ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
182, 563-572
(1977)
Inhibition of Phosphofructokinase by Fructose 1,6-diphosphatase in Mammalian Systems: Protein-Protein Interaction or Fructose 1,6diphosphate Trapping?’ H. D. SOLING,’ Abteilung
fiir
Klinische
Biochemie,
G. BERNHARD, Medizinische Gdttingen, Received
A. KUHN, Universititsklinik Germany
November
AND Giittingen,
H.-J.
LUCK
Humboldtallee
1, D-34
16, 1976
The aim of the investigation was to discriminate between a direct inhibitory effect of fructose 1,6-diphosphatase (FDPase) (EC 3.1.3.11) on phosphofructokinase (PFK) (EC 2.7.1.11) and an inhibition of the PFK-catalyzed reaction by removal of FDP. The following results strongly support the second hypothesis. (i) Inhibition of PFK by FDPase directly follows the decrease of the actual concentrations of FDPase. (ii) FDPase has no inhibitory effect as long as the concentration of FDP is kept above 2 pM. (iii) FDPase has no inhibitory effect when PFK activation is achieved by glucose 1,6diphosphate, which is not split by FDPase. (iv) FDPase has an inhibitory effect when the activation is achiered by sedoheptulose 1,7-diphosphate, which is split by FDPase. (v) High concentrations of aldolase inhibited the PFK-catalyzed reaction. The degree of inhibition showed a similar correlation with the level of FDP in the assay system when FDPase instead of aldolase was used. (vi) FDP-equilibrated PFK, but not glucose 1,6diphosphate-equilibrated PFK, is inactivated by FDPase. (vii) Muscle and liver PFK react in the same way with either muscle or liver FDPase.
Recently, Uyeda and Luby (1) have described experiments from which they deduced the existence of a direct interaction between FDPase3 and PFK. FDPase led to an inhibition of the PFK-catalyzed reaction. They discussed the possibility that FDPase could have inhibited this reaction by decreasing the steady-state concentration of FDP, an activator of PFK, but they believed that they ruled out this possibility in their experiments. Emerk and Frieden (2>, on the other hand, stress the important role of the level of FDP for evaluating PFK activity in the usual assay sys-
terns and question whether the results obtained by Uyeda and Luby (1) and recently by Proffitt et al. (3) might still result from changes in the concentration of FDP. However, in all of these experiments, FDP concentrations in the various test systems have never been examined and correlated with the actual PFK activity. Therefore, we measured FDP concentrations under a variety of experimental conditions to discriminate between a direct effect of FDPase on PFK and an indirect effect based on changes in the concentration of FDP. Moreover, experiments with glucose 1,6-diphosphate and sedoheptulose 1,7-diphosphate were performed. The conclusion drawn from the various types of experiments is that FDPase affects PFK not by a protein-protein interaction but by changing the concentration of FDP.
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H. D. Soling. * Address all correspondence to Professor H. D. Soling. 3 Abbreviations used FDPase, fructose 1,6-d& phosphatase (EC 3.1.3.11); PFK, phosphofructokinase (EC 2.7.1.11); F-6-P, fructose 6-phosphate; FDP, fructose 1,6-diphosphate; G-1,6-DP, glucose 1,6-diphosphate; SDP, sedoheptulose 1,7-diphosphate; G-6-P, glucose 6-phosphate; SDS, sodium dodecyl sulfate.
MATERIALS Rabbit auxiliary
AND
muscle PFK, rabbit enzymes, and all
METHODS muscle FDPase, coenzymes and
all sub-
563 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
564
SOLING
strates except SDP were obtained from BoehringerMannheim Corp., Mannheim, Germany. SDP and rotenone were from Sigma Chemie, Munich, Germany; all other chemicals (analytical grade) were from E. Merck, A.G., Darmstadt, Germany.
Enzyme Activity Enzyme units (u) are defined as micromoles substrate converted per minute at 25°C.
Enzyme Preparations
of
Used
Rabbit muscle PFK was obtained as a suspension in 3.2 M (NH&SO, containing 10 mM potassium phosphate and a mixture of adenosine phosphates (1 mM) from Boehringer-Mannheim Corp. It had a specific activity of 85 to 90 U. mg of protein-’ in the “optimal test system,” which was performed under the following conditions (final concentrations): triethanolamine-Cl (pH 8.0), 50 mM; MgCl*, 3mM; mercaptoethanol, 5 mM; 5’-AMP, 2.4 mM; NADH, 0.34 mM; F-6-P, 2.4 mM; ATP, 0.6 mM; aldolase, 0.11 U/ml; glycerophosphate dehydrogenase, 0.31 U/ml; triosephosphate isomerase, 5.6 U/ml. Initially, rabbit muscle PFK was freed from adenine nucleotides by charcoal treatment according to Emerk and Frieden (2). However, since removal of endogenously bound ATP did not affect the results obtained, PFK was subsequently only freed from (NH&SO, and unbound adenine nucleotides by dialysis against 10 mM potassium phosphate buffer, pH 7.4. Rat liver PFK4 was purified according to Brand and Soling (4). The enzyme had a specific activity of 75 U.mg-’ under the same conditions as mentioned for muscle PFK. The preparation used was suspended in a glycerol-phosphate medium (glycerol, 6 M; potassium phosphate, 10 mM; MgCl*, 0.25 mM; mercaptoethanol, 2.5 mM; F-6-P,, 0.5 mM; pH 7.0). The enzyme was not treated with charcoal. Rabbit muscle FDPase was obtained as a suspension in 3.3 M (NH&SO, from Boehringer-Mannheim Corp. It had a specific activity of 12 U.mg of protein-’ in the following test system (final concentrations): triethanolamine-Cl (pH 8.0), 75 mM; EDTA, 2.2 mM; M&X2, 9.4 mM; FDP, 1 mM; NADP, 0.5 mM; phosphohexose isomerase, 2.2 U/ml; glucose 6-phosphate dehydrogenase (yeast), 0.35 U/ml. The enzyme preparation contained 0.12 U. mg-’ of pyruvate kinase activity. Rat liver FDPase was purified by a modification of the method of Pontremoli et al. (5), the main differences being that the heat step was not performed at pH 4.2 but at pH 7.0 and that the substrate-facilitated elution from carboxymethyl cellulose was performed at pH 6.1. The enzyme had a
ET
AL.
specific activity of 24 U. mg of protein-’ in the test system described for the muscle enzyme. It was homogenous after SDS-polyacrylamide gel electrophoresis and exhibited a pH optimum at pH 7.9 and a ratio of activity at pH 7.9 to activity at pH 9.0 of 2.35 with Mg2+ as the divalent cation. Unless otherwise mentioned, the enzyme was suspended in 5 mM sodium malonate buffer, pH 6.2. Rabbit muscle aldolase was obtained as a suspension in 3.2 M (NH&SO, from Boehringer-Mannheim Corp. and had a specific activity of 9 U’mg of protein’ according to the manufacturer.
Preparation of Decomplemented Anti-Rat Liver FDPase Antiserum Anti-rat liver FDPase antiserum was obtained by injecting into rabbits about 2 mg of purified rat liver FDPase together with complete Freund’s adjuvant at multiple sites intracutaneously. This procedure was repeated after 2 weeks. Blood was taken another 2 weeks later and tested for FDPase antibodies and their specificity by the Ouchterlony double-diffusion test and by immunotitration. Control serum was obtained from untreated rabbits of the same strain. Anti-rat liver FDPase antiserum and control serum were decomplemented by heating at 50°C for 30 min. Denatured protein was removed by centrifugation. The supernatant was brought to 50% saturation by the addition of solid (NH&SO,. After centrifugation, the sediment was dialyzed against 150 mM NaCl and resuspended in the original serum volume.
Determination
Assay
system
I (pyruvate
kinase-coupled
assay).
The assay mixture was as follows (final concentrations): triethanolamine-Cl, 50 mM; Mg(CH3C00)2, 5 mM; KCl, 25 mM; NH,Cl, 0.5 mM; EDTA, 1 mM; dithiothreitol, 0.2 mM; phosphoenolpyruvate, 0.2 mM; ATP, 0.5 mM; fructose g-phosphate, 0.1 mM; glucose B-phosphate, 0.35 mM; NADH, 0.35 mM; phosphohexose isomerase, 5.3 U/ml; pyruvate kinase, 24 U/ml; lactate dehydrogenase, 24 U/ml. The final pH was 7.2, and the temperature was 25°C. The auxiliary enzymes had been freed from (NH&SO, by filtration on a Sephadex G-25 column equilibrated with 50 mM sodium phosphate (pH 7.1) containing 1 mM EDTA. Unless otherwise mentioned, the reaction was initiated by the addition of appropriate amounts of PFK. Assay
4 Rat liver phosphofi-uctokinase was given to us by Dr. Inge Brand and was purified in our laboratory.
of PFK Activity
In the experiments in which the interaction between FDPase or aldolase and PFK was examined, one of the following test systems was used (not the optimal test system already described).
system
II (pyruvate
kinuse-coupled
assay).
This system is identical to that given by Proffitt et al. (3) except that the auxiliary enzymes were desalted over Sephadex G-25 equilibrated with 50 mM
PHOSPHOFRUCTOKINASE
INHIBITION
Tris-Cl, pH 7.2, instead of being dialyzed against 10 mM Tris-Cl, pH 7.5. Assay system III (aldolase-coupled assay). The assay mixture contained (final concentrations): triethanolamine-Cl, 50 mM; ATP, 0.5 mM; F-6-P, 0.1 mM; Mg(CH,COO),, 5 mM; NADH, 0.2 mM; triosephosphate isomerase, 5 U/ml; glycerophosphate dehydrogenase, 0.6 U/ml; varying amounts of aldolase and phosphofructokinase. The final pH was 7.2, and the temperature was 25°C. Aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase had been freed from (NH&SO, prior to use by gel filtration over Sephadex G-25 equilibrated with 50 mM Tris-Cl (pH 7.1) containing 1 mM EDTA. Assay system IV (aldolase-coupled assay). The assay mixture contained (final concentrations): triethanolamine-Cl, 50 mM; Mg(CH,C0012, 10 mM; KCl, 25 mM; NADH, 0.2 mM; ATP, 0.5 mM; AMP, 0.8 mM; rotenone, 3 pg/ml; (NH&SO,, 90 mM; triose phosphate isomerase, 5 U/ml; glycerophosphate dehydrogenase, 0.6 U/ml; appropriate amounts of PFK activity from rat liver 100,OOOg supernatant which had been pretreated as will be mentioned below. The reaction was started with F-6-P (0.1 mM). The final pH was 7.2, and the temperature was 30°C. Endogenous aldolase activity in the 100,OOOg supernatant was used. The other auxiliary enzymes were freed from (NH&SO, as mentioned for assay system III.
FDP Infusion
into Assay
System
I
Experiments in which FDP was infused into the photometer cuvette during the assay were performed as follows. The Aminco DW-2 dual-wavelength spectrophotometer was used in the dualwavelength mode (wavelength pair, 375/350 nm). The cuvette lifter was removed and replaced by a rotating rod driven by an electromotor at about 500 rpm. A small Teflon-coated stirring bar was placed into the cuvette. It did not interfere with the optical measurement. FDP was infused via polyethylene tubing (0.5-mm internal diameter) by a UNITA III (Braun-Melsungen, Germany) infusion pump at a rate of 5 to 30 &min.
Multiple Determinations PFK-Activity Assays
of FDP
during
For this purpose, a large volume of the assay mixture was prepared (5-10 times the volume needed for one cuvette). From this mixture, a cuvette was tilled and transferred to the DW-2 spectrophotometer. The cuvette and the larger rest of the assay mixture were started simultaneously. Aliquots for the determination of FDP were taken from the larger incubation volume at appropriate time points, while the reaction was followed in the photometer. Since both assay mixtures had the same concentration of PFK and were run at exactly the same temperature, the results of both types of meas-
BY
FRUCTOSE
urements (FDP could be directly
565
1,6-DIPHOSPHATASE concentration versus reaction related to one another.
Preparation of the FDPase-Free Super-n&ant from Rat Liver
rate)
100,OOOg
Rat liver was homogenized (Potter homogenizer, Teflon pestle) with 2 vol (v/w) of the following homogenization medium (final concentrations): sucrose, 200 mM; MgCl,, 0.5 mM; mercaptoethanol, 5 mM; pH 7.0. After centrifugation (100,000g for 1 h), an aliquot of the supernatant was filtered through a Sephadex G-25 column equilibrated with the homogenization medium. To 0.8 ml of the filtrate 0.08 ml of decomplemented anti-rat liver FDPase antiserum was added, followed by an incubation for 45 min at 37°C. After centrifugation at 30,OOOg for 10 min, the supernatant was tested for FDPase activity. FDPase had completely disappeared. For controls, the same filtrate was treated in the same way except that decomplemented control serum instead of antiserum was used. PFK activity was measured in the optimal test system and in assay system IV.
Interaction between PFK from Rabbit Muscle
and
FDPase
Rabbit muscle PFK (20 mg) was sedimented by centrifugation (12,000g for 10 min). The sediment was suspended in 1 ml of 50 mM sodium phosphate (pH 7.4) containing 2 mM dithiothreitol and freed from (NH&SO, by gel filtration over Sephadex G-25 which had been equilibrated with the same medium. The PFK-containing fraction was brought to 2.5 ml and divided into two portions of 1.25 ml each. One portion was brought to 2.0 mM (final concentration) with respect to FDP (sodium salt); the other portion was brought to the same concentration with respect to G-1,8DP (tricyclohexylammonium salt). The mixtures were incubated at 4°C for 12 h and then dialyzed for 6 h against 500 ml of the initial suspension medium. This dialysis step was repeated once for 12 h, another time twice for 6 h, and finally once for another 12 h. Under these conditions, 13.6 nmol.mg of protein-’ of G-1,6-DP and 18.1 nmol . mg of protein’ of FDP stayed bound to PFK. About the same amount of sugar diphosphates bound was found before the last 12-h dialysis, indicating that any free sugar diphosphates had been removed. PFK equilibrated in this way with either FDP or G-1,8DP was incubated with rabbit muscle FDPase as described in the legends to Figs. 5 and 6. The FDPase had been freed from (NH&SO, by gel tiltration over Sephadex G-25 which had been equilibrated with 5 mM sodium malonate buffer, pH 6.2. The incubation conditions (PFK + FDPase) were similar to those described by Proffitt et al. (3). The incubation mixture contained (final concentrations): Tris-Cl, 50 mM; potassium phosphate, 2 mM; dithio-
566
SOLING
threitol, 0.2 my. The final pH was 7.5, and the incubation temperature was 25°C. Control incubations contained PFK but no FDPase. To correct for the effects of FDPase carried over from the incubation mixture into the test system (assay system II), tests were also performed in which the test mixture contained that amount of FDPase which would have been carried over from the incubation mixture (PFK and FDPase); in this case, the assay was started with PFK from the control incubation not containing FDPase.
Determination phate
of Fructose
1,6-diphos-
The concentration of FDP was determined in the PFK assay system under a variety of different test conditions. For this purpose, aliquots were removed from the assay mixture and added to one-quarter of the volume of 6 N HClO,. The HClO&reated sample was brought to 50°C for 5 min to destroy enzyme activities (especially aldolase) which would remain after deproteinization with cold HClO,. The heat treatment did not lead to measurable losses of FDP, F-6-P, or G-6-P. After the heat treatment, the sample was cooled at 0°C for 10 min and centrifuged (20,OOOg for 10 min). The supernatant was brought to pH 6-6.5 with KOH, and KClO, was removed by centrifugation. The supernatant was used for the determination of FDP. Although the determination of FDP via aldolase, triosephosphate isomerase, and o-glycerophosphate dehydrogenase results in the formation of 2 mol of NADH per mole of FDP split in the aldolase reaction, this assay proceeds extremely slowly when the concentration of FDP is low. Therefore, FDP was determined in the same coupled reaction sequence as that used for the determination of FDPase activity. The assay mixture contained (final concentrations): triethanolamine-Cl, 60 mM; MgCl,, 8 mM; EDTA, 0.8 mM; NADP, 0.66 mM; appropriate amounts of neutralized HClO, extract. The pH was 8.0, and the temperature was 32°C. The reaction was started by the addition of glucose 6-phosphate dehydrogenase (0.24 U/ml) and phosphohexose isomerase (1.5 U/ml). When all of the G-6-P and F-6-P had been converted to 6-phosphogluconate and the reaction had come to a complete stop, FDPase was added (0.14 U/ml of purified rat liver FDPase) and the increase in the formation of NADPH was measured. It was established in control experiments that this assay system allows the determination of small amounts of FDP in the presence of a lOO- to 500-fold excess of glucose 6-phosphate and/or fructose 6-phosphate. Since the concentrations of FDP measured in our experiments were rather low, the assays were performed in an Aminco DW-2 dual-wavelength spectrophotometer, which allowed a 200-fold amplification of the signal in the dual mode (wavelength
ET AL. pair, 375/350 nm). Glucose I-phosphate dehydrogenase and phosphohexose isomerase were not freed from (NH&SO, prior to use.
Reaction of Glucose 1,6-diphosphate with FDPase and Purity of Glucose 1,6-diphosphate The assay system described for the determination of FDPase activity was used. The reaction mixture contained 0.12 U/ml of purified rat liver FDPase. The reaction was started by adding G-1,6-DP (final concentration, 1 m&f). The rate was less than 3% of the rate with the same concentration of FDP and was not significantly different from the control value without substrate. The amount of FDP in the G-1,6-DP preparation was determined in the sensitive FDP assay system already described. The G-1,6-DP preparation used contained 1.2% FDP on a molar basis. RESULTS
When PFK activity was measured using assay system I (pyruvate kinase coupled), the reaction rate after the addition of PFK was not linear from the beginning, but started slowly and reached linearity only after several minutes. The increase in reaction velocity occurred together with an increase in the FDP concentration in the assay system (Fig. 1 curve A). When 20 PM FDP (final concentration) was added before starting the reaction, the reaction rate was linear from the start with PFK and remained so (Fig. 1). When under these conditions FDPase was added immediately after the addition of PFK, the reaction slowed down within a few minutes, again in parallel with a decrease in the concentration of FDP in the assay system (Fig. 1, curve B). In this respect, there was no difference between rat liver FDPase and identical activities of rabbit muscle FDPase . To discriminate between a direct inhibition of PFK by FDPase on one hand and an inhibition of PFK by draining off the positive allosteric effector FDP on the other hand, another experimental approach was carried out: The reaction was initiated by PFK in the presence of 20 PM FDP and 20 PM FDPase. At the same time, FDP was infused into the assay cuvette at varying rates to avoid or to inhibit the drop in concentration of FDP due to FDPase. As shown in Fig. 2, curve A, the inhibition by
PHOSPHOFRUCTOKINASE
INHIBITION
BY FRUCTOSE
1,8DIPHOSPHATASE
FIG. 1. Correlation between the activity of phosphofructokinase and the concentration of FDP. PFK activity was measured using assay system I (see Materials and Methods). In curve A (control), no exogenous FDP was added. The reaction was started by the addition of 0.75 pg of ATP-free purified rabbit muscle PFK. In curve B, FDP (final concentration, 20 pM) was added to the assay mixture before starting the reaction by the addition of 0.75 pg of ATP-free purified rabbit muscle PFK. Two seconds later, 3.2 pg of purified rat liver FDPase was added. The total assay volume in A and B was 2.1 ml. Samples for the determination of FDP were taken from larger assay mixtures which were run in parallel to the reaction in the photometer cuvette, as described under Materials and Methods. The reaction was followed in an Aminco-Chance DW2 spectrophotometer at the wavelength pair 275/250 nm. The arrows and numbers indicate the time points of measurement and the concentrations of FDP in the reaction mixture, respectively.
FIG. 2. Effects of a continuous infusion of varying amounts of FDP into the reaction cuvette on the inhibitory effect of FDPase on phosphofructokinase. Phosphofructokinase was measured using assay system I (see Materials and Methods). Immediately before starting the reaction, FDP (final concentration, 20 PM) was added to the assay system. The reaction was initiated by the addition of 0.8 pg of purified ATP-free rabbit muscle phosphofructokinase. One second later, purified rat liver FDPase was added. The amount of FDPase added catalyzed the conversion of 18 nmol of FDP.min-’ under the conditions of assay system I and a FDP concentration of 20 PM. (A) represents a control assay without infusion of FDP; 11.8 (B), 17.6 (0, and 23.5 (Dl nmol of FDP.min’ were continuously infused into the reaction cuvette. The reaction was stopped for the analysis of FDP 10 min atier the addition of PFK. The reaction was measured at the wavelength pair 375/350 nm in an Aminco-Chance DW-2 dual-wavelength spectrophotometer.
567
568
SOLING
FDPase was clearly visible when the FDP concentration had fallen to 1.3 PM. When the concentration was set to 40.9 or 13.6 PM (Fig. 2, curves C and D), the reaction was linear from the start and remained so in spite of the presence of FDPase. Even at a very low infusion rate, which was only able to keep the concentration of FDP at 2.6 PM, the reaction rate was only slightly lowered by FDPase (Fig. 2, curve B). This fits well with the observation (Fig. 1) that without an infusion of FDP the reaction rate in the presence of FDPase continued to proceed linearly until the FDP concentration had fallen below 2.5 FM. G-1,6-DP is able to activate PFK but is not split by FDPase. When FDPase was added to G-1,6-DP-activated PFK, the reaction rate was not significantly affected (Fig. 3, curve B) in contrast to the system in which PFK was activated by FDP (Fig. 3, curve A). When a preincubation was carried out in the presence of PFK, FDPase, and G-1,6DP, the addition of F-6-P initiated immediately the PFK-catalyzed reaction and the rate was linear from the beginning (Fig. 3, curve D). However, when the preincubation system contained FDP instead of G-1,6-DP, no reaction could be initiated by the addition of F-6-P (Fig. 3,
ET AL.
curve C) due to the fact that all FDP had been split by FDPase during the preincubation period. Again, FDPases from rat liver and from rabbit muscle behaved identically in this respect. Sedoheptulose 1,7-diphosphate activates PFK, although less than FDP or G-1,6-DP (Fig. 4). In the presence of 20 or 40 PM
FIG. 4. Stimulation of rat liver PFK by sedoheptulose 1,7-diphosphate (SDP) and inhibition of this effect by purified rat liver FDPase. The activity was measured using assay system I (see Materials and Methods). The test system contained 0.24 pg.ml-’ of PFK and 1.9 pg.ml-’ of FDPase. (B and 0. In B and C, SDP was added to the test mixture before starting the reaction. The SDP concentration was 20 pM in C and 40 ~,LM in D. Curve A represents a control experiment without added SDP.
FIG. 3. Effects of purified rat liver FDPase on the reaction catalyzed by purified rat liver phosphofi-uctokinase in the presence of either 10 pM fructose 1,6diphosphate (A and C) or 80 pM glucose 1,6-diphosphate (B and D). PFK activity was measured in assay system I (see Materials and Methods). The test system contained 0.24 pg.ml-’ of PFK. The amount of FDPase added was 1.9 pg.ml-‘. The sequence of addition was: (A) PFK, FDP, F-6-P, FDPase; (B) PFK, G-1,6-DP, F-6-P, FDPase; (Cl F-6-P, FDP, FDPase, PFK: (D) F-6-P, G-1,6-DP, FDPase, PFK.
PHOSPHOFRUCTOKINASE
INHIBITION
SDP, a linear reaction rate was reached earlier than in its absence. Apparently, the activating effect of SDP at the concentrations used was not sufficient to activate PFK fully, so that complete activation was only reached after additional FDP had been formed during the reaction. Upon addition of FDPase, the reaction rate was almost abolished within a few minutes (Fig. 4, curves B and C). Since it is known from the work of Bonsignore et al. (6) and Pontremoli et al. (7) that FDPase splits not only FDP but also SDP, it seems rather clear that the inhibitory effect of FDPase is again brought about by removal of SDP as well as FDP and not by a direct proteinprotein interaction. The inhibition of the PFK-catalyzed reaction by higher activities of aldolase has already been reported by El-Badry et al. (8) and was attributed to the removal of FDP. However, FDP concentrations had not been measured. Table I shows that, with increasing concentrations of aldolase in assay system III, the flux through the TABLE CORRELATION FDP IN THE
CONCENTRATION
Concentration of aldolase (pg.ml-‘) 0.14 0.34 0.54 0.67 0.81 0.94 1.01
I
BETWEEN THE CONCENTRATION TEST SYSTEM, REACTION RATE,
OF AND
OF ALWLASE”
Reaction rate after reaching linearity (AE ‘10 min-‘) 0.148 0.260 0.260 0.260 0.225 0.163 0.095
Concentration of FDP (pm01 . liter’) 17.55 5.83 2.77 1.29 0.53 0.44 0.27
a Assay system III (see Materials and Methods) was used. Rabbit muscle PFK pretreated as described under Materials and Methods was used (2.7 pg/ml). The rabbit muscle aldolase used had been freed from ammonium sulfate by gel filtration on Sephadex G-25 equilibrated with 50 mM Tris-Cl (pH 7.1) containing 1 mM EDTA. The reaction rates given were taken from the linear portion of the reaction curve. Linearity was reached after 1 to 5 min depending on the concentration of the aldolase. Aliquota for determination of the concentration of FDP were taken 2 min after linearity had been reached.
BY
FRUCTOSE
1,8DIPHOSPHATASE
569
coupled enzyme system first increased, but started to decrease when the concentration of aldolase exceeded 0.67 pg * ml-‘. At the lowest concentration of aldolase, the highest concentration of FDP was found, as is to be expected since under these circumstances aldolase and not PFK was rate limiting. When the concentration of aldolase was raised, PFK became rate limiting as indicated by the constant overall rate between 0.34 and 0.67 ,ug of aldolase-ml-‘. When the concentration of aldolase exceeded 0.67 pg. ml-‘, the overall rate started to decrease. This decrease was associated with a decrease in the steadystate concentration of FDP below 1.29 j-&M. This is in good agreement with the results presented in Fig. 1, where an inhibition of the reaction by FDPase was not seen before the concentration of FDP had fallen below 2.5 PM. Thus, for a given concentration of FDP, there is no difference between the action of FDPase and aldolase on PFK. All of these results made it rather likely that the inactivating effect of FDPase on PFK described by Proffitt et al. (3) was also due to removal of FDP from PFK. Therefore, rabbit muscle PFK equilibrated with either FDP or G-1,8DP was incubated with FDPase in the same system as that described by Proffitt et al. (31, the main difference being that rabbit muscle FDPase instead of rabbit liver FDPase was used. After incubation, PFK was tested in the same test system as that given by Proffitt et al. (3). The results are given in Figs. 5and6. The effect of FDPase on PFK which had been equilibrated with FDP is seen from the time interval between the start of the reaction in the test system and the point at which the reaction became linear. When the ratio PFK/FDPase was l/z (w/w), the time until linearity was reached was prolonged when the activity was tested 30 s after the addition of FDPase (Fig. 5A). After 10 min of incubation, it lasted more than 18 min until linearity was reached (Fig. 5B). When the ratio PFK/FDPase was lowered to l/3, the effect of FDPase was seen 30 s after the addition of FDPase. At greater than 20 min, the PFK reaction did not start (Fig. 5C). The same result
570
SOLING ET AL.
FIG. 5. Effects of preincubation of FDP-equilibrated rabbit muscle PFK with rabbit muscle FDPase on PFK activity. Phosphofructokinase was equilibrated with FDP under the conditions described under Materials and Methods. It contained 18.1 nmol of FDP mg of protein-‘. PFK activity in assay system II (see Materials and Methods) is shown. The reaction was initiated by the addition of an aliquot of the incubation mixture. (Al and (Bl refer to the same incubation. This incubation contained, in a final volume of 0.2 ml, 90 pg of FDP-equilibrated PFK and 188 pg of FDPase. The interaction of the two enzymes was started by the addition of FDPase. The incubation was carried out as described under Materials and Methods. After 30 s and after 20 min, aliquots were removed from the incubation and tested for PFK activity. (Cl and (D) refer to another incubation of the same FDP-equilibrated PFK with FDPase. In this case, only 60 pg of the PFK was incubated with 188 pg of FDPase; otherwise the conditions were the same as those of (A) and (B). In (Cl and (D), FDPase-treated PFK remained inactive in assay system II even after more than 30 to 40 min. To the control incubations, instead of FDPase, only the same volume of 5 mM sodium malonate (pH 6.2) was added.
was seen after 20 min of incubation (Fig. 5D). In contrast, G-1,6-DP-equilibrated PFK was not significantly inactivated (Fig. 6) even at a concentration of FDPase which was about four times higher than that of PFK (w/w). In comparison to control incubations, the reaction reached linearity about 2 min later. This resulted from the transfer of some FDPase activity from the incubation medium into the test system. There was no difference whether G-1,6-DP-equilibrated PFK was incubated for 30 s or for 20 min with FDPase (Figs. 6A and 6B). All of these incubations were performed in the absence of Mg2+ and EDTA. When FDPase activity was tested under these conditions, FDPase activity could still be measured although the activity was only about 3.5% of the activity obtained in the presence of Mg2+ and EDTA. However, this residual activity was sufficient to split all or most of the FDP associated with FDP-equilibrated PFK.
Effects of Removal of FDPase on PFK Activity in Rat Liver 100,OOOg Supernatant Due to dilution during homogenization, gel filtration, and immunoprecipitation, the final activity of PFK in the extract was rather low but still measurable without difficulty when a lo-fold scale expension was used during the photometric assay (assay system IV). The removal of FDPase by precipitation with anti-rat liver FDPase antiserum increased the apparent PFK activity (Table II). This increase was associated with an increase in the concentration of FDP in the assay system (Table II). DISCUSSION
According to our results, the findings used by Uyeda and Luby ( 1) and by Proffitt et al. (3) as an argument for a direct inhibition of PFK by FDPase can be explained by removal of FDP from the PFK protein.
PHOSPHOFRUCTOKINASE
INHIBITION
FIG. 6. Effects of preincubation of G-1,8DPequilibrated rabbit muscle PFK with rabbit muscle FDPase on PFK activity. The conditions of the experiment were the same as those of Fig. 5 except that PFK which had been equilibrated with glucose 1,6-diphosphate was used. The PFK contained 13.6 nmol of glucose 1,6-diphosphate ‘mg of protein-‘. The incubation mixture contained, in a final volume of 0.2 ml, 65 pg of glucose 1,6-diphosphate-equilibrated PFK and 260 pg of FDPase. PFK activity in assay system II is shown. The reaction was initiated by the addition of aliquots of the incubation mixture after 30 s (A) and after 20 min (B) of incubation. TABLE
II
EFFECTS OF REMOVAL OF FDPase ACTIVITY FROM RAT LIVER 100,OOOg SUPERNATANT (ES,) BY IMMUNOPRECIPITATION ON THE ACTIVITY OF PFK FROM THE SAME HOMOGENATE” Experiment
I
II
Experimental tion
condi-
S, treated with control serum S, treated with antiFDPase antiserum S3 treated with control serum S, treated with antiFDPase antiserum
PFK activity W’wj
Concentration of FDP (pm01 . liter’)
0.040
0.37
0.062
0.64
0.050
0.28
0.088
0.76
’ Assay system IV (see Materials and Methods) was used. No exogenous aldolase was added since endogenous activity proved to be sufficient. The concentrations of FDP were determined in samples taken from the assay mixture when the reaction rate had become linear.
El-Badry et al. (8) suspected that the inactivation of sheep heart PFK by aldolase and FDPase resulted from the removal of FDP from PFK rather than from a direct protein-protein interaction. A similar con-
BY
FRUCTOSE
1.6-DIPHOSPHATASE
571
elusion was drawn by Emerk and Frieden (2). The direct measurement of the concentration of FDP during inactivation of PFK by FDPases from the different sources as well as by aldolase (Table I) shows clearly that the degree of inhibition correlates with the concentration of FDP but does not exhibit specificity with respect to either aldolase or FDPase. Moreover, FDPase exerts no inhibitory effect when the decrease of FDP is avoided (e.g., by infusion of FDP) or when FDP is replaced by G-1,6DP, a metabolite not split by FDPase. In accordance with Proffitt et al. (31, we found an immediate inactivation of rabbit muscle PFK by FDPase. In contrast to Proffitt et al. (31, we used rabbit muscle FDPase instead of rabbit liver FDPase in order to work with a more physiological system. This rapid inactivation occurred only with untreated PFK or with FDPequilibrated PFK and not with G-1,6-DPequilibrated PFK. The inactivation of untreated FDP-equilibrated PFK was seen in the absence of exogenous Mg*+ and EDTA, both known activators of FDPase. Profftt et al. (3) concluded, therefore, that FDPase must have been completely inactive under those conditions, and, hence, the inactivation must have proceeded via direct protein-protein interaction. This argument is not valid since, according to our measurements, the FDPase present in the incubation mixture was still active, although much less than seen after the addition of Mg2+ and EDTA. Moreover, when FDPase is added at rather high concentrations, it most probably can remove some FDP from PFK due to its high affinity for FDP. This high affinity of FDPase for FDP, among other findings, is illustrated by the fact that, in experiments where FDP was added at an initial concentration of 20 PM, added FDPase was able to drop the concentration of FDP very rapidly to values below 0.2 PM (the limit of our method). The rabbit muscle FDPase contained small amounts of pyruvate kinase (see Materials and Methods). However, even under the assumption that all binding sites of this contaminating pyruvate kinase would have been satu-
572
SOLING
rated with FDP, this would account for only less than 1% of the FDP initially bound to PFK. Proffitt et al. (3) state that the addition of 10 PM FDP prevented the FDPase-induced inactivation of PFK, and one wonders why they did not arrive at the conclusions that the availability of FDP and not the mere presence of FDPase determined PFK activity. Furthermore, any protein-protein interaction between FDPase and PFK in muscle must be considered to be of little or no physiological importance since the amount of FDPase protein in skeletal muscle is less than 1% of that of PFK and a proteinprotein interaction between FDPase and PFK could only occur, if at all, in liver where the amount of FDPase protein is considerably higher than that of PFK protein. A homologous liver system was not used by Proffitt et al. (3) but was examined by Uyeda and Luby (1). We believe that our findings can also explain the results of Uyeda and Luby (1) with one exception: In the experiments of Uyeda and Luby (11, the inhibitory effect of FDPase on PFK was abolished when the FDPase had been activated by prior treatment with homocysteine according to Nakashima et al. (9). Since we were unable to activate purified “neutral” rat liver FDPase or rabbit muscle FDPase by homocysteine in the system described by Nakashima et al. (9), we
ET
AL.
could not repeat these experiments. However, it is possible that in the experiments of Uyeda and Luby (1) the modification of FDPase by homocysteine had been associated with a decreased affinity of FDP for FDPase, which of course is not visible if FDPase activity is tested under V conditions. An increased KmmFDP for FDPase would lead to an increased steady-state concentration of FDP in the combined PFK-FDPase system, resulting in no or a diminished inhibition of PFK. REFERENCES 1. UYEDA, K., AND LUBY, L. J. (1974) J. Bill. Chem. 249,4562-4570. 2. EMERK, K., AND FRIEDEN, C. (1975) Arch. Biothem. Biophys. 168, 210-218. 3. PROFFITT, R. T., SANKAVAN, L., AND POGELL, B. M. (1976) Biochemistry 15, 2918-2925. 4. BRAND, I., AND SOLING, H. D. (1974) J. Bid. Chem. 249, 7824-7831. 5. PONTREMOLI, S., TRANIELLO, S., LUPIS, B., AND WOOD, W. A. (1965)J. Biol. Chem. 240, 34593463. 6. BONSIGNORE, A., MANCIAROITI, G., MANGIAROITI, M. A., DE FLORA, A., AND PONTREMOLI, S. (1963) J. Biol. Chem. 238, 3151-3154. 7. PONTREMOLI, S., LUPIS, B., Wool, W. A., TRANIELLO, S., AND HORECKER, B. L. (1965) J. Biol. Chem. 240, 3464-3468. 8. EGBADRY, A. M., OTANI, A., AND MANSOLJR, T. E. (1973) J. Bid. Chem. 248, 557-563. 9. NAKA~HIMA, K., HORECKER, B. L., TRANIELLO, S., AND PONTREMOLI, S. (1970)Arch. Biochem. Biophys. 139, 190-199.
OF BIOCHEMISTRY
AND
BIOPHYSICS
182, 563-572
(1977)
Inhibition of Phosphofructokinase by Fructose 1,6-diphosphatase in Mammalian Systems: Protein-Protein Interaction or Fructose 1,6diphosphate Trapping?’ H. D. SOLING,’ Abteilung
fiir
Klinische
Biochemie,
G. BERNHARD, Medizinische Gdttingen, Received
A. KUHN, Universititsklinik Germany
November
AND Giittingen,
H.-J.
LUCK
Humboldtallee
1, D-34
16, 1976
The aim of the investigation was to discriminate between a direct inhibitory effect of fructose 1,6-diphosphatase (FDPase) (EC 3.1.3.11) on phosphofructokinase (PFK) (EC 2.7.1.11) and an inhibition of the PFK-catalyzed reaction by removal of FDP. The following results strongly support the second hypothesis. (i) Inhibition of PFK by FDPase directly follows the decrease of the actual concentrations of FDPase. (ii) FDPase has no inhibitory effect as long as the concentration of FDP is kept above 2 pM. (iii) FDPase has no inhibitory effect when PFK activation is achieved by glucose 1,6diphosphate, which is not split by FDPase. (iv) FDPase has an inhibitory effect when the activation is achiered by sedoheptulose 1,7-diphosphate, which is split by FDPase. (v) High concentrations of aldolase inhibited the PFK-catalyzed reaction. The degree of inhibition showed a similar correlation with the level of FDP in the assay system when FDPase instead of aldolase was used. (vi) FDP-equilibrated PFK, but not glucose 1,6diphosphate-equilibrated PFK, is inactivated by FDPase. (vii) Muscle and liver PFK react in the same way with either muscle or liver FDPase.
Recently, Uyeda and Luby (1) have described experiments from which they deduced the existence of a direct interaction between FDPase3 and PFK. FDPase led to an inhibition of the PFK-catalyzed reaction. They discussed the possibility that FDPase could have inhibited this reaction by decreasing the steady-state concentration of FDP, an activator of PFK, but they believed that they ruled out this possibility in their experiments. Emerk and Frieden (2>, on the other hand, stress the important role of the level of FDP for evaluating PFK activity in the usual assay sys-
terns and question whether the results obtained by Uyeda and Luby (1) and recently by Proffitt et al. (3) might still result from changes in the concentration of FDP. However, in all of these experiments, FDP concentrations in the various test systems have never been examined and correlated with the actual PFK activity. Therefore, we measured FDP concentrations under a variety of experimental conditions to discriminate between a direct effect of FDPase on PFK and an indirect effect based on changes in the concentration of FDP. Moreover, experiments with glucose 1,6-diphosphate and sedoheptulose 1,7-diphosphate were performed. The conclusion drawn from the various types of experiments is that FDPase affects PFK not by a protein-protein interaction but by changing the concentration of FDP.
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H. D. Soling. * Address all correspondence to Professor H. D. Soling. 3 Abbreviations used FDPase, fructose 1,6-d& phosphatase (EC 3.1.3.11); PFK, phosphofructokinase (EC 2.7.1.11); F-6-P, fructose 6-phosphate; FDP, fructose 1,6-diphosphate; G-1,6-DP, glucose 1,6-diphosphate; SDP, sedoheptulose 1,7-diphosphate; G-6-P, glucose 6-phosphate; SDS, sodium dodecyl sulfate.
MATERIALS Rabbit auxiliary
AND
muscle PFK, rabbit enzymes, and all
METHODS muscle FDPase, coenzymes and
all sub-
563 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
564
SOLING
strates except SDP were obtained from BoehringerMannheim Corp., Mannheim, Germany. SDP and rotenone were from Sigma Chemie, Munich, Germany; all other chemicals (analytical grade) were from E. Merck, A.G., Darmstadt, Germany.
Enzyme Activity Enzyme units (u) are defined as micromoles substrate converted per minute at 25°C.
Enzyme Preparations
of
Used
Rabbit muscle PFK was obtained as a suspension in 3.2 M (NH&SO, containing 10 mM potassium phosphate and a mixture of adenosine phosphates (1 mM) from Boehringer-Mannheim Corp. It had a specific activity of 85 to 90 U. mg of protein-’ in the “optimal test system,” which was performed under the following conditions (final concentrations): triethanolamine-Cl (pH 8.0), 50 mM; MgCl*, 3mM; mercaptoethanol, 5 mM; 5’-AMP, 2.4 mM; NADH, 0.34 mM; F-6-P, 2.4 mM; ATP, 0.6 mM; aldolase, 0.11 U/ml; glycerophosphate dehydrogenase, 0.31 U/ml; triosephosphate isomerase, 5.6 U/ml. Initially, rabbit muscle PFK was freed from adenine nucleotides by charcoal treatment according to Emerk and Frieden (2). However, since removal of endogenously bound ATP did not affect the results obtained, PFK was subsequently only freed from (NH&SO, and unbound adenine nucleotides by dialysis against 10 mM potassium phosphate buffer, pH 7.4. Rat liver PFK4 was purified according to Brand and Soling (4). The enzyme had a specific activity of 75 U.mg-’ under the same conditions as mentioned for muscle PFK. The preparation used was suspended in a glycerol-phosphate medium (glycerol, 6 M; potassium phosphate, 10 mM; MgCl*, 0.25 mM; mercaptoethanol, 2.5 mM; F-6-P,, 0.5 mM; pH 7.0). The enzyme was not treated with charcoal. Rabbit muscle FDPase was obtained as a suspension in 3.3 M (NH&SO, from Boehringer-Mannheim Corp. It had a specific activity of 12 U.mg of protein-’ in the following test system (final concentrations): triethanolamine-Cl (pH 8.0), 75 mM; EDTA, 2.2 mM; M&X2, 9.4 mM; FDP, 1 mM; NADP, 0.5 mM; phosphohexose isomerase, 2.2 U/ml; glucose 6-phosphate dehydrogenase (yeast), 0.35 U/ml. The enzyme preparation contained 0.12 U. mg-’ of pyruvate kinase activity. Rat liver FDPase was purified by a modification of the method of Pontremoli et al. (5), the main differences being that the heat step was not performed at pH 4.2 but at pH 7.0 and that the substrate-facilitated elution from carboxymethyl cellulose was performed at pH 6.1. The enzyme had a
ET
AL.
specific activity of 24 U. mg of protein-’ in the test system described for the muscle enzyme. It was homogenous after SDS-polyacrylamide gel electrophoresis and exhibited a pH optimum at pH 7.9 and a ratio of activity at pH 7.9 to activity at pH 9.0 of 2.35 with Mg2+ as the divalent cation. Unless otherwise mentioned, the enzyme was suspended in 5 mM sodium malonate buffer, pH 6.2. Rabbit muscle aldolase was obtained as a suspension in 3.2 M (NH&SO, from Boehringer-Mannheim Corp. and had a specific activity of 9 U’mg of protein’ according to the manufacturer.
Preparation of Decomplemented Anti-Rat Liver FDPase Antiserum Anti-rat liver FDPase antiserum was obtained by injecting into rabbits about 2 mg of purified rat liver FDPase together with complete Freund’s adjuvant at multiple sites intracutaneously. This procedure was repeated after 2 weeks. Blood was taken another 2 weeks later and tested for FDPase antibodies and their specificity by the Ouchterlony double-diffusion test and by immunotitration. Control serum was obtained from untreated rabbits of the same strain. Anti-rat liver FDPase antiserum and control serum were decomplemented by heating at 50°C for 30 min. Denatured protein was removed by centrifugation. The supernatant was brought to 50% saturation by the addition of solid (NH&SO,. After centrifugation, the sediment was dialyzed against 150 mM NaCl and resuspended in the original serum volume.
Determination
Assay
system
I (pyruvate
kinase-coupled
assay).
The assay mixture was as follows (final concentrations): triethanolamine-Cl, 50 mM; Mg(CH3C00)2, 5 mM; KCl, 25 mM; NH,Cl, 0.5 mM; EDTA, 1 mM; dithiothreitol, 0.2 mM; phosphoenolpyruvate, 0.2 mM; ATP, 0.5 mM; fructose g-phosphate, 0.1 mM; glucose B-phosphate, 0.35 mM; NADH, 0.35 mM; phosphohexose isomerase, 5.3 U/ml; pyruvate kinase, 24 U/ml; lactate dehydrogenase, 24 U/ml. The final pH was 7.2, and the temperature was 25°C. The auxiliary enzymes had been freed from (NH&SO, by filtration on a Sephadex G-25 column equilibrated with 50 mM sodium phosphate (pH 7.1) containing 1 mM EDTA. Unless otherwise mentioned, the reaction was initiated by the addition of appropriate amounts of PFK. Assay
4 Rat liver phosphofi-uctokinase was given to us by Dr. Inge Brand and was purified in our laboratory.
of PFK Activity
In the experiments in which the interaction between FDPase or aldolase and PFK was examined, one of the following test systems was used (not the optimal test system already described).
system
II (pyruvate
kinuse-coupled
assay).
This system is identical to that given by Proffitt et al. (3) except that the auxiliary enzymes were desalted over Sephadex G-25 equilibrated with 50 mM
PHOSPHOFRUCTOKINASE
INHIBITION
Tris-Cl, pH 7.2, instead of being dialyzed against 10 mM Tris-Cl, pH 7.5. Assay system III (aldolase-coupled assay). The assay mixture contained (final concentrations): triethanolamine-Cl, 50 mM; ATP, 0.5 mM; F-6-P, 0.1 mM; Mg(CH,COO),, 5 mM; NADH, 0.2 mM; triosephosphate isomerase, 5 U/ml; glycerophosphate dehydrogenase, 0.6 U/ml; varying amounts of aldolase and phosphofructokinase. The final pH was 7.2, and the temperature was 25°C. Aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase had been freed from (NH&SO, prior to use by gel filtration over Sephadex G-25 equilibrated with 50 mM Tris-Cl (pH 7.1) containing 1 mM EDTA. Assay system IV (aldolase-coupled assay). The assay mixture contained (final concentrations): triethanolamine-Cl, 50 mM; Mg(CH,C0012, 10 mM; KCl, 25 mM; NADH, 0.2 mM; ATP, 0.5 mM; AMP, 0.8 mM; rotenone, 3 pg/ml; (NH&SO,, 90 mM; triose phosphate isomerase, 5 U/ml; glycerophosphate dehydrogenase, 0.6 U/ml; appropriate amounts of PFK activity from rat liver 100,OOOg supernatant which had been pretreated as will be mentioned below. The reaction was started with F-6-P (0.1 mM). The final pH was 7.2, and the temperature was 30°C. Endogenous aldolase activity in the 100,OOOg supernatant was used. The other auxiliary enzymes were freed from (NH&SO, as mentioned for assay system III.
FDP Infusion
into Assay
System
I
Experiments in which FDP was infused into the photometer cuvette during the assay were performed as follows. The Aminco DW-2 dual-wavelength spectrophotometer was used in the dualwavelength mode (wavelength pair, 375/350 nm). The cuvette lifter was removed and replaced by a rotating rod driven by an electromotor at about 500 rpm. A small Teflon-coated stirring bar was placed into the cuvette. It did not interfere with the optical measurement. FDP was infused via polyethylene tubing (0.5-mm internal diameter) by a UNITA III (Braun-Melsungen, Germany) infusion pump at a rate of 5 to 30 &min.
Multiple Determinations PFK-Activity Assays
of FDP
during
For this purpose, a large volume of the assay mixture was prepared (5-10 times the volume needed for one cuvette). From this mixture, a cuvette was tilled and transferred to the DW-2 spectrophotometer. The cuvette and the larger rest of the assay mixture were started simultaneously. Aliquots for the determination of FDP were taken from the larger incubation volume at appropriate time points, while the reaction was followed in the photometer. Since both assay mixtures had the same concentration of PFK and were run at exactly the same temperature, the results of both types of meas-
BY
FRUCTOSE
urements (FDP could be directly
565
1,6-DIPHOSPHATASE concentration versus reaction related to one another.
Preparation of the FDPase-Free Super-n&ant from Rat Liver
rate)
100,OOOg
Rat liver was homogenized (Potter homogenizer, Teflon pestle) with 2 vol (v/w) of the following homogenization medium (final concentrations): sucrose, 200 mM; MgCl,, 0.5 mM; mercaptoethanol, 5 mM; pH 7.0. After centrifugation (100,000g for 1 h), an aliquot of the supernatant was filtered through a Sephadex G-25 column equilibrated with the homogenization medium. To 0.8 ml of the filtrate 0.08 ml of decomplemented anti-rat liver FDPase antiserum was added, followed by an incubation for 45 min at 37°C. After centrifugation at 30,OOOg for 10 min, the supernatant was tested for FDPase activity. FDPase had completely disappeared. For controls, the same filtrate was treated in the same way except that decomplemented control serum instead of antiserum was used. PFK activity was measured in the optimal test system and in assay system IV.
Interaction between PFK from Rabbit Muscle
and
FDPase
Rabbit muscle PFK (20 mg) was sedimented by centrifugation (12,000g for 10 min). The sediment was suspended in 1 ml of 50 mM sodium phosphate (pH 7.4) containing 2 mM dithiothreitol and freed from (NH&SO, by gel filtration over Sephadex G-25 which had been equilibrated with the same medium. The PFK-containing fraction was brought to 2.5 ml and divided into two portions of 1.25 ml each. One portion was brought to 2.0 mM (final concentration) with respect to FDP (sodium salt); the other portion was brought to the same concentration with respect to G-1,8DP (tricyclohexylammonium salt). The mixtures were incubated at 4°C for 12 h and then dialyzed for 6 h against 500 ml of the initial suspension medium. This dialysis step was repeated once for 12 h, another time twice for 6 h, and finally once for another 12 h. Under these conditions, 13.6 nmol.mg of protein-’ of G-1,6-DP and 18.1 nmol . mg of protein’ of FDP stayed bound to PFK. About the same amount of sugar diphosphates bound was found before the last 12-h dialysis, indicating that any free sugar diphosphates had been removed. PFK equilibrated in this way with either FDP or G-1,8DP was incubated with rabbit muscle FDPase as described in the legends to Figs. 5 and 6. The FDPase had been freed from (NH&SO, by gel tiltration over Sephadex G-25 which had been equilibrated with 5 mM sodium malonate buffer, pH 6.2. The incubation conditions (PFK + FDPase) were similar to those described by Proffitt et al. (3). The incubation mixture contained (final concentrations): Tris-Cl, 50 mM; potassium phosphate, 2 mM; dithio-
566
SOLING
threitol, 0.2 my. The final pH was 7.5, and the incubation temperature was 25°C. Control incubations contained PFK but no FDPase. To correct for the effects of FDPase carried over from the incubation mixture into the test system (assay system II), tests were also performed in which the test mixture contained that amount of FDPase which would have been carried over from the incubation mixture (PFK and FDPase); in this case, the assay was started with PFK from the control incubation not containing FDPase.
Determination phate
of Fructose
1,6-diphos-
The concentration of FDP was determined in the PFK assay system under a variety of different test conditions. For this purpose, aliquots were removed from the assay mixture and added to one-quarter of the volume of 6 N HClO,. The HClO&reated sample was brought to 50°C for 5 min to destroy enzyme activities (especially aldolase) which would remain after deproteinization with cold HClO,. The heat treatment did not lead to measurable losses of FDP, F-6-P, or G-6-P. After the heat treatment, the sample was cooled at 0°C for 10 min and centrifuged (20,OOOg for 10 min). The supernatant was brought to pH 6-6.5 with KOH, and KClO, was removed by centrifugation. The supernatant was used for the determination of FDP. Although the determination of FDP via aldolase, triosephosphate isomerase, and o-glycerophosphate dehydrogenase results in the formation of 2 mol of NADH per mole of FDP split in the aldolase reaction, this assay proceeds extremely slowly when the concentration of FDP is low. Therefore, FDP was determined in the same coupled reaction sequence as that used for the determination of FDPase activity. The assay mixture contained (final concentrations): triethanolamine-Cl, 60 mM; MgCl,, 8 mM; EDTA, 0.8 mM; NADP, 0.66 mM; appropriate amounts of neutralized HClO, extract. The pH was 8.0, and the temperature was 32°C. The reaction was started by the addition of glucose 6-phosphate dehydrogenase (0.24 U/ml) and phosphohexose isomerase (1.5 U/ml). When all of the G-6-P and F-6-P had been converted to 6-phosphogluconate and the reaction had come to a complete stop, FDPase was added (0.14 U/ml of purified rat liver FDPase) and the increase in the formation of NADPH was measured. It was established in control experiments that this assay system allows the determination of small amounts of FDP in the presence of a lOO- to 500-fold excess of glucose 6-phosphate and/or fructose 6-phosphate. Since the concentrations of FDP measured in our experiments were rather low, the assays were performed in an Aminco DW-2 dual-wavelength spectrophotometer, which allowed a 200-fold amplification of the signal in the dual mode (wavelength
ET AL. pair, 375/350 nm). Glucose I-phosphate dehydrogenase and phosphohexose isomerase were not freed from (NH&SO, prior to use.
Reaction of Glucose 1,6-diphosphate with FDPase and Purity of Glucose 1,6-diphosphate The assay system described for the determination of FDPase activity was used. The reaction mixture contained 0.12 U/ml of purified rat liver FDPase. The reaction was started by adding G-1,6-DP (final concentration, 1 m&f). The rate was less than 3% of the rate with the same concentration of FDP and was not significantly different from the control value without substrate. The amount of FDP in the G-1,6-DP preparation was determined in the sensitive FDP assay system already described. The G-1,6-DP preparation used contained 1.2% FDP on a molar basis. RESULTS
When PFK activity was measured using assay system I (pyruvate kinase coupled), the reaction rate after the addition of PFK was not linear from the beginning, but started slowly and reached linearity only after several minutes. The increase in reaction velocity occurred together with an increase in the FDP concentration in the assay system (Fig. 1 curve A). When 20 PM FDP (final concentration) was added before starting the reaction, the reaction rate was linear from the start with PFK and remained so (Fig. 1). When under these conditions FDPase was added immediately after the addition of PFK, the reaction slowed down within a few minutes, again in parallel with a decrease in the concentration of FDP in the assay system (Fig. 1, curve B). In this respect, there was no difference between rat liver FDPase and identical activities of rabbit muscle FDPase . To discriminate between a direct inhibition of PFK by FDPase on one hand and an inhibition of PFK by draining off the positive allosteric effector FDP on the other hand, another experimental approach was carried out: The reaction was initiated by PFK in the presence of 20 PM FDP and 20 PM FDPase. At the same time, FDP was infused into the assay cuvette at varying rates to avoid or to inhibit the drop in concentration of FDP due to FDPase. As shown in Fig. 2, curve A, the inhibition by
PHOSPHOFRUCTOKINASE
INHIBITION
BY FRUCTOSE
1,8DIPHOSPHATASE
FIG. 1. Correlation between the activity of phosphofructokinase and the concentration of FDP. PFK activity was measured using assay system I (see Materials and Methods). In curve A (control), no exogenous FDP was added. The reaction was started by the addition of 0.75 pg of ATP-free purified rabbit muscle PFK. In curve B, FDP (final concentration, 20 pM) was added to the assay mixture before starting the reaction by the addition of 0.75 pg of ATP-free purified rabbit muscle PFK. Two seconds later, 3.2 pg of purified rat liver FDPase was added. The total assay volume in A and B was 2.1 ml. Samples for the determination of FDP were taken from larger assay mixtures which were run in parallel to the reaction in the photometer cuvette, as described under Materials and Methods. The reaction was followed in an Aminco-Chance DW2 spectrophotometer at the wavelength pair 275/250 nm. The arrows and numbers indicate the time points of measurement and the concentrations of FDP in the reaction mixture, respectively.
FIG. 2. Effects of a continuous infusion of varying amounts of FDP into the reaction cuvette on the inhibitory effect of FDPase on phosphofructokinase. Phosphofructokinase was measured using assay system I (see Materials and Methods). Immediately before starting the reaction, FDP (final concentration, 20 PM) was added to the assay system. The reaction was initiated by the addition of 0.8 pg of purified ATP-free rabbit muscle phosphofructokinase. One second later, purified rat liver FDPase was added. The amount of FDPase added catalyzed the conversion of 18 nmol of FDP.min-’ under the conditions of assay system I and a FDP concentration of 20 PM. (A) represents a control assay without infusion of FDP; 11.8 (B), 17.6 (0, and 23.5 (Dl nmol of FDP.min’ were continuously infused into the reaction cuvette. The reaction was stopped for the analysis of FDP 10 min atier the addition of PFK. The reaction was measured at the wavelength pair 375/350 nm in an Aminco-Chance DW-2 dual-wavelength spectrophotometer.
567
568
SOLING
FDPase was clearly visible when the FDP concentration had fallen to 1.3 PM. When the concentration was set to 40.9 or 13.6 PM (Fig. 2, curves C and D), the reaction was linear from the start and remained so in spite of the presence of FDPase. Even at a very low infusion rate, which was only able to keep the concentration of FDP at 2.6 PM, the reaction rate was only slightly lowered by FDPase (Fig. 2, curve B). This fits well with the observation (Fig. 1) that without an infusion of FDP the reaction rate in the presence of FDPase continued to proceed linearly until the FDP concentration had fallen below 2.5 FM. G-1,6-DP is able to activate PFK but is not split by FDPase. When FDPase was added to G-1,6-DP-activated PFK, the reaction rate was not significantly affected (Fig. 3, curve B) in contrast to the system in which PFK was activated by FDP (Fig. 3, curve A). When a preincubation was carried out in the presence of PFK, FDPase, and G-1,6DP, the addition of F-6-P initiated immediately the PFK-catalyzed reaction and the rate was linear from the beginning (Fig. 3, curve D). However, when the preincubation system contained FDP instead of G-1,6-DP, no reaction could be initiated by the addition of F-6-P (Fig. 3,
ET AL.
curve C) due to the fact that all FDP had been split by FDPase during the preincubation period. Again, FDPases from rat liver and from rabbit muscle behaved identically in this respect. Sedoheptulose 1,7-diphosphate activates PFK, although less than FDP or G-1,6-DP (Fig. 4). In the presence of 20 or 40 PM
FIG. 4. Stimulation of rat liver PFK by sedoheptulose 1,7-diphosphate (SDP) and inhibition of this effect by purified rat liver FDPase. The activity was measured using assay system I (see Materials and Methods). The test system contained 0.24 pg.ml-’ of PFK and 1.9 pg.ml-’ of FDPase. (B and 0. In B and C, SDP was added to the test mixture before starting the reaction. The SDP concentration was 20 pM in C and 40 ~,LM in D. Curve A represents a control experiment without added SDP.
FIG. 3. Effects of purified rat liver FDPase on the reaction catalyzed by purified rat liver phosphofi-uctokinase in the presence of either 10 pM fructose 1,6diphosphate (A and C) or 80 pM glucose 1,6-diphosphate (B and D). PFK activity was measured in assay system I (see Materials and Methods). The test system contained 0.24 pg.ml-’ of PFK. The amount of FDPase added was 1.9 pg.ml-‘. The sequence of addition was: (A) PFK, FDP, F-6-P, FDPase; (B) PFK, G-1,6-DP, F-6-P, FDPase; (Cl F-6-P, FDP, FDPase, PFK: (D) F-6-P, G-1,6-DP, FDPase, PFK.
PHOSPHOFRUCTOKINASE
INHIBITION
SDP, a linear reaction rate was reached earlier than in its absence. Apparently, the activating effect of SDP at the concentrations used was not sufficient to activate PFK fully, so that complete activation was only reached after additional FDP had been formed during the reaction. Upon addition of FDPase, the reaction rate was almost abolished within a few minutes (Fig. 4, curves B and C). Since it is known from the work of Bonsignore et al. (6) and Pontremoli et al. (7) that FDPase splits not only FDP but also SDP, it seems rather clear that the inhibitory effect of FDPase is again brought about by removal of SDP as well as FDP and not by a direct proteinprotein interaction. The inhibition of the PFK-catalyzed reaction by higher activities of aldolase has already been reported by El-Badry et al. (8) and was attributed to the removal of FDP. However, FDP concentrations had not been measured. Table I shows that, with increasing concentrations of aldolase in assay system III, the flux through the TABLE CORRELATION FDP IN THE
CONCENTRATION
Concentration of aldolase (pg.ml-‘) 0.14 0.34 0.54 0.67 0.81 0.94 1.01
I
BETWEEN THE CONCENTRATION TEST SYSTEM, REACTION RATE,
OF AND
OF ALWLASE”
Reaction rate after reaching linearity (AE ‘10 min-‘) 0.148 0.260 0.260 0.260 0.225 0.163 0.095
Concentration of FDP (pm01 . liter’) 17.55 5.83 2.77 1.29 0.53 0.44 0.27
a Assay system III (see Materials and Methods) was used. Rabbit muscle PFK pretreated as described under Materials and Methods was used (2.7 pg/ml). The rabbit muscle aldolase used had been freed from ammonium sulfate by gel filtration on Sephadex G-25 equilibrated with 50 mM Tris-Cl (pH 7.1) containing 1 mM EDTA. The reaction rates given were taken from the linear portion of the reaction curve. Linearity was reached after 1 to 5 min depending on the concentration of the aldolase. Aliquota for determination of the concentration of FDP were taken 2 min after linearity had been reached.
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coupled enzyme system first increased, but started to decrease when the concentration of aldolase exceeded 0.67 pg * ml-‘. At the lowest concentration of aldolase, the highest concentration of FDP was found, as is to be expected since under these circumstances aldolase and not PFK was rate limiting. When the concentration of aldolase was raised, PFK became rate limiting as indicated by the constant overall rate between 0.34 and 0.67 ,ug of aldolase-ml-‘. When the concentration of aldolase exceeded 0.67 pg. ml-‘, the overall rate started to decrease. This decrease was associated with a decrease in the steadystate concentration of FDP below 1.29 j-&M. This is in good agreement with the results presented in Fig. 1, where an inhibition of the reaction by FDPase was not seen before the concentration of FDP had fallen below 2.5 PM. Thus, for a given concentration of FDP, there is no difference between the action of FDPase and aldolase on PFK. All of these results made it rather likely that the inactivating effect of FDPase on PFK described by Proffitt et al. (3) was also due to removal of FDP from PFK. Therefore, rabbit muscle PFK equilibrated with either FDP or G-1,8DP was incubated with FDPase in the same system as that described by Proffitt et al. (31, the main difference being that rabbit muscle FDPase instead of rabbit liver FDPase was used. After incubation, PFK was tested in the same test system as that given by Proffitt et al. (3). The results are given in Figs. 5and6. The effect of FDPase on PFK which had been equilibrated with FDP is seen from the time interval between the start of the reaction in the test system and the point at which the reaction became linear. When the ratio PFK/FDPase was l/z (w/w), the time until linearity was reached was prolonged when the activity was tested 30 s after the addition of FDPase (Fig. 5A). After 10 min of incubation, it lasted more than 18 min until linearity was reached (Fig. 5B). When the ratio PFK/FDPase was lowered to l/3, the effect of FDPase was seen 30 s after the addition of FDPase. At greater than 20 min, the PFK reaction did not start (Fig. 5C). The same result
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FIG. 5. Effects of preincubation of FDP-equilibrated rabbit muscle PFK with rabbit muscle FDPase on PFK activity. Phosphofructokinase was equilibrated with FDP under the conditions described under Materials and Methods. It contained 18.1 nmol of FDP mg of protein-‘. PFK activity in assay system II (see Materials and Methods) is shown. The reaction was initiated by the addition of an aliquot of the incubation mixture. (Al and (Bl refer to the same incubation. This incubation contained, in a final volume of 0.2 ml, 90 pg of FDP-equilibrated PFK and 188 pg of FDPase. The interaction of the two enzymes was started by the addition of FDPase. The incubation was carried out as described under Materials and Methods. After 30 s and after 20 min, aliquots were removed from the incubation and tested for PFK activity. (Cl and (D) refer to another incubation of the same FDP-equilibrated PFK with FDPase. In this case, only 60 pg of the PFK was incubated with 188 pg of FDPase; otherwise the conditions were the same as those of (A) and (B). In (Cl and (D), FDPase-treated PFK remained inactive in assay system II even after more than 30 to 40 min. To the control incubations, instead of FDPase, only the same volume of 5 mM sodium malonate (pH 6.2) was added.
was seen after 20 min of incubation (Fig. 5D). In contrast, G-1,6-DP-equilibrated PFK was not significantly inactivated (Fig. 6) even at a concentration of FDPase which was about four times higher than that of PFK (w/w). In comparison to control incubations, the reaction reached linearity about 2 min later. This resulted from the transfer of some FDPase activity from the incubation medium into the test system. There was no difference whether G-1,6-DP-equilibrated PFK was incubated for 30 s or for 20 min with FDPase (Figs. 6A and 6B). All of these incubations were performed in the absence of Mg2+ and EDTA. When FDPase activity was tested under these conditions, FDPase activity could still be measured although the activity was only about 3.5% of the activity obtained in the presence of Mg2+ and EDTA. However, this residual activity was sufficient to split all or most of the FDP associated with FDP-equilibrated PFK.
Effects of Removal of FDPase on PFK Activity in Rat Liver 100,OOOg Supernatant Due to dilution during homogenization, gel filtration, and immunoprecipitation, the final activity of PFK in the extract was rather low but still measurable without difficulty when a lo-fold scale expension was used during the photometric assay (assay system IV). The removal of FDPase by precipitation with anti-rat liver FDPase antiserum increased the apparent PFK activity (Table II). This increase was associated with an increase in the concentration of FDP in the assay system (Table II). DISCUSSION
According to our results, the findings used by Uyeda and Luby ( 1) and by Proffitt et al. (3) as an argument for a direct inhibition of PFK by FDPase can be explained by removal of FDP from the PFK protein.
PHOSPHOFRUCTOKINASE
INHIBITION
FIG. 6. Effects of preincubation of G-1,8DPequilibrated rabbit muscle PFK with rabbit muscle FDPase on PFK activity. The conditions of the experiment were the same as those of Fig. 5 except that PFK which had been equilibrated with glucose 1,6-diphosphate was used. The PFK contained 13.6 nmol of glucose 1,6-diphosphate ‘mg of protein-‘. The incubation mixture contained, in a final volume of 0.2 ml, 65 pg of glucose 1,6-diphosphate-equilibrated PFK and 260 pg of FDPase. PFK activity in assay system II is shown. The reaction was initiated by the addition of aliquots of the incubation mixture after 30 s (A) and after 20 min (B) of incubation. TABLE
II
EFFECTS OF REMOVAL OF FDPase ACTIVITY FROM RAT LIVER 100,OOOg SUPERNATANT (ES,) BY IMMUNOPRECIPITATION ON THE ACTIVITY OF PFK FROM THE SAME HOMOGENATE” Experiment
I
II
Experimental tion
condi-
S, treated with control serum S, treated with antiFDPase antiserum S3 treated with control serum S, treated with antiFDPase antiserum
PFK activity W’wj
Concentration of FDP (pm01 . liter’)
0.040
0.37
0.062
0.64
0.050
0.28
0.088
0.76
’ Assay system IV (see Materials and Methods) was used. No exogenous aldolase was added since endogenous activity proved to be sufficient. The concentrations of FDP were determined in samples taken from the assay mixture when the reaction rate had become linear.
El-Badry et al. (8) suspected that the inactivation of sheep heart PFK by aldolase and FDPase resulted from the removal of FDP from PFK rather than from a direct protein-protein interaction. A similar con-
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1.6-DIPHOSPHATASE
571
elusion was drawn by Emerk and Frieden (2). The direct measurement of the concentration of FDP during inactivation of PFK by FDPases from the different sources as well as by aldolase (Table I) shows clearly that the degree of inhibition correlates with the concentration of FDP but does not exhibit specificity with respect to either aldolase or FDPase. Moreover, FDPase exerts no inhibitory effect when the decrease of FDP is avoided (e.g., by infusion of FDP) or when FDP is replaced by G-1,6DP, a metabolite not split by FDPase. In accordance with Proffitt et al. (31, we found an immediate inactivation of rabbit muscle PFK by FDPase. In contrast to Proffitt et al. (31, we used rabbit muscle FDPase instead of rabbit liver FDPase in order to work with a more physiological system. This rapid inactivation occurred only with untreated PFK or with FDPequilibrated PFK and not with G-1,6-DPequilibrated PFK. The inactivation of untreated FDP-equilibrated PFK was seen in the absence of exogenous Mg*+ and EDTA, both known activators of FDPase. Profftt et al. (3) concluded, therefore, that FDPase must have been completely inactive under those conditions, and, hence, the inactivation must have proceeded via direct protein-protein interaction. This argument is not valid since, according to our measurements, the FDPase present in the incubation mixture was still active, although much less than seen after the addition of Mg2+ and EDTA. Moreover, when FDPase is added at rather high concentrations, it most probably can remove some FDP from PFK due to its high affinity for FDP. This high affinity of FDPase for FDP, among other findings, is illustrated by the fact that, in experiments where FDP was added at an initial concentration of 20 PM, added FDPase was able to drop the concentration of FDP very rapidly to values below 0.2 PM (the limit of our method). The rabbit muscle FDPase contained small amounts of pyruvate kinase (see Materials and Methods). However, even under the assumption that all binding sites of this contaminating pyruvate kinase would have been satu-
572
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rated with FDP, this would account for only less than 1% of the FDP initially bound to PFK. Proffitt et al. (3) state that the addition of 10 PM FDP prevented the FDPase-induced inactivation of PFK, and one wonders why they did not arrive at the conclusions that the availability of FDP and not the mere presence of FDPase determined PFK activity. Furthermore, any protein-protein interaction between FDPase and PFK in muscle must be considered to be of little or no physiological importance since the amount of FDPase protein in skeletal muscle is less than 1% of that of PFK and a proteinprotein interaction between FDPase and PFK could only occur, if at all, in liver where the amount of FDPase protein is considerably higher than that of PFK protein. A homologous liver system was not used by Proffitt et al. (3) but was examined by Uyeda and Luby (1). We believe that our findings can also explain the results of Uyeda and Luby (1) with one exception: In the experiments of Uyeda and Luby (11, the inhibitory effect of FDPase on PFK was abolished when the FDPase had been activated by prior treatment with homocysteine according to Nakashima et al. (9). Since we were unable to activate purified “neutral” rat liver FDPase or rabbit muscle FDPase by homocysteine in the system described by Nakashima et al. (9), we
ET
AL.
could not repeat these experiments. However, it is possible that in the experiments of Uyeda and Luby (1) the modification of FDPase by homocysteine had been associated with a decreased affinity of FDP for FDPase, which of course is not visible if FDPase activity is tested under V conditions. An increased KmmFDP for FDPase would lead to an increased steady-state concentration of FDP in the combined PFK-FDPase system, resulting in no or a diminished inhibition of PFK. REFERENCES 1. UYEDA, K., AND LUBY, L. J. (1974) J. Bill. Chem. 249,4562-4570. 2. EMERK, K., AND FRIEDEN, C. (1975) Arch. Biothem. Biophys. 168, 210-218. 3. PROFFITT, R. T., SANKAVAN, L., AND POGELL, B. M. (1976) Biochemistry 15, 2918-2925. 4. BRAND, I., AND SOLING, H. D. (1974) J. Bid. Chem. 249, 7824-7831. 5. PONTREMOLI, S., TRANIELLO, S., LUPIS, B., AND WOOD, W. A. (1965)J. Biol. Chem. 240, 34593463. 6. BONSIGNORE, A., MANCIAROITI, G., MANGIAROITI, M. A., DE FLORA, A., AND PONTREMOLI, S. (1963) J. Biol. Chem. 238, 3151-3154. 7. PONTREMOLI, S., LUPIS, B., Wool, W. A., TRANIELLO, S., AND HORECKER, B. L. (1965) J. Biol. Chem. 240, 3464-3468. 8. EGBADRY, A. M., OTANI, A., AND MANSOLJR, T. E. (1973) J. Bid. Chem. 248, 557-563. 9. NAKA~HIMA, K., HORECKER, B. L., TRANIELLO, S., AND PONTREMOLI, S. (1970)Arch. Biochem. Biophys. 139, 190-199.
