscle only 30-60s) it activated phosphofructokinase from the lipogenic tissues, adipose and liver, 3- and 5.7-fold. Because
REGULATION TABLE
OF PHOSPHOFRUCTOKINASE I
ACTIVATION CONSTANTS FOR 6-PHOSPHOGLUCONATE AND FRUCTOSE 2,6-BISPHOSPHATE OF PHOSPHOFRUCTOKINASE FROM RAT TISSUES”
Tissue Liver Kidney Adipose Muscle
6-Phosphogluconate (PM) 40-60 15-65 10-13 ND*
Fructose 2,6-bisphosphate (nM) 80 21-25 20-28 5-8
“Activation constants are defined as the concentration of etiector which produces one-half of the maximum activation. The values for 6-phosphogluconate were estimated from plots of l/velocity vs l/ [6-phosphogluconate]. The activation constants for fructose 2,gbisphosphate were estimated from plots of velocity vs [fructose 2,6-bisphosphate]. For phosphofructokinase from kidney, adipose, and skeletal muscle, the lower values of the ranges of activation constants were estimated from assays done with mixtures of adenine nucleotides (ATP, ADP, AMP) which are as follows (mM): kidney (2.3, 1.3, 0.33), adipose (4.7, 1.2, 0.95), and muscle (3.2, 1.1, 0.10). The concentraton of adenine nucleotides in kidney and muscle are averages of those reported by Williamson and Brosnan (X3), and those for adipose were reported by Saggerson and Greenbaum (19). The higher values were obtained from assays done with 1.0 mN ATP. Estimates of the activation constant for 6-phosphogluconate of phosphofructokinase from liver were obtained from data in our previous report (1) and from the data in Fig. 2 with 0.1 mN ATP. *Because the effect of 6-phosphogluconate on phosphofructokinase from skeletal muscle is small and not statistically significant (see Table II), we did not calculate an activation constant.
these assays of phosphofructokinase were done with extracts of tissues, the differences in the relative activation of the enzyme from different tissues could he due, in part, to influences of endogenous effectors. The activation of phosphofructokinase from rat liver by 6-phosphogluconate is influenced by fructose 2,6-bisphosphate (l), a potent activator of phosphofructokinase from many sources (9-11). The influence of fructose 2,6-bisphosphate on phospho-
FROM
RAT
581
TISSUES
fructokinase from mammalian tissues has been studied by others with the enzyme from liver or skeletal muscle (9-11). Fructose 2,6-bisphosphate also activates phosphofructokinase from adipose tissue and from kidney (Fig. 3). The response of phosphofructokinase to fructose 2,6-bisphosphate is slightly sigmoidal when the enzyme from either tissue is assayed with 1 mM ATP. In the presence of a mixture of adenine nucleotides, however, the response of the enzyme from adipose tissue is hyperbolic with a lower activation constant (Fig. 3 and Table I). This probably results from the influence of AMP on the activation of phosphofructokinase by fructose 2,gbisphosphate (12). Maximum activation was obtained with 50 nM fructose 2,6-bisphosphate. Activation constants for fructose 2,6-bisphosphate of phosphofructokinase from four tissues are listed in Table I. It is apparent that, based on activation constants, fructose 2,6-bisphosphate is a more potent activator of phosphofructokinase than is 6-phosphogluconate. In fact, fructose 2,6-bisphosphate is the most potent activator of phosphofructokinase that is known (9-11). The potency of fructose 2,6-bisphosphate has led some to suggest
.-f 6.2 t 5a .o 4.: 3:: 2-
0.2
0.4
[ATPI,
0.6
LO
1
mM
FIG. 2. The influence of ATP on the activation of phosphofructokinase by 6-phosphogluconate. Phosphofructokinase in an extract of liver was assayed with various concentrations of ATP and 6-phosphogluconate: 0, none; A, 40 PM; A, 200 pM; and n , 1000 PM. The concentration of fructose 6-phosphate was 100 pi%
SOMMERCORN, TABLE
STEWARD,
II
ACTIVATION OF PHOSPHOFRUCTOKINASE FROM RAT TISSUESBY 6-PHOSPHOGLUCONATEa
Tissue Adipose
6-P-GlcA (PM) 0 200
Liver
0 200
Kidney
0 200
Muscle
0 200
PFK activity (nmol min-’ mg protein-‘) 1.75 + 0.45
Ratio of activities 3.0
5.30 + 0.98 0.23 +- 0.14 1.32 f 0.42
5.7
1.55 + 0.45
1.6
2.50 f 0.45 214 f. 80 362 + 115
1.3
a Phosphofructokinase in extracts of tissues was assayed in the presence of 1.0 mM ATP and either 50 pM (adipose, kidney and skeletal muscle) or 100 gM (liver) fructose g-phosphate. The concentrations of fructose B-phosphate are the approximate values of the S0.5for fructose B-phosphate in the absence of 6phosphogluconate. Values are means + SE. The number of determinations (n) for phosphofructokinase from adipose, kidney and muscle was 8; n = 3 for the enzyme from liver. The effects of 6-phosphogluconate on phosphofructokinase from adipose, liver, and kidney were statistically significant (P < 0.05); the effect on the enzyme from muscle was not. Statistical significance was assessed with Student’s t test (20). The ratio is the activity in the presence of 6-phosphogluconate divided by the activity without B-phosphogluconate.
that it is the most important regulator of the enzyme. However, the extreme potency of fructose 2,6-bisphosphate could argue against its importance in the physiological regulation of phosphofructokinase. This is because the concentration of fructose 2,6bisphosphate in tissues is approximately lOOO-fold higher than the apparent activation constant for fructose 2,6-bisphosphate of phosphofructokinase. For example, the concentration of fructose 2,6-bisphosphate in liver ranges from 2 to 34 nmol/g (13, 14); the activation constant is approximately 80 nM (Table I) and the maximum effect on hepatic phosphofructokinase is obtained with approximately 400 nM fructose 2,6-bisphosphate (15, 16).
AND
FREEDLAND
The concentrations of fructose 2,6-bisphosphate in adipose tissue, kidney, and skeletal muscle are, respectively, 6.0, 8.0, and 2.6 nmol/g tissue (13). These concentrations are all much higher than the activation constants for fructose 2,6-bisphosphate of phosphofructokinase from those tissues (Table I). Despite the discrepancy between its concentration in tissues and its apparent activation constant for phosphofructokinase, there is substantial evidence that fructose 2,&bisphosphate contributes to the physiological regulation of hepatic phosphofructokinase (9-11). It is possible that fructose 2,6-bisphosphate is less potent in vivo than it is in v&o. Uyeda et al. (17) showed that, if hepatic phosphofructokinase is assayed in the presence of physiological concentrations of
iFIG. 3. Activation of phosphofructokinase from adipose tissue and from kidney by fructose 2,6-bisphosphate. Phosphofructokinase in extracts of either adipose tissue or kidney was assayed with various concentrations of fructose 2,6-bisphosphate in the presence of either 1 mM ATP (0) or a mixture of adenine nucleotides (4.7 mM ATP, 1.2 mM ADP, and 0.95 mM AMP) (X). The concentration of fructose 6phosphate was 50 PM.
REGULATION
OF PHOSPHOFRUCTOKINASE
ATP and fructose 6-phosphate, the apparent activation constant for fructose 2,6bisphosphate is 2.5 pM, which is near the range of its concentration in liver. The activation constants for 6-phosphogluconate agree very well with the concentration of 6-phosphogluconate in tissues. We found that its concentration in liver ranged from 10 to 135.4 nmol/g (unpublished work). Phosphofructokinase from liver responds to 6-phosphogluconate in the range of 10 to 200 pM (l), with an activation constant of 40-60 pM (Table I). The concentration of 6-phosphogluconate in other mammalian tissues is also in the micromolar range (18). Thus, changes in the intracellular concentration of 6-phosphogluconate could alter the activity of phosphofructokinase. Activation of phosphofructokinase by 6phosphoglueonate is enhanced by fructose 2,6-bisphosphate when the enzyme is assayed with low concentrations of fructose 6-phosphate. With higher concentrations of fructose 6-phosphate, the effects of 6phosphogluconate and fructose 2,6-bisphosphate are additive (1). We found the same results with the enzyme from kidney (Fig. 1), adipose tissue, and skeletal muscle. The activation is at least additive both when the concentrations of effeetors are near their respective activation constants (Fig. 1) and when they are present at concentrations which give maximal individual effects (1). Thus, it is likely that the two effecters act at different sites on the enzyme. Additional support for this interpretation comes from the fact that fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP (9-11) and that 6-phosphogluconate does not. Regulation of phosphofructokinase by 6phosphogluconate may provide a means of coordinating the disposition of glucose 6phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis. Because these two pathways can contribute substrates for the synthesis of fatty acids, the influence of 6-phosphogluconate on phosphofructokinase may be important during lipogenesis. This hypothesis
FROM
RAT
533
TISSUES
is supported by the fact that the relative activation of phosphofructokinase by 6phosphogluconate is much greater with the enzyme from the lipogenic tissues, liver and adipose, than it is with the enzyme from nonlipogenic tissues. Furthermore, the activation constants for 6-phosphogluconate of phosphofructokinase from liver and adipose correspond with the concentrations of 6-phosphogluconate in tissues. We found that the concentration of 6-phosphogluconate in liver is associated with the lipogenic nature of diets which were fed to rats (unpublished work). 6Phosphogluconate also activates pyruvate kinase (3,4), the second regulated enzyme of glycolysis, and the magnitude of the activation is greater with pyruvate kinase from livers of rats fed a lipogenic diet (3). Thus, 6-phosphogluconate may also contribute to the coordination of activities of phosphofructokinase and pyruvate kinase during lipogenesis. ACKNOWLEDGMENT We thank Mr. Ernest these studies.
Avery
for his assistance
in
REFERENCES 1. SOMMERCORN, J., AND FREEDLAND, Biol Chem 257, 9424-9428. 2. SOMMERCORN,
R. A. (1982)
J., AND FREEDLAND,
R. A.
J,
(1981)
Biochem. Biophys. Res. Cwmmun 99,563-567. 3. SMITH, S. B., AND FREEDLAND, R. A. (1979) J. BioL them 254, 10644-10648. 4. SMITH, S. B., AND FREEDLAND, J. Physiol 240, E279-E285. 5. KAGIMOTO,
R. A. (1981)
K. (1979) J. Bid
T., AND UYEDA,
A-. Chem
254, 5584-5587. 6. LOWRY, 0. H., ROSEBROLJGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem 193,
265-275. 7. PASSONNEAU,
J. V.,
AND
LOWRY,
0.
H.
(1963)
Biochem. Biophys. Res Commun. 13, 372-379. 8. UYEDA, K. (1979) Adv. Ewymol. Ret! Areas Mol. Biol 48, 193-244. 9. HERS,
H.-G.,
Biochem
AND VAN
SCHAFTINGEN,
E. (1982)
J. 206, 1-12.
10. UYEDA, K., FURYUA, E., RICHARDS, C. S., AND YoKOYAMA, M. (1982) Mol. Cell. Biochem. 48, 97120. 11. PILKIS, S. J., EL-MAGHRABI, M. R., MCGRANE, M.,
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STEWARD,
PILKIS, J., Fox, E., AND CLAUS, T. H. (1982) &fol CelL EndocrirwL 25,245-266. 12. VAN SCHAFTINGEN, E., JEIT, M.-F., HUE, L., AND HERS, H.-G. (1981) Proc. NatL Acud &i. USA 78.3483-3486.
AND
17. 18.
13. KUWAJIMA, M., AND UYEDA, K. (1982) Bioch.em Biophgs. Res. Cwrnmun 104, 84-88. 14. NEELY, P., EL-MAGHRABI, M. R., PILKIS, S. J., AND CLAUS, T. H. (1981) Diabdes 30, 1062-1064.
19.
15. VAN SCHAFTINGEN, E., HUE, L., AND HERS, H.-G. (1980) Biochem J. 192,897~901.
20.
16. PILKIS, S. J., EL-MAGHRABI,
M. R., PILKIS, J.,
FREEDLAND
CLAUS, T. H., AND CUMMING, D. A. (1981) J. BioL Chem. 256, 3171-31’74. UYEDA, K., FURUYA, E., AND LUBY, L. J. (1981) J. BioL Chem 256, 8394-8399. WILLIAMSON, D. H., AND BROSNAN, J. T. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd ed., Vol. 4., pp. 2266-2302, Academic Press, New York. SAGGERSON, E. D., AND GREENBAUM, A. L. (1970) B&hem J. 119,193-219. STEEL, R. G. D., AND TORRIE, J. H. (1960) Principles and Procedures of Statistics, pp. 72-75, McGraw-Hill, New York.
Activation of Phosphofructokinase from Rat Tissues by 6-Phosphogluconate and Fructose 2,6-Bisphosphate’ JAMES SOMMERCORN,2 TINA STEWARD, Department
AND
RICHARD A. FREEDLAND
of Physiol&d Sciences, S&ml of Veterinary Medicine, 95616 University of California, Davis, Cal&rnio
Received February
2, 1934, and in revised form April
2, 1934
6-Phosphogluconate activates phosphofructokinase from liver, adipose tissue, kidney, and skeletal muscle by decreasing the apparent S0.5for fructose 6-phosphate without affecting the maximum velocity. The response of phosphofructokinase to 6-phosphogluconate is hyperbolic, with apparent activation constants similar to concentrations of 6-phosphogluconate in tissues. Phosphofructokinase from these tissues is also activated by fructose 2,6-bisphosphate, but the apparent activation constants are much less than the concentrations of fructose 2,6-bisphosphate in tissues. Under most conditions, the effects of 6-phosphogluconate and fructose 2,6-bisphosphate are additive. However, with low concentrations of fructose 6-phosphate there is synergism between the effecters. Whereas fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP, 6-phosphogluconate does not. Thus, the effecters probably act at different sites on the enzyme. The relative effect of 6-phosphogluconate is much greater on phosphofructokinase from the lipogenic tissues, adipose, and liver, than it is on the enzyme from kidney or skeletal muscle. Thus, the influence of 6-phosphogluconate on phosphofructokinase, which could coordinate the disposition of glucose 6phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis, may be important for lipogenesis. 6-Phosphogluconate, an intermediate of the oxidative branch of the hexosemonophosphate pathway, activates both hepatic phosphofructokinase (ATP:D-fructoseSphosphate-1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis (1,2) and pyruvate kinase, the second regulated glycolytic enzyme (3,4). The influence of 6-phosphogluconate on these two enzymes may provide a means of coordinating fluxes through two pathways which contribute substrates for the syni The work was supported by U. S. Public Health Service Grant AM23319. a Present address: Howard Hughes Medical Institute, SL-15 University of Washington, Seattle, Wash. 98195. a To whom correspondence should be addressed.
thesis of fatty acids. To assess the importance to lipogenesis of the influence of 6phosphogluconate on phosphofructokinase, we have studied the effect on the enzyme from adipose, another lipogenic tissue, and on the enzyme from kidney and skeletal muscle, tissues which do not synthesize fatty acids. MATERIALS
AND
METHODS
Male Sprague-Dawley rats were either purchased from Hilltop Lab Animals, Inc. (Scottsdale, Pa.) or they were obtained from a colony which is kept by the Department of Animal Science on campus. The animals ranged in weight from 150 to 540 g, and they were fed a commercial diet (Rat Chow, Ralston Purina Co., St. Louis, MO.) ad Zibitum Sodium pentobarbital was injected intraperitoneally into the rats to induce general anesthesia. They were then decapitated, and
579
0003-9861/34 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
580
SOMMERCORN,
STEWARD,
the liver, skeletal muscle (latissimus dorsi), kidneys, and abdominal adipose tissue were removed and placed in cold saline on ice. After they had cooled, the tissues were placed in 4 vol of buffer which comprised 50 mM triethanolamine/HCl (pH 7.5), 250 mrd sucrose, 100 mM NaF, and 1.0 mM EDTA, and they were disrupted, at 4’C, with a Potter-Elvehjem tissue homogenizer. The homogenates were sedimented at 27,OOOgfor 30 min at 4”C, and the resultant supernates were assayed for phosphofructokinase activity as described previously (1, 5). Extracts from the various tissues were diluted with homogenizing buffer to give similar rates of oxidation of NADH. Protein was assayed by the method of Lowry et al. (6) using bovine serum albumin as a standard. Metabolites and coupling enzymes were obtained from Sigma Chemical Company (St. Louis, MO.).
RESULTS
AND
DISCUSSION
6-Phosphogluconate activated phosphofructokinase in extracts of all the tissues we tested. The effect was achieved by decreasing the S0.5for fructose 6-phosphate without affecting the maximum velocity. 6-Phosphogluconate also made the response of phosphofructokinase to fructose 6-phosphate more hyperbolic. These results, which agree with those obtained with phosphofructokinase from liver (1, 2), are shown in Fig. 1 with the enzyme from kidney.4 The activity of phosphofructokinase from liver is a hyperbolic function of the concentration of 6-phosphogluconate (1). This is also true of phosphofructokinase from adipose, kidney, and skeletal muscle. Apparent activation constants are listed in Table I. The ability of 6-phosphogluconate to activate phosphofructokinase is influenced by adenine nucleotides. Increasing concentrations of ATP progressively decrease the magnitude of the activation of phosphofructokinase by 6-phosphogluconate (Fig. 2). In the presence of 2.5 mM ATP, which is a physiological concentration (l), 6phosphogluconate had very little influence on enzyme activity. Although 6-phosphogluconate does not completely reverse the ’ 6-Phosphogluconate also activates phosphofructokinase from brain and heart muscle. We have not examined further the influence of 6-phosphogluconate on phosphofructokinase from these two tissues.
AND
FREEDLAND
4o c KIDNEY ’
PFK ’
’
0.2 [Fru-6-P],mM
’
;-I
0.3
2.0
FIG. 1. Activation of phosphofructokinase by 6phosphogluconate and fructose 2,6-bisphosphate. Phosphofructokinase in an extract of kidney was assayed in the presence of 2.3 mM ATP, 1.3 rnM ADP and 0.33 mM AMP. The units of specific activity are nanomoles minute-’ milligram protein-‘. The symbols refer to assays with 0, no effecters; 0,30 PM 6-phosphogluconate; A, 30 nM fructose 2,6-bisphosphate; and X, both effecters.
inhibition of the enzyme by high concentrations of ATP, it does activate phosphofructokinase assayed in the presence of a mixture of adenine nucleotides which contains a high concentration of ATP (Table I and Ref. (1)). It is likely that this results from the fact that AMP and ADP overcome the inhibition of phosphofructokinase by ATP (7, 8). In Table II, the activation by 6-phosphogluconate of phosphofructokinase from several tissues is shown. The relative effect of 6-phosphogluconate, expressed as the ratio of enzyme activity in the presence of 200 I.LM 6-phosphogluconate divided by the activity without 6-phosphogluconate, was greater on phosphofructokinase from liver and adipose tissue than on the enzyme from kidney or muscle. Whereas 200 PM 6-phosphogluconate, a concentration which provides the maximum activation of phosphofructokinase from liver (1) as well as from the other tissues (data not shown), increased the activity of phosphofructokinase from kidney and muscle only 30-60s) it activated phosphofructokinase from the lipogenic tissues, adipose and liver, 3- and 5.7-fold. Because
REGULATION TABLE
OF PHOSPHOFRUCTOKINASE I
ACTIVATION CONSTANTS FOR 6-PHOSPHOGLUCONATE AND FRUCTOSE 2,6-BISPHOSPHATE OF PHOSPHOFRUCTOKINASE FROM RAT TISSUES”
Tissue Liver Kidney Adipose Muscle
6-Phosphogluconate (PM) 40-60 15-65 10-13 ND*
Fructose 2,6-bisphosphate (nM) 80 21-25 20-28 5-8
“Activation constants are defined as the concentration of etiector which produces one-half of the maximum activation. The values for 6-phosphogluconate were estimated from plots of l/velocity vs l/ [6-phosphogluconate]. The activation constants for fructose 2,gbisphosphate were estimated from plots of velocity vs [fructose 2,6-bisphosphate]. For phosphofructokinase from kidney, adipose, and skeletal muscle, the lower values of the ranges of activation constants were estimated from assays done with mixtures of adenine nucleotides (ATP, ADP, AMP) which are as follows (mM): kidney (2.3, 1.3, 0.33), adipose (4.7, 1.2, 0.95), and muscle (3.2, 1.1, 0.10). The concentraton of adenine nucleotides in kidney and muscle are averages of those reported by Williamson and Brosnan (X3), and those for adipose were reported by Saggerson and Greenbaum (19). The higher values were obtained from assays done with 1.0 mN ATP. Estimates of the activation constant for 6-phosphogluconate of phosphofructokinase from liver were obtained from data in our previous report (1) and from the data in Fig. 2 with 0.1 mN ATP. *Because the effect of 6-phosphogluconate on phosphofructokinase from skeletal muscle is small and not statistically significant (see Table II), we did not calculate an activation constant.
these assays of phosphofructokinase were done with extracts of tissues, the differences in the relative activation of the enzyme from different tissues could he due, in part, to influences of endogenous effectors. The activation of phosphofructokinase from rat liver by 6-phosphogluconate is influenced by fructose 2,6-bisphosphate (l), a potent activator of phosphofructokinase from many sources (9-11). The influence of fructose 2,6-bisphosphate on phospho-
FROM
RAT
581
TISSUES
fructokinase from mammalian tissues has been studied by others with the enzyme from liver or skeletal muscle (9-11). Fructose 2,6-bisphosphate also activates phosphofructokinase from adipose tissue and from kidney (Fig. 3). The response of phosphofructokinase to fructose 2,6-bisphosphate is slightly sigmoidal when the enzyme from either tissue is assayed with 1 mM ATP. In the presence of a mixture of adenine nucleotides, however, the response of the enzyme from adipose tissue is hyperbolic with a lower activation constant (Fig. 3 and Table I). This probably results from the influence of AMP on the activation of phosphofructokinase by fructose 2,gbisphosphate (12). Maximum activation was obtained with 50 nM fructose 2,6-bisphosphate. Activation constants for fructose 2,6-bisphosphate of phosphofructokinase from four tissues are listed in Table I. It is apparent that, based on activation constants, fructose 2,6-bisphosphate is a more potent activator of phosphofructokinase than is 6-phosphogluconate. In fact, fructose 2,6-bisphosphate is the most potent activator of phosphofructokinase that is known (9-11). The potency of fructose 2,6-bisphosphate has led some to suggest
.-f 6.2 t 5a .o 4.: 3:: 2-
0.2
0.4
[ATPI,
0.6
LO
1
mM
FIG. 2. The influence of ATP on the activation of phosphofructokinase by 6-phosphogluconate. Phosphofructokinase in an extract of liver was assayed with various concentrations of ATP and 6-phosphogluconate: 0, none; A, 40 PM; A, 200 pM; and n , 1000 PM. The concentration of fructose 6-phosphate was 100 pi%
SOMMERCORN, TABLE
STEWARD,
II
ACTIVATION OF PHOSPHOFRUCTOKINASE FROM RAT TISSUESBY 6-PHOSPHOGLUCONATEa
Tissue Adipose
6-P-GlcA (PM) 0 200
Liver
0 200
Kidney
0 200
Muscle
0 200
PFK activity (nmol min-’ mg protein-‘) 1.75 + 0.45
Ratio of activities 3.0
5.30 + 0.98 0.23 +- 0.14 1.32 f 0.42
5.7
1.55 + 0.45
1.6
2.50 f 0.45 214 f. 80 362 + 115
1.3
a Phosphofructokinase in extracts of tissues was assayed in the presence of 1.0 mM ATP and either 50 pM (adipose, kidney and skeletal muscle) or 100 gM (liver) fructose g-phosphate. The concentrations of fructose B-phosphate are the approximate values of the S0.5for fructose B-phosphate in the absence of 6phosphogluconate. Values are means + SE. The number of determinations (n) for phosphofructokinase from adipose, kidney and muscle was 8; n = 3 for the enzyme from liver. The effects of 6-phosphogluconate on phosphofructokinase from adipose, liver, and kidney were statistically significant (P < 0.05); the effect on the enzyme from muscle was not. Statistical significance was assessed with Student’s t test (20). The ratio is the activity in the presence of 6-phosphogluconate divided by the activity without B-phosphogluconate.
that it is the most important regulator of the enzyme. However, the extreme potency of fructose 2,6-bisphosphate could argue against its importance in the physiological regulation of phosphofructokinase. This is because the concentration of fructose 2,6bisphosphate in tissues is approximately lOOO-fold higher than the apparent activation constant for fructose 2,6-bisphosphate of phosphofructokinase. For example, the concentration of fructose 2,6-bisphosphate in liver ranges from 2 to 34 nmol/g (13, 14); the activation constant is approximately 80 nM (Table I) and the maximum effect on hepatic phosphofructokinase is obtained with approximately 400 nM fructose 2,6-bisphosphate (15, 16).
AND
FREEDLAND
The concentrations of fructose 2,6-bisphosphate in adipose tissue, kidney, and skeletal muscle are, respectively, 6.0, 8.0, and 2.6 nmol/g tissue (13). These concentrations are all much higher than the activation constants for fructose 2,6-bisphosphate of phosphofructokinase from those tissues (Table I). Despite the discrepancy between its concentration in tissues and its apparent activation constant for phosphofructokinase, there is substantial evidence that fructose 2,&bisphosphate contributes to the physiological regulation of hepatic phosphofructokinase (9-11). It is possible that fructose 2,6-bisphosphate is less potent in vivo than it is in v&o. Uyeda et al. (17) showed that, if hepatic phosphofructokinase is assayed in the presence of physiological concentrations of
iFIG. 3. Activation of phosphofructokinase from adipose tissue and from kidney by fructose 2,6-bisphosphate. Phosphofructokinase in extracts of either adipose tissue or kidney was assayed with various concentrations of fructose 2,6-bisphosphate in the presence of either 1 mM ATP (0) or a mixture of adenine nucleotides (4.7 mM ATP, 1.2 mM ADP, and 0.95 mM AMP) (X). The concentration of fructose 6phosphate was 50 PM.
REGULATION
OF PHOSPHOFRUCTOKINASE
ATP and fructose 6-phosphate, the apparent activation constant for fructose 2,6bisphosphate is 2.5 pM, which is near the range of its concentration in liver. The activation constants for 6-phosphogluconate agree very well with the concentration of 6-phosphogluconate in tissues. We found that its concentration in liver ranged from 10 to 135.4 nmol/g (unpublished work). Phosphofructokinase from liver responds to 6-phosphogluconate in the range of 10 to 200 pM (l), with an activation constant of 40-60 pM (Table I). The concentration of 6-phosphogluconate in other mammalian tissues is also in the micromolar range (18). Thus, changes in the intracellular concentration of 6-phosphogluconate could alter the activity of phosphofructokinase. Activation of phosphofructokinase by 6phosphoglueonate is enhanced by fructose 2,6-bisphosphate when the enzyme is assayed with low concentrations of fructose 6-phosphate. With higher concentrations of fructose 6-phosphate, the effects of 6phosphogluconate and fructose 2,6-bisphosphate are additive (1). We found the same results with the enzyme from kidney (Fig. 1), adipose tissue, and skeletal muscle. The activation is at least additive both when the concentrations of effeetors are near their respective activation constants (Fig. 1) and when they are present at concentrations which give maximal individual effects (1). Thus, it is likely that the two effecters act at different sites on the enzyme. Additional support for this interpretation comes from the fact that fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP (9-11) and that 6-phosphogluconate does not. Regulation of phosphofructokinase by 6phosphogluconate may provide a means of coordinating the disposition of glucose 6phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis. Because these two pathways can contribute substrates for the synthesis of fatty acids, the influence of 6-phosphogluconate on phosphofructokinase may be important during lipogenesis. This hypothesis
FROM
RAT
533
TISSUES
is supported by the fact that the relative activation of phosphofructokinase by 6phosphogluconate is much greater with the enzyme from the lipogenic tissues, liver and adipose, than it is with the enzyme from nonlipogenic tissues. Furthermore, the activation constants for 6-phosphogluconate of phosphofructokinase from liver and adipose correspond with the concentrations of 6-phosphogluconate in tissues. We found that the concentration of 6-phosphogluconate in liver is associated with the lipogenic nature of diets which were fed to rats (unpublished work). 6Phosphogluconate also activates pyruvate kinase (3,4), the second regulated enzyme of glycolysis, and the magnitude of the activation is greater with pyruvate kinase from livers of rats fed a lipogenic diet (3). Thus, 6-phosphogluconate may also contribute to the coordination of activities of phosphofructokinase and pyruvate kinase during lipogenesis. ACKNOWLEDGMENT We thank Mr. Ernest these studies.
Avery
for his assistance
in
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