The Effect of Natural and Synthetic D-Fructose 2,6-Bisphosphate on the Regulatory Kinetic Properties of Liver and Muscle Phosphofructokinases*

Transcription

1 THE JOURNAL OF k%iologlcal CHEMISTRY Vol. 256, No. 16. Issue of August 25. pp Printed in U.S.A. The Effect of Natural and Synthetic D-Fructose 2,6-Bisphosphate on the Regulatory Kinetic Properties of Liver and Muscle Phosphofructokinases* Kosaku Uyeda, Eisuke Furuya, and Lynne (Received for publication, February 23, 1981, and in revised form, May 8, 1981) J. Luby From the Pre-Clinical Science Unit, Veterans Administration Medical Center, and Department of Biochemistry, Uniuersity of Texas Health Science Center at Dallas, Dallas, Texas The effect of natural activation factor and synthetic fructose-2,6-pz on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-pz show identical effects on the allosteric kinetic proper- ties of both enzymes. Auctose-2,6-Pz counteracts inhibition byatpand citrate and decreases the K,,, for fructose-6-p. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The K,,, values of liver andmusclephosphofructokinasefor purified rat liver enzyme under near physiological conditions. The results of their study indicate that the activity of isolated fructose-2,6-pz in the presence of 1.26 IMI ATP are 12 phosphofructokinase is insufficient to account for the necesmilliunits/ml (or 24 lu~r)and 5 milliunits/ml (or 10 m), respectively. At near physiological concentrations of sary cellular activity due to its high K,,, for fructose-6-p (6 ATP (3111~) and fructose-6-p (0.2 m), however, the K,,, mm) even in the presence of known positive effectors of the values for fructose-2,6-pz are increased to 12 PM and 0.8 enzyme including AMP, fructose-1,6-p2, and Pi. However, we pt for liver and muscle enzymes, respectively. Thus, have shown more recently that activation factor in less than fructose-2,6-pz is the most potent activator of the en- physiological concentrations can activate the enzyme suffizyme compared to other known activators such as fruc- ciently to account for the enzyme activity in vivo (1). tose-1,6-pz. The rates of the reaction catalyzed by the In this report, we present a detailed study of the effect of enzymes under the above conditions are nonlinear: the D-fNCtOSe-2,6-P2, both synthetic and natural, on the allosteric rates decelerate in the absence or in the presence of kinetics of liver and muscle phosphofructokinase. lower concentrations of fructose-2,6-pz, but the rates become linear in the presence of higher concentrations MATERIALS AND METHODS of fructose-2,6-pz. Fructose-2,6-Pz also protects phos- phofructokinase against inactivation by heat. Fructose- 2,6-Pz, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues. Recently, we have isolated from rat liver an activator of phosphofructokinase termed activation factor which is extremely effective in releasing the ATP inhibition of the liver enzymes (1). We have shown that glucagon administration results in rapid decrease in the level of this factor while glucose stimulates its synthesis (2). Moreover, phosphorylated phosphofructokinase shows a weaker affinity for the factor than the dephospho enzymes (3). Thus, this factor appears to play an important role in regulation of phosphofructokinase. During their study of the fructose-6-p/fructose-1,6-p~ cycle using hepatocytes, van Schaftingen and Hers (4) isolated a similar factor. Based on the observation that it is acid-labile and that the acid hydrolysis products were fructose-6-p and Pi, they tentatively suggested that the product is fructose-2,6- Pz (5). We have synthesized the activation factor in pure form * This work was supported by the Veterans Administration and by United States Public Health Service grant AM The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact by alkaline hydrolysis of D-fructose-1,2-cyclic 6-Pz (6). Chemical characterization, chromatographic analyses, and NMR studies led us to characterize the factor as P-D-fructose-2,6-Pz (6). The kinetic properties of liver phosphofructokinase with varying degrees of punty from various sources (7-14, see also reviews by Uyeda (15)) have been investigated. Recently, Reinhart and Lardy (16) examined the allosteric properties of Activation factor was isolated from rat liver (6) and ~-hctose-2,6- PZ was synthesized from D-fructose-12-cyclic 6-Pz (6). The solution of D-fructose-2,6-P~ was usually made in 0.01 N NaOH. All other chemicals were reagent grade and obtained from commercial sources. Muscle phosphofructokinase was prepared from rabbit muscle as before (17). Rat liver phosphofructokinase was partially purified as described previously (l), with the following modification. The crude extract of rat liver after heating and ammonium sulfate fractionation was precipitated with a polyethylene glycol concentration between 4.5% to 5.6% and the precipitate was chromatographed on a Sepharose 4B column. The specific activity of the enzyme after gel filtration was approximately 50 units/mg and was completely free of bound activation factor. The assay for the activation factor and synthetic fructose-2,6-p* using rabbit muscle phosphofructokinase has been modified slightly from the previously published method (Z), as follows. The reaction mixture contained in a find volume of 1 ml: 50 mm 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (ph 7.4),0.2 mm EDTA, 5 mm MgClp, 1 mm NHd.3, 0.16 mm NADH, 2.5 mm dithiothreitol, 1 mm fructose- 6-P, desalted aldolase (0.4 unit), triose-p-isomerase (2.4 units, and a- glycero-p dehydrogenase (0.4 unit). The activation factor was added to the reaction mixture, followed by addition of 15 milliunits of purified muscle phosphofructokinase, and after 2 min, the reaction was initiated by the addition of 1.25 mm ATP. One unit of activation factor is defined as the amount of the factor which increases phosphofructokinase activity by 1 unit under these conditions. There is no phosphofructokinase activity in the absence of activation factor under these assay conditions. The concentration of fructose-2,6-pz was determined after acid treatment under which fructose-2,6-p2 was converted quantitatively to fructose-6-p (6), and fructose-6-p was determined enzymatically as described previously (6). The specific activity of the natural and

2 Effect of Fructose-L?,6-Pz on Phosphofructokinase 8395 synthetic fructose-2,6-p* varied from 480 units/pmol to 540 units/ pmol depending upon preparation. It is more convenient to determine the concentration in units of activity rather than micromoles because the latter assay method requires relatively large amounts of the sample. Allosteric kinetic properties of phosphofructokinase were determined in a reaction mixture containing 50 mm 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid (ph 7.4), 0.2 mm EDTA, 5 mm MgC12, 1 mm NH4Cl, 0.16 mm NADH, 2.5 mm dithiothreitol, 1 mm fructose- 6-P, desalted aldolase (0.4 unit), triose-p-isomerase (2.4 units), and a-glycero-p dehydrogenase (0.4 unit) in a final volume of 1 ml. The activation factor was added to the reaction mixture followed by addition of 15 milliunits of phosphofructokinase, and after 2 min, the reaction was initiated by the addition of indicated amounts of ATP. Assays were performed at 25 "C at a fiied concentration of 1.25 mm ATP and 1 mm fructose-6-p unless otherwise stated. The activity is expressed as v/vmar where u is the activity under the above assay conditions and VmaX is the optimum activity determined at ph 8 (1). RESULTS Activation by D -Fructose-2,6-P2 Rates of the Reaction-Assays for allosteric kinetics of phosphofructokinase demonstrate complex hysteretic behavior and rates of the reaction are usually nonlinear. Reinhart and Lardy (16) have analyzed the property extensively using rat liver phosphofructokinase, and our results obtained in the absence of fructose-2,6-p2 confim their observation. The effect of fructose-2,6-p2 on the rate of the reaction catalyzed by muscle and liver phosphofructokinases are shown in Fig. 1. The muscle enzyme shows nonlinear decelerating rates of reaction in the absence or in the presence of less than 2.6 milliunits of fructose-2,6-pz. Above 4.4 milliunits of the factor the rate becomes linear. Similar deceleration of the reaction rates is seen with liver enzyme, but the activation by fructose-2,6-p2 requires much higher concentration than that for muscle enzymes; even in the presence of 87 milliunits of the factor, the reaction is nonlinear. Under these conditions, the reactions initiated with additions of ATP show generally a high rate of initial reaction but decelerated after 1 to 2 min. When the reaction in the absence of fructose-2,6-p2 was initiated with addition of the enzyme with subsequent addition of the factor, a long lag period which lasts more than 6 to 10 rnin resulted (data not shown). If the factor was added within 30 s after the initiation of the reaction, however, the lag period was less than 2 min. After the lag period under these conditions, the reaction approaches a comparable rate to that in which the factor was present prior to initiation of the reaction. Effect of Different Concentrations of Fructose-2,6-P2 The effect of varying concentrations of natural and synthetic fructose-2,6-p2 at 1.25 mm ATP on liver and muscle phosphofructokinase is shown in Fig. 2. Neither muscle nor liver phosphofructokinase shows activity in the absence of - V i I I I I I I O. I t j $ f,,,,, , ACTIVATION FACTQR (munltr) I FRUCTOSE-2.6-?2 (pm) FIG. 2. Influence of varying natural activation factor or synthetic fructose-2,6-pz on the activities of muscle and liver phosphofructokinase (PFK). 0 and A, natural activation factor; 0 and A, synthetic fructose-2,6-p2. The experimental conditions are described under "Materials and Methods." I " " " " " I h o.2 t I - LlVtM O' i 01 J TIME (min) FIG. 1. Typical tracings of the phosphofructokinase (PFK) reaction in the presence and absence of fructose-2,6-pz. The reaction mixture contained all the components as described under O.' t "Materials and Methods" and indicated amounts of fructose-2,6-p2. At zero time the reaction was initiated with addition of 1.25 mm ATP. 1

3 8396 Effect of Fructose-2,6-P2 on Phosphofructokinase fructose-2,6-p~ under the experimental conditions employed. Both synthetic and natural fructose-2,6-p2 show nearly identical saturation curves. The K0.s values for muscle and liver enzymes, however, are different; these values are 12 milliunits (or PM) and 5 milliunits/ml (or 0.01 p ~ for ) liver and muscle phosphofructokinase, respectively. These differences in the Ko.~ for fructose-2,6-p~ between these enzymes appear to be due to their differences in the sensitivity to ATP inhibition, as discussed below. Van Schaftingen et al. (4) have reported that a half-maximum activation of crude liver enzyme was obtained with o O FRUCTOSE-2,6-PZ (ym) FIG. 3. Influence of varying fructose-2,6-p~ on the activities of muscle and liver phosphofructokinase (PFK), under physiological conditions. The experimental conditions were same as in Fig. 2 except ATP = 3 mm, and fructose-6-p = 0.1 mm. ATP (mm) p~ fructose-2,6-p~ in the presence of1.5 mm ATP. These results suggest that fructose-2,6-p~ is the most potent activator of the enzyme. Fructose-l,6-P~ and glucose-1,6-p~ are also strong activators. Under the same conditions, the for glucose-1,6-p2 is 5 PM for the liver enzyme indicating that K,,, for fructose-2,6-pz is 50-fold lower than that for glucose-1,6- PP. The above experiments were performed at 1.25 mm ATP and 1 mm fructose-6-p which is not within physiological conditions. The saturation curves for fructose-2,6-pz under near physiological concentrations of ATP (3 mm) and fructose- 6-P (0.1 mm) were determined, and the results (Fig. 3) show that Ko.5 values for fructose-2,6-p~ are increased to 0.6 p~ and 2.5 PM for muscle and liver phosphofructokinase, respectively. The curves also indicate that the liver enzyme shows a high degree of positive cooperativity for fructose-2,6-p2 while only a slight cooperativity is observed with the muscle phosphofructokinase. The Hill coefficients calculated from these re- sults are 4.5 and 1.4 for liver and muscle phosphofructokinases, respectively. Release of ATP and Citrate Inhibition by Fructose-2,6-P~ The effect of the natural and synthetic fructose-2,6-p~ on the ATP inhibition of muscle and liver phosphofructokinase is shown in Fig. 4. The ATP inhibition of the enzymes from both sources is greatly released by 13 milliunits of fructose- 2,6-P~. Both the synthetic and the natural fructose-2,6-p~ are equally effective. Although the liver enzyme is more sensitive to the inhibition than the muscle enzyme as shown previously (IO), the degree of activation of both enzymes by the factor is similar. Furthermore, the maximum u/vmaa obtained in the presence of the factor is also comparable, 0.63 and 0.7 at 0.3 mm and 0.7 mm ATP for muscle and liver phosphofructokinase, respectively. Citrate inhibition of the liver enzyme is also relieved with increasing concentrations of natural fructose-2,6-p2 (Fig. 5). Similar results were obtained with synthetic fructose-2,6-p2 and also with muscle phosphofructokinase (data not shown). Effect of Ko.~ for Fructose-6-P The influence on the activity of muscle and liver phosphofructokinases at varying concentrations of fructose-6-p in the presence of several fixed concentrations of fructose-2,6-p* is shown in Fig. 6. In the absence of the activator, the KO, of the liver enzyme for fructose-6-p is 6 mm. The addition of increasing concentrations of fructose-2,6-pz: 2,4,16, and 35 milliunits/ml decrease the Ko.~ values to 3.5 PM, 2.1 mm, 1 mm and 0.6 mm, respectively, which corresponds to a 10-fold decrease in the in the presence of 0.07 pm (35 mnits/d) Fru-2,6-Pz ATP (mm) FIG. 4. Effect of activation factor and fructose-2,6-p~ on ATP inhibition of liver and muscle phosphofructokinase (PFK). 0, no factor; 0, fructose-2,6-p2; A, activation factor (Am. CITRATE (CY! FIG. 5. Effect of natural activation factor on citrate inhibition of liver phosphofructokinase. The reaction mixture was same as in Fig. 4 excedt ATP = 0.25 mm. Varving concentrations of citrate and indicated amounts of activation fa&-(af) were added.

4 Effect of Fructose-2,6-Pz on Phosphofructokinase 8397 compared to that in the absence. The degree of cooperativity (Table I) as measured by the HiU coefficient, is also reduced from 4.5 in the absence to in the presence of 2 to 35 milliunits/ml of fructose-2,6-pz. A simiiar decrease in the K0.5 for fructose-6-p is observed with muscle enzyme. In the absence of the activator, KO, for the substrate is 1.4 mm but the presence of 1.1 mdliunits/mi and 3.2 milliunits/ml of the factor decreases the K0.5 to 0.8 mm and 0.3 mm, respectively. The Hill coefficient of muscle phosphofructokinase is also decreased from 3.1 to 2.0 by 3.2 milliunits/ml of fructose-2,6-pz (Table I). Synergism of the Activation by AMP and Fructose-2,6-P2 AMP is known to be an effective activator of all mammalian phosphofructokinases. The effect of fructose-2,6.-p2 on the AMP activation of phosphofructokinases is shown in Fig. 7. Under these conditions the K0.5 of liver phosphofructokinase for AMP in the absence of fructose-2,6-pz appears to be at " - "mor LIVER PFK roo 250 AMP (pm1 - AF ImumW V - "mox I ' 0.5. AF (munits) 'I O AMP 60 OJMI FIG. 7. Effect of fructose-2,6-p~ and varying AMP on the activation of liver and muscle phosphofructokinase (PFK). The reaction mixture was the same as in Fig. 1 except that varying concentrations of AMP and indicated amounts of fructose-2,6-p2 (AF) were added o FRUCTOSE-6-P (mm FRUCTOSE-6-P (mm) FIG. 6. Influence of fructose-2,6-pz on the activities of liver and muscle phosphofructokinases (PFK) with varying fructose-6-p. The reaction was carried out as in Fig. 4 except that the concentration of fructose-6-p was varied at a fixed concentration of fructose-2,6-p* (AF). TABLE I Hill coefficients for fructose-6-p The Hill coefficients were calculated from the data in Fig Phosphofructokinase Fructose-2,6-P2 Hill coefficient milliunits nm Muscle Liver least 150 PM. The presence of increasing Concentrations of fructose-2,6-p2 (up to 8 miuiunits/ml, VM) greatly decreases the K0.5 to less than 10 PM. The presence of16 milliunits/nd (0.032 PM) of fructose-2,6-p2 alone activates the enzyme to 80% of the maximum with only a small additional activation by AMP (0). These results clearly demonstrate the striking synergistic activation of liver phosphofructokinase by both fructose-2,6-p* and AMP. A similar but less striking activation of muscle enzyme by these effectors is shown in the lower figure. At low concentrations of fructose-2,6-p2 (less than 2 milliunits/ml), the synergism with AMP is observed but no effect of AMP is detectable at 4 and 8 milliunits/ml of fructose-2,6-p2 with this enzyme. Activation by Fructose-2,6-P2 and AMP under Physiological Conditions It is of interest to examine the influence of a combination of these effectors on the for fructose-6-p under physiological conditions. The effect on the liver enzyme of three different concentrations of fructose-2,6-p~ and 1 PM AMP as a function of fructose-6-p concentration at 3 mm ATP is shown in Fig. 8. In the presence of 1 PM AMP and in the absence of fructose- 2,6-P2 (A), the enzyme shows the KO., for fructose-6-p of above 6 m (data not shown) (16). The however, is decreased to 0.14 mm in the presence of 2 units of fructose-2,6-pz alone (A), which is close to the physiological concentration of this factor in rat liver (1, 2). The value for the K0.5 for fructose-6- P is further decreased to 0.06 mm and 0.04 mm in the presence of both AMP (1 PM) and fructose-2,6-p2 (2 and 3 units), respectively. Thus, these results demonstrate that fructose- 2,6-P2 and AMP at physiological concentrations are able to reduce the X, for fructose-6-p (10 to 100 p ~ ) a, concentration which is in the range of that found in liver (18, 19).

5 8398 Effect of Fructose-2,6-P2 Phosphofructokinase on - V "max 08- F-2,6-c+AMP(lpM) "A r, r 04- /ltyf-2,6-% (2 units) FRUCTOSE-6-P (mm) FIG. 8. Influence of fructose-2,6-pz and AMP and varying concentrations of fructose-6-p on liver phosphofructokinase. A, fructose-2,6-p~ (F-2,6-&) (2 units), A, AMP (1 ELM); 0, fructose-2,6- Pp (1 unit) + AMP (1 ELM); 0, fructose-2,6-p2 (2 units) + AMP (1 PM); 0, fructose-2,6-p2 (3 units) + AMP (1 ELM). ATP was at 3 mm in all the reaction mixtures. FIG. 9. Heat inactivation of liver phosphofructokinase. Liver phosphofructokinase (6 units/ml) in 50 mm Tris-P,, ph 8, containing the indicated Concentrations of fructose-2,6-p2 (AF) was incubated at 50 "C. Aliquots were removed at given timed intervals and assayed for phosphofructokinase activity. Protection against Heat Inactivation by Fructose-2,6-P2 Liver phosphofructokinase is inactivated rapidly at 50 "C (Fig. 9) approximately 50% of the enzyme is denatured within 3 min. Addition of10 milliunits/ml of the factor, however, results in a partial protection against this heat inactivation, and full protection is achieved using 31 milliunits/ml of fructose-2,6-p~. DISCUSSION Kinetic and equilibrium binding studies have shown that fructose-1,6-p2 is a potent activator of muscle phosphofructokinase among the known positive effectors including AMP and Pi. Hill and Hammes (20) using the.muscle enzyme have obtained a binding constant for fructose-1,6-p2 of approximately 4 to 28 ~ Lat M ph 7 in the presence of varying phosphate concentration. Hood and Hollaway (21) showed that 5 p~ fructose-1,6-p2 is sufficient to eliminate a lag period exhibited by ATP-inhibited phosphofructokinase. Fructose-2,6-P2, however, is a more potent activator of the enzyme than fructosel,6-p2 or glucose-1,6-pz. Although the K0.5 for this activator varies with ATP concentration, at 1.25 mm ATP, the values for muscle and liver phosphofructokinase are 10 nm and 25 nm respectively (Fig. 2), which is at least 100 times lower than the binding constant for fructose-1,6-p2. Van Schaftingen et QZ. (4) also obtained similar value (0.1 p ~ with ) crude liver enzyme under their assay conditions. Even at physiological concentrations of ATP (3 mm) and fructose-6-p (0.1 mm), the values for muscle and liver enzymes are 0.6 p~ and 2.5 p ~ respectively, (Fig. 3). Under similar conditions, fructose- 1,6-P2 (50 p ~ failed ) to show any activation. Thus, fructose- 2,6-P2 is the most potent activator of the enzyme thus far discovered. The results reported here suggest that the action of fructose-2,6-pz on phosphofructokinase is similar to that of fructose-1,6-p2 or glucose-1,6-pz. Fructose-2,6-P* activates the enzyme activity by (a) counter-acting inhibition by ATP and citrate, (b) decreasing the Ko.~ for fructose-6-p at least 10 times, (e) acting synergistically with AMP by lowering the for AMP, (d) lowering the for Pi (data not shown). Moreover, we found that at a relatively low concentration of fructose-2,6-p~, no additional activation was observed when the enzyme was already activated by glucose-1,6-p2. The effect of fructose-2,6-pz on the rates of the reaction under ATPinhibited conditions also provides additional supportive evidence. A lag period was observed when the reaction was initiated with the enzyme in the absence of fructose-2,6-p2 but the lag can be eliminated by the addition of fructose-2,6-p2. This result is similar to the effect of fructose-1,6-p~ on the enzyme observed by others (21). The observed effect of fructose-2,6-p2 on the rate of the reaction (Fig. 1) also provides additional information about the mechanism of its action. The reaction rate becomes con- stant or linear in the presence of fructose-2,6-p2 while it decreases in its absence. Such a nonlinear transient kinetics of liver phosphofructokinase have been observed by Ramaiah and Tejwani (8) and Reinhart and Lardy (16). The exact mechanism of the transient kinetics is not known but could be interpreted in terms of the allosterism model of Monod et al. (22). The enzyme could exist in two conformational states, a fructose-6-p-induced state and an ATP-inhibited state, with slow interconversion between these two states producing the transients. Fructose-2,6-Pz effectively binds to the active state and protects the enzyme against ATP-induced conformational changes to the inhibited state. Other explanations, however, are also possible, and the elucidation of the exact mechanism of fructose-2,6-p2 action requires additional experiments. Muscle phosphofructokinase also shows similar transient kinetics, but less dramatically than the liver enzyme (Fig. 1). Nonlinear kinetics were observed with the muscle enzyme only in the presence of less than 2.6 milliunits of fructose-2,6-ps, above this concentration the enzyme shows linear rates. Regulatory kinetic properties of phosphofructokinase have been extensively investigated because the enzyme catalyzes a key step in glycolysis. The investigation has revealed a multitude of metabolites which modulate the activity of the enzyme (see recent review in Ref. 15). These metabolites include ATP, citrate, and triose phosphates as inhibitors; and fructose-6-p, fructose-1,6-p2, AMP, ADP, and Pi as activators. Attempts to rationalize the regulation of phosphofructokinase in vivo based on the in vitro results, however, have not been satisfactory. One difficulty is that the concentrations of these metabolites do not change significantly, especially in liver. For example, the ATP and citrate concentrations in liver usually remain constant at 3 pmol/g and 0.3 pmol/g, respectively, under various nutritional states (19, 23). Similarly the amounts of the activators, such as AMP and fructose-1,6-p~, do not fluctuate. Another problem with the metabolite control

6 Effect of Fructose-2,6-P~ on Phosphofructokinase 8399 mechanism is the observation that the K, for fructose-6-p is 6 mm (16, Fig. 5) while its concentration in vivo is less than 0.1 mm (24). Similarly, the AMP concentration in cytoplasm has been estimated to be 1 pm, while that of fructose-1,6-p2 is 10 to 25 PM (25). Although it is true that AMP, fructose-l,6- P2, and Pi act synergistically in activating phosphofructokinase, the amounts of these effectors are too low to activate the enzyme sufficiently. Reinhart and Lardy (26) also recognized this difficulty and sought for an alternative mechanism. They argued that the enzyme concentration in uiuo (20 to 50 pg/ml) is considerably higher than that usually used for enzyme assays (lzss than 1 pg/ml). Thus, the enzyme may exist as a large aggregate in uiuo, and the large polymers may show a lower K, for fructose-6-p. Since the K,,, for fructose-6- P of the large polymers has not been determined, it is difficult to decide whether the K,,, is sufficiently decreased (from 6 to 0.1 mm) to account for the enzyme s activity in uiuo. Thus, these proposed mechanisms involving regulation by wellknown metabolites or the aggregation states of phosphofructokinase are not convincing and may not apply to the in uiuo conditions. One alternative mechanism is activation by fructose-2,6-p2. We feel that this factor may be the most important allosteric activator of phosphofructokinase in liver for the following reasons: (a) its concentration changes rapidly under glucagon stimulation or glucose administration (2); (b) it has a very high affinity for the enzyme, at least 1 order of magnitude higher than fructose-1,6-p2 under physiological conditions; (c) it acts synergistically with AMP and Pi in counteracting ATP and citrate inhibition; and (d) it decreases the KO s for fructose-6-p to a more physiological concentration of this substrate in the presence of in vivo levels of ATP, AMP, and fructose-2,6-p2 (Fig. 8). Thus, all these observations indicate the importance of fructose-2,6-p* in regulation of phosphofructokinase in liver. The importance of fructose-2,6-p2 may not be limited to the liver, since as shown in this report, muscle phosphofructokinase is also activated by this compound. Moreover, the presence of fructose-2,6-p2 is not confined to the liver, but occurs in all other tissues we have examined thus far. Thus, fructose-2,6-p2 may play an important role in the regulation of phosphofructokinase in all tissues. Fructose-2,6-P2 also seems to play an important role in hormonal regulation of phosphofructokinase in liver. We have shown that the concentration of fructose-2,6-p2 decreases within 2 min after glucagon administration to hepatocytes and increases rapidly with glucose stimulation (2). In addition to reducing the fructose-2,6-p2 level, glucagon also stimulates the phosphorylation of phosphofructokinase (27), and the phosphorylated enzyme shows a decreased affinity for fructose-2,6-p2 (3). The combination of these two effects induced by glucagon, namely the decreased amount of fructose-2,6-p~ and the reduced affinity of phosphorylated phosphofructokinase for fructose-2,6-p2 results in an extremely efficient means to inhibit the enzyme, and thus inhibit glycolysis in liver. REFERENCES 1. Furuya, E., and Uyeda, K. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, Richards, C. S., and Uyeda, K. (1980) Biochem. Biophys. Res. Commun. 97, Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255, Van Schaftingen, E., Hue, L., and Hers, H. G. (1980) Biochem. J. 192, Van Schaftingen, E., and Hers, H. G. (1980) Biochem. Biophys. Res. Commun. 96, Uyeda, K., Furuya, E., and Sherry, A. D. (1981) J. Biol. Chem. 256, in press 7. Newsholme, E. A., and Start, C. (1973) Regulation in Metabolism, pp , John Wiley and Sons, New York 8. Ramaiah, A., and Tejwani, G. A. (1973) Eur. J. Biochem. 39, Kono, N., and Uyeda, K. (1974) J. BioE. Chem. 249, Tsai, M. Y., and Kemp, R. G. (1974) J. Biol. Chem. 249, Dunnaway, G. A., Jr., and Weber, G. (1974) Arch. Biochem. Biophys. 162, Brand, I. A., and Soling, H.-D. (1979) J. Biol. Chem. 249, Colombo, G., Tate, P. W., Girotti, A. W., and Kemp, R. G. (1975) J. Biol. Chem. 250, Pettigrew, D. W., and Frieden, C. (1979) J. Biol. Chem. 254, Uyeda, K. (1979) Adu. Enzymol. Relat. Areas Mol. Biol. 48, Reinhart, G. D., and Lardy H. A. (1980) Biochemistry 19, Uyeda, K. (1969) Biochemistry 8, Clark, M. G., Kneer, N. M., Bosch, A. L., and Lardy, H. A. (1974) J. Biol. Chem. 249, Veech, R. L., Lawson, J. W. R., Cornell, N. W., and Krebs, H. A. (1979) J. Biol. Chem. 254, Hill, D. E., and Hames, G. G. (1975) Biochemistry 14, Hood, K., and Hollaway, M. R. (1976) FEBS Lett. 68, Monod, J., Wyman, J., and Changeaux, J. P. (1965) J. Mol. Biol. 12, Guynn, R. W., Veloso, D., and Veech, R. L. (1972) J. Biol. Chem. 247, Lawson, J. W. R., Guynn, R. W., Cornell, N., and Veech, R. L. (1976) in Gluconeogenesis (Mehlman, M. A,, and Hauson, R., eds) John Wiley and Sons, New York 25. Veech, R. L., Raijman, L., Dalziel, K., and Krebs, H. A. (1969) Biochem. J. 115, Reinhart, G. D., and Lardy, H. A. (1980) Biochemistry 19, Kagimoto, T., and Uyeda, K. (1979) J. Biol. Chem. 254,