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Evidence for an interaction between fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase
ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 19’7,No. 1, October 1, pp. 356-363, 1979
Evidence
for an Interaction between Fructose 1,6-Bisphosphatase and Fructose 1,6-Bisphosphate Aldolase
S. PONTREMOLI,” E. MELLONI,” F. SALAMINO,” B. SPARATORE,* M. MICHETTI,” V. N. SINGH,? AND B. L. HORECKERt *Institute of Biological Chemistry, University of Genoa, Genoa, Italy, and tRoche Institute of Molecular Biology, Nutley, New Jersey 07110
Received April 11, 19’79;revised May 25, 1979 Three distinct lines of evidence suggest interaction and possible complex formation between fructose l&biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn *+ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.
There has been considerable discussion of the possibility that in the cell the “soluble” cytoplasmic enzymes may be bound to cellular matrices or organized into multienzyme complexes (for reviews and discussion of the problem see Refs. (l-3)). A multienzyme complex containing all of the glycolytic enzyme activities has been detected in extracts of Escherichia coli (41, and both kinetic and physicochemical evidence for an interaction of fructose 1,6-biphosphate aldolase and glyceraldehyde 3-phosphate dehydrogenase isolated from rabbit muscle has been reported (5, 6). Direct evidence for an interaction of glycolytic or gluconeogenie enzymes involving weak noncovalent forces is difficult to obtain by the methods usually employed. Recently Fahien and Smith (‘7) have used a technique based on the extent of penetration of the enzyme proteins into Sephadex G-200 to demonstrate an association between glutamateoxaloacetate transaminase and glutamic dehydrogenase. 0003-9861/79/l10356-08$02.00/O Copyright 0 1979by AcademicPress, Inc. AII rights of reproductionin any form reserved.
Our interest in a possible interaction between rabbit liver fructose l,gbisphosphate aldolase and rabbit liver fructose 1,6bisphosphatase (Fru-P,ase)‘, which catalyze successive reaction in gluconeogenesis, arose from the observation that rabbit liver aldolase tended to copurify with rabbit liver Fru-Pzase when the livers were obtained from fed, but not from fasted rabbits (8). In the present paper we report evidence for an interaction between the two enzymes based on changes in physicochemical properties of each enzyme caused by the presence of the other enzyme protein. We have also observed that the ability of liver Fru-Pzase and liver aldolase to penetrate into Ultrogel AcA34 was decreased when both enzymes were present. These effects were observed only with mixtures of enzymes isolated from the same tissue (i.e., I Abbreviations used: Fru-Po, fructose 1,6-bisphosphate; Fru-P*ase, fructose 1,6-bisphosphatase; PEP, phosphoenol pyruvate.
356
INTERACTION
BETWEEN
Fru-P,ase AND Fru-P,ase ALDOLASE
liver), but not when one of the proteins was replaced by the corresponding enzyme purified from muscle, MATERIALS Fru-P,ase was purified from rabbit liver as previously described (9). Muscle Fru-P,ase was purified from skeletal muscle of commercial field-grown brown rabbits by the procedure described by Black et al. (lo), as modified by Pontremoli et al. (11) except that a column of Ultrogel AcA44 was employed instead of Sephadex G-100 for the final filtration. The purified enzyme was stored at 2°C as a precipitate in 80% saturated ammonium sulfate solution. The activity assayed according to the procedure of Black et al. (10) was 15.6 pmol Fru-PP hydrolyzed/min/mg of protein. In polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, the preparation showed a single band with electrophoretic mobility identical to that of purified rabbit liver FruP,ase with an approximate subunit molecular weight of 36,000. Fructose 1,6-biphosphate aldolase (EC 4.1.2.13) was purified from rabbit liver by the following procedure carried out at 0-4°C: 100 g of fresh liver was homogenized in 10-g batches with 45-ml quantities of 0.25 M sucrose containing 1 mM EDTA, pH 7.0, using a Potter-Elvehjem homogenizer with a Teflon pestle and a clearance of 0.2 mm. The combined homogenates were centrifuged for 30 min at 15,000g and the supernatant solution (500 ml) was treated with 10 g of moist phosphocellulose Pll (Whatman Biochemicals Ltd., Springfield Mill, England) which had been prepared and washed as previously described (12). During the addition the pH was maintained at 7.0 with 2 N NaOH. The suspension was filtered and the clear filtrate was treated with additional phosphocellulose, maintaining the pH at 6.4 with 2 N NaOH, until 80-96% of the aldolase activity was absorbed. The resin was then collected by vacuum filtration, washed with 1 liter of 0.15 M Na-acetate buffer, pH 6.0, containing 0.1 mM EDTA, and transferred to a column (approximately 3 x 15 cm) where washing was continued with 0.2 M Na-acetate buffer in 0.1 mM EDTA, pH 6.1, until the absorbance at 280 nm was less than 0.04. Aldolase was then eluted with the last washing buffer containing 2 mM Fru-P,. The fractions containing aldolase were combined and precipitated with 4 vol of saturated (NH&SO,. The specific activity was 2 units/mgof protein and the recovery was 60-80% of the activity present in the first supernatant solution. Before use, enzyme solutions were filtered through a column of Sephadex G-100 equilibrated with 0.01 M Na-acetate, 0.1 mM EDTA, pH 6.5, to remove Fru-P,, and triose phosphates. Rabbit muscle fructose 1,6-biphosphate aldolase, pyruvate kinase, myokinase, glycerophosphate dehy-
357
drogenase and triose-phosphate isomerase, ATP, AMP, and PEP were purchased from BoehringerMannheim, Mannheim, German Federal Republic. FIX-P, and NADP were obtained from Sigma Chemical Company, St. Louis, Missouri. Ultrogel AcA44 and Ultrogel AcA34 were obtained from LKB, Bromma 1, Sweden. All other chemicals were reagent grade. Radioactive 65ZnCl, (carrier free, 100 mCi/mg) was obtained from Radiochemical Centre Ltd., Amersham, England. Antiserum against rabbit liver aldolase, prepared in the guinea pig, or serum from nonimmunized guinea pigs were coupled (13) to BrCN-activated Sepharose (Pharmacia Fine Chemicals). The resin was then washed with 0.1 M NaHCO,, pH 9.0, and 20 mM Naphosphate, pH 7.0, containing 0.15 M NaCl and finally equilibrated with 20 mM diethanolamine-20 mM triethanolamine, pH 8.5. METHODS Fru-P,ase activity was measured in a spectrophotometric assay system based on reduction of NA.DP at pH 7.5 in the presence of 2 mM MgCl, (19, or by the release of inorganic phosphate, using the method of Tashima and Yoshimura (14) modified as previously described (9). Fructose 1,6-bisphosphate aldolase was assayed as described by Gracy et al. (15). Measurement of the binding of 65Zn*+to Fru-P,ase was carried out by the gel-filtration technique previously described (9), using Sephadex G-50 columns (1.2 x 54 cm) equilibrated with 20 mM diethanolamine-20 mM triethanolamine at the indicated pH and a flow rate of 0.8 ml/min. Fractions of 1 ml were collected in plastic tubes and radioactivity content was measured in a Packard auto-counter Model 5110. The protein concentration was determined by the absorbance at 280 nm using a value of 0.73 for the absorbance of a solution containing 1 mg/ml. When mixtures of Fru-P2ase and aldolase were used, the aldolase content of each fraction was calculated from the aldolase activity, using a specific activity of 2 units/mg for the liver enzyme and 10 units/mg for the muscle enzyme. The molecular weights of liver and muscle Fru-P,ase were taken as 144,000(12) and 142,000 (10) and those of liver and muscle aldolase as 158,000 (15) and 160,000 (16), respectively. Unless indicated, incubation of Fru-P,ase with aldolase was carried out at room temperature (202PC). Fluorescence measurements were carried out in an Aminoc Bowman spectrophofluorometer with a 10 x lo-mm cell and right angle optical geometry. Fluorescence spectra were recorded using an excitation wavelength of 280 nm. All experiments were carried out at 20°C in 20 mM diethanolamine-20 mM triethanolamine, pH 8.5. Gel penetration experiments. These experiments were carried out according to the procedure of Ackers
358
PONTREMOLI
ET AL.
(17) as described by Fahien and Smith (7). Two milliliters of packed Ultrogel AcA34, washed three times in plastic tubes with 5 mM diethanolamine-5 mM triethanolamine, pH 3.5, was mixed with 2 ml of the same buffer solution containing either Fru-P,ase alone or aldolase alone or mixtures of the two enzymes. The suspensions were stirred for 2 h at 4°C. At the end of this time the suspensions were centrifuged (5009) and aliquots of the clear supernatant were removed for determination of FruP,ase activity. The distribution coefficient (K,) was defined as: K = Vf - va D v, -v, where VI is the penetration volume of Fru-P,ase calculated from the total units of Fru-P,ase added, divided by the units per milliliter of Fru-Ppase recovered in the clear supernatant, V, is the total volume of the system. V,, the volume of the aqueous phase, was calculated from a parallel experiment with the same quantity of Ultrogel AcA34 and the same volume of buffer containing blue Dextran 2000 as a marker, by dividing the total amount of blue Dextran added by the concentration of blue Dextran recovered in the clear supernatant. RESULTS
Effect of Rabbit Liver Fru-P,ase on the Fluorescence Emission Spectrum of Rabbit Liver Aldolase The addition of rabbit liver Fru-P,ase to a solution of rabbit liver aldolase caused the fluorescence emission spectrum of the latter to increase in intensity by nearly twofold, with a shift in the emission maximum from 336 to 330 nm (Fig. 1, solid curves). FruP,ase also largely prevented the red shift that is observed when the emission spectrum of aldolase is measured in the presence of nondissociating concentrations of urea (Fig. 1, broken curves). The blue shift in the fluorescence emission spectrum of aldolase was dependent on the concentration of FruPease and reached a maximum when the molar ratio of the Fru-P,ase to aldolase was 2:l (Fig. 2). The effects of 2 eq of liver Fru-P,ase on the location of the emission maximum of liver aldolase are summarized in Table I. The addition of liver Fru-Pzase to a solution of liver aldolase caused a blue shift of 6 nm in the absence of urea, and of 9 nm in the presence of 3 M urea. The red shift observed when the emission spectrum of liver al-
300
320
340
WAVELENGTH
380
380
(nm)
FIG. 1. Effect of rabbit liver Fru-P,ase on the fluorescence emission spectrum of rabbit liver aldolase in the presence or absence of 3 M urea. The solutions contained 0.6 nmol of rabbit liver aldolase in 1.5 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5, or a mixture of 0.6 nmol of rabbit liver aldolaae and 1.2 nmol of rabbit liver Fru-Pzase. They were incubated for 10 min at 20°C and the fluorescence spectra were recorded as reported under Methods. A solution (0.65 ml) containing 10 M urea dissolved in the same buffer was then added to each enzyme solution and the fluorescence spectra were again recorded. The broken lines, representing the spectra recorded after the addition of urea, were corrected for dilution by the urea solution. A solution containing 1.2 nmol of rabbit liver Fru-P,ase alone showed only a small emission peak at 322 nm, with relative fluorescence intensity at that wavelength of approximately 1.3 (not plotted).
dolase was measured in 3 M urea was reduced from 4 to 1 nm by the addition of 2 eq of liver Fru-P,ase. No change in the intensity of fluorescence (data not shown) or in the position of the emission maxima (Table I) was observed when rabbit muscle FruP,ase was added instead of rabbit liver FruP,ase. The effects were therefore specific for the enzymes isolated from the same tissue, and were not due to the catalytic activity of Fru-P2ase. Similar, but smaller, effects on the fluorescence spectrum of rabbit muscle aldolase were observed when the spectra were measured in the presence of rabbit muscle Fru-Pzase; but the emission spectrum was not affected by Fru-P,ase from liver (Table I).
INTERACTION
BETWEEN
336 I
nmol
I / 1 2 Fru-P2asepNette
I 3
359
Fru-P,ase AND Fru-P,ase ALDOLASE
z
J
FIG. 2. Changes in the fluorescence emission spectrum of rabbit liver aldolase as a function of the quantity of rabbit liver Fru-P,ase added. The spectra were recorded as described in the legend to Fig. 1, with the indicated amounts of purified rabbit liver Fru-P,ase added to 0.6 nmol of rabbit liver aldolase, in a total volume of 1.5 ml.
Effect of Rabbit Liver and Muscle Aldolases on the Binding of Zn2+ by Rabbit Liver Fm-P,ase We have previously reported (9) that rabbit liver Fru-P*ase binds 3 eq of Zn2+
per subunit; 2 with high affinity in the absence of the substrate, fructose 1,6-bisphosphate, and the third, with lower affinity, whose binding requires the presence of the substrate. Binding of Zn2+ to the first site inhibits the activity of the enzyme. The binding of Zn2+ to this site, measured in the presence of 0.05 )(LM Zn2+, was found to be decreased by nearly one-half when rabbit liver aldolase was present (Fig. 3). No decrease in the binding of Zn2+ to Fru-P,ase was detected when rabbit muscle aldolase was added in place of rabbit liver aldolase. At this concentration of Zn2+, no binding to either liver or muscle aldolase alone was detected. Similar binding experiments were carried out with varying concentrations of Zn2+ in the column buffer ranging from 40 InM to 10 ,XM and the data plotted according to Scatchard (Fig. 4). These experiments confirmed that the binding of Zn2+ to the first and second sets of binding sites of rabbit liver Fru-P,ase was decreased when liver aldolase was present. The concentrations of Zn2+ required to fill half of the first set of four binding sites were calculated to be 0.018 and 0.09 PM, in the absence or presence of aldolase, respectively. The value of
TABLE I EFFECTOFFru-P,ase ONTHEFLUORESCENCEEMISSIONSPECTRUMOFRABBITLIVERANDMUSCLEALDOLASES Emission maxima and blue shift (-1 in enzyme mixture” No urea
+3 M urea
Red shift produced by 3 M urea’
(nm)
(nm)
(nm)
Liver aldolase +Liver Fru-P,ase +Muscle Fru-Pzase
336 330 (-6) 336 (0)
340 331 (-9) 340 (0)
+4 +1 +4
Muscle aldolase + Muscle Fru-Pzase + Liver Fru-P,ase
331 329 (-2) 331 (0)
336 331 c-51 336 (0)
+5 +2 +5
Additions”
B Purified rabbit liver aldolase (0.6 nmol) or purified rabbit muscle aldolase (0.6 nmol) was incubated with or without 1.2 nmol of purified rabbit liver Fru-P2ase or purified rabbit muscle Fru-P,ase for 10 min at 20°C in 1.5 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5. b The fluorescence spectra were recorded as described in the legend to Fig. 1 before and after the addition to the enzyme solution of 0.65 ml of 10 M urea dissolved in the same buffer. The numbers in parentheses represent the blue shift observed in the presence of Fru-P2ase. c The red shift was calculated by subtracting the maximum emission wavelength of the sample without urea from the maximum emission wavelength of the sample in 3 M urea.
360
PONTREMOLI
ET AL.
KD for the second set of sites was similarly increased from 0.3 PM in the absence of aldolase to 0.8 PM in its presence (Fig. 43). Binding of Zn*+ to the third set of sites, which depends on the presence of FruPz, was not affected and the value of K, was estimated to be 3 PM (Fig. 4B) in agreement with the value reported previously (9). Does Aldolase Affect the Binding of Mg2+ to the First Two Sets of Binding Sites? We have previously reported that the addition of Mg2+ reduces the apparent affinity of the first two sets of binding sites for Zn2+ by approximately one order of magnitude (9) and we employed this property to evaluate the effect of aldolase on the binding of Mg 2+ to these sites. The concentration of Zn2+ required to fill nearly half of the first set of sites was increased lo-fold in the presence of 2 MM Mg and the same ratio was observed when aldolase was present, al-
10
20
30 FRACTION
40
so
00
70
NUMBERS
FIG. 3. Binding of Zn*+ to rabbit liver Fru-P,ase and the effect of rabbit liver aldolase or rabbit muscle aldolase. The binding experiments were carried out as described under Methods. Before filtration, 1.7 nmol of purified rabbit liver Fru-P,ase was incubated for 10 min at 20°C in 0.2 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5, containing 7 nmol of 65Zn2+,either alone or together with 0.85 nmol of purified rabbit liver aldolase or 0.35 nmol of purified rabbit muscle aldolase. In other experiments, as indicated, 1.7 nmol of either rabbit liver aldolase or rabbit muscle aldolase was incubated with Zn*+ in the same conditions. Each sample was filtered through a Sephadex G-50 column equilibrated with the same buffer solution containing 0.05 pM s5Zn2+.The speciAc radioactivity of ssZne+was 12 cpm/pmol.
FIG. 4. Scatchard (19) plots for the binding of 6sZn2+to rabbit liver Fru-P,ase in the presence or in the absence of rabbit liver aldolase. The binding experiments were carried out as described under Methods and in the legend to Fig. 3. The quantity of 65Zn2+in the enzyme mixtures before filtration was 7 nmol in A and 20 nmol in B. The concentrations of 6sZn2+used to equilibrate and develop the columns ranged from 0.04 to 0.25 @Min A and from 0.4 to 10 pM in B. In B 0.1 mM Fru-P, was also added to the incubation mixtures and to the buffers used to equilibrate the columns. The specific radioactivity of 6sZnz+ varied from 12 cpm/pmol at the lower concentrations to 2.5 cpmlpmol at the higher concentrations. The amount of 65Zn*+bound to Fru-P,ase was calculated from the excess radioactivity in the protein peak after subtracting the radioactivity in the same volume of equilibrating buffer. The values (V) are expressed as mol of 65Zn2+boundlmol of Fru-P,ase. When mixtures of Fru-P,ase and aldolase were used the amount of aldolase in the protein peak was estimated from its catalytic activity, using a specific activity of 2 unitslmg for the liver enzyme, and 10 units/mg for the muscle enzyme. The amount of Fru-P,aae was established by the absorbance at 280 nm and by measurement of catalytic activity of the enzyme in each fraction of the protein peak by the spectrophotometric assay system in the presence of 0.1 mM EDTA as reported under Methods, using a specific activity of 14.6 units/mg.
though in each case the concentration of Zn2+ required was increased by 2-fold (Table II, compare lines 1 and 2). Similarly when the concentration of Zn2+ was increased and that of Mg2+ decreased, to permit measurements of binding to the second set of sites, the ratio of Zn2+ concentrations required was increased by 1.5fold both in the presence and absence of aldolase. Although the experiments were indirect the results suggest that the binding of Mg*+ to the first two sets of sites is modified to the same extent as is the binding of Zn2+.
INTERACTION
BETWEEN
361
Fru-P,ase AND Fru-P,ase ALDOLASE TABLE II
EFFECTOFALDOLASE ONTHE RATIOOF%?+ BOUNDIN THE PRESENCEANDABSENCEOF Mg*+ Equilibrium Zn*+ concentration (PM)' Experiment 1 2 3 4
Conditions”
Equivalents of Zn2+ bound”
No Mg*+ (A)
+Mg2+ (B)
Ratio WA)
No Aldolase +Aldolase No Aldolase +Aldolase
1.57 1.55 6.6 6.6
0.02 0.04 1.6 3.5
0.2 0.44 2.5 5.0
10 11 1.56 1.51
a The binding experiments were carried out as described under Methods, using 1.7 nmol of Fru-P*ase mixed, where indicated, with 0.85 nmol of aldolase. Before filtration 7 nmol of 65Zn2Cwas added in experiments 1 and 2 and 14 nmol in experiments 3 and 4. b Determined as described under Methods and in the legend to Fig. 4. c The concentrations of ZnZ+ required in the equilibrating buffers to achieve the amount of binding shown in the second column. The equilibrating buffers also contained 2 mM Mg*+ in experiments 1 and 2; 0.1 mM in experiments 3 and 4.
Correlation of Zn2+ Binding and Inhibition by Zn2+ of Fru-Ppse Activity at Low Concentrations of Zn2f
conditions (Table III). The results obtained indicated that the decreased binding of Znzf to rabbit liver Fru-P*ase in the presence of rabbit liver aldolase correlates with the deThe binding of 65Zn2+was measured in creased inhibition of Fru-P,ase activity. the presence of 2 mM MgClz and the activity The plot of these data revealed that the of the enzyme measured under the same experimental values fall on a straight line TABLE III EFFECT OF RABBIT LIVER ALDOLASE ON THE BINDING AND INHIBITION BY asZn2+OF RABBIT LIVER Fru-Ppase IN THE PRESENCE0~2 mM MgCl,
Experiment” 1 2 3 4 5
Equivalents of 6sZnZ+bound (mol/mol of Fru-P,ase)D
Inhibition of Fru-P,ase activity (%Y
6sZnZ+added to the equilibrating buffer (PM)
No addition
+Aldolase
No addition
+Aldolase
0.2 0.4 0.6 0.8 1.2
0.88 2.16 2.4 3.0 3.62
0.31 1.04 1.28 1.81 2.57
21 55 63 78 92
6 22 32 49 70
n The binding experiments were carried out as described under Methods and in the legends to Figs. 3 and 4. Before filtration 2.6 nmol of Fru-Pzase was incubated for 10 min at 20°C with 1.3 nmol of aldolase in 0.25 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5, containing 2 mM MgCl, and 11 nmol of 65Zn2+. The enzyme mixtures were applied to a Sephadex C-50 column equilibrated with the same buffer solution containing the indicated amounts of ssZn2+and 2 mM MgCl,. The specific radioactivity of 6sZn2+was 8.6 cpm/pmol. b The amount of 6sZn2+bound was calculated as described under Methods and in the legend to Fig. 4. c Fru-P,ase activity was measured on aliquots (0.05 ml) of the fractions containing the enzyme protein (from 0.09 to 0.06 mg/ml) diluted loo-fold with the buffer that had eluted from the column before the protein peak. To 0.25 ml of the diluted enzyme solution, 1~1 of 25 mM EDTA and 1~1 of 25 mM Fru-P, or 1~1 of 25 mM Fru-P, alone were added. After 10 min the inorganic phosphate released was determined as described under Methods. The extent of inhibition was calculated from the aetivity determined in the presence of Fru-P, alone and the activity determined in the presence of Fru-P, and EDTA, with the latter taken as 100%.
362
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ET AL.
TABLE
IV
DISTRIBUTION COEFFICIENTS(K,) OF RABBIT LIVER Fru-P,ase INTO ULTROGEL AcA34”
KLI
Addition and concentration Liver Fru-P,ase (nmol)
Liver aldolase (nmol)
Liver Fru-P,ase
Liver aldolase
1.5 0 1.5 1.5
0 1.5 0.75 1.5
0.84 0.50 0.50
-b 0.81 0.52
Muscle Fru-P,ase (nmol)
Liver aldolase (nmol)
1.5 1.5 1.5
0 0.75 1.5
Muscle Fru-P,ase 0.83
-
0.83 0.83
-
a The experimental procedure and the calculations are described under Methods. 6 Not measured.
that extrapolated to 100% inhibition with 4 eq of Zn2+ bound; this supports the previously proposed model in which the inhibitory effects of Zn*+ were related to its binding to the first set of sites (9). In the presence of rabbit liver aldolase the decreased binding of Zn2+ is correlated with the decreased inhibition of catalytic activity. Reversibility
of the Effect of Aldolase
When the mixture containing rabbit liver Fru-P2ase and rabbit liver aldolase was incubated for 10 min and the aldolase removed by passage through a column containing anti-rabbit liver aldolase serum coupled to Sepharose the affinity of the FruPzase emerging from the column for Zn2+ was the same as that of the native enzyme (data not shown). Thus the effect of aldolase on the affinity of Fru-Pzase for Zn2+ is fully reversible. Gel Filtration Equilibration of Fru-P,ase in the Presence and in the Absence of Rabbit Liver Aldolase The penetration of rabbit liver Fru-P2ase into Ultrogel AcA34 was examined in the absence and in the presence of rabbit liver aldolase (Table IV). The distribution coefficients of rabbit liver Fru-P2ase and rabbit liver aldolase were both significantly decreased in a mixture of the two enzymes, but the distribution coefficient for rabbit
muscle Fru-Pzase was not altered by the addition of rabbit liver aldolase. In similar experiments, muscle aldolase was shown to have no effect on the distribution coefficient of liver Fru-P,ase (data not shown). DISCUSSION
The present results provide evidence for an interaction between liver Fru-Pzase and liver aldolase that is specific for the enzymes isolated from the same tissue. The maximum interaction appears to occur in mixtures containing 2 eq of Fru-P2ase tetramer per equivalent of aldolase tetramer. The reciprocal effects do not appear to be due to the catalytic activities of the enzymes, for example to the removal of traces of Fru-P, that might be present in the enzyme preparations. Thus an equal concentration of muscle aldolase had no effect on the binding of Zn2+ to rabbit liver Fru-P,ase, although its intrinsic specific activity is eightfold greater. Futhermore the effect of aldolase was reversed when it was removed by absorption to specific antiserum coupled to Sepharose. Direct evidence for the formation of a complex could not be obtained. In sucrose gradient sedimentation experiments with a mixture of rabbit liver Fru-P2ase and rabbit liver aldolase each sedimented at the same rate as in the absence of the other proteins. No differences could be observed when the individual enzymes or mixtures of
INTERACTION
BETWEEN
Fru-P,ase AND Fru-P,ase ALDOLASE
the two were filtered through Sephadex G-200 columns. If a complex of these two enzymes were to exist in the cell it might be expected to favor the flux in the direction of gluconeogenesis, since the product of the reaction catalyzed by aldolase, Fru-P,, is the substrate for Fru-Pzase. In addition, the interaction of aldolase and Fru-P*ase might contribute to the regulation of Fru-P2ase activity by reducing the binding of Zn*+ to the sites of high or intermediate affinity. We have previously reported (9) for the rabbit liver enzyme that binding of Zn2+ to the high affinity sites results in decreased activity, whereas binding of Zn2+ to the second set of sites results in enhanced activity. Thus, depending on the concentration of Zn2+ ions, which could be influenced by the levels of endogenous chelating agents, such as histidine (18), the interaction between FruP,ase and aldolase may contribute to enhanced or diminished Fru-P2ase activity. The effect of liver Fru-Pzase on the tryptophan emission spectrum of liver aldolase suggests that the conformation of the latter enzyme is also changed. Thus far, however, we have been unable to detect any effect on the catalytic properties of aldolase in the presumed complex. ACKNOWLEDGMENT The Institute of Biological Chemistry, University of Genoa, acknowledges support from the Italian CNR. REFERENCES 1. MASTERS, C. J. (1977) in Current Topics in Cellular Regulation (Horeeker, B. L., and Stadtman, E. R., eds.), Vol. 12, pp. 75-105, Academic Press, New York. 2. OTTAWAY,J. H., AND MOWBRAY,J. (1977)in Cur-
3.
4. 5. 6.
7.
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rent Topics in Cellular Regulation (Horeeker, B. L., and Stadtman, E. R., eds.), Vol. 12, pp. 107-208, Academic Press, New York. KELETI, T., BATKE, J., OVADI, J., JANCSIK, V., AND BARTHA, F. (1977)in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 15, pp. 233-265, Academic Press, New York. MOWBRAY,J. AND MOSES,V. (1976) Eur. J. Biothem. 66, 25-36. OVADI, J., AND KELETI, T. (1978) Eur. J. Biothem. 85, 157-161. OVADI, J., SALERNO, C., KELETI, T., AND FASELLA, P. (1978) Eur. J. Biochm. 90, 499-503. FAHIEN, L. A., AND SMITH, S. E. (1974) J. Biol.
Chem. 249, 2696-2703. 8. PONTREMOLI, S., MELLONI, E., SALAMINO, F., MICHETTI, M., BOTELHO, L. H., EL-DORRY, H. A., CHU, D. K., ISAACS, C. E., AND HORECKER, B. L. (1978) Arch. Biochem. Biophys. 191, 825-827. 9. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1978) Arch. Biochem. Biophys. 188, 90-97. 10. BLACK, W. J., VAN TOL, A., FERNANDO, J., AND HORECKER, B. L. (1972) Arch. Biochem. Biophys. 151, 576-590. 11. PONTREMOLI, S., SPARATORE, B., SALAMINO, F., MELLONI, E., AND HORECKER, B. L. (1979) Arch. Biochem. Biophys. 194,481-485. 12. TRANIELLO, S., MELLONI, E., PONTREMOLI, S., SIA, C. L., AND HORECKER, B. L. (1972)Arch. Biochem. Biophys. 149,222-231.
13. AXEN, R., PORATH,J., AND ERNBACK, S. (1967) Nature (London) 214, 1302-1304. 14. TASHIMA, Y., AND YOSHIMURA, N. (1975) J. Biochem. 78, 1161- 1169. 15. GRACY, R. W., LACKO, A. G., AND HORECKER, B. L. (1969) J. Biol. Chem. 244, 3913-3919. 16. SIA, C. L., AND HORECKER, B. L. (1968) Arch. Biochem. Biophys. 123, 186-194. 17. ACKERS, G. K. (1964) Biochemistry 3, 723-730. 18. PONTREMOLI, S., MELLONI, E., DE FLORA, A., AND HORECKER, B. L. (1974) Proc. Nat. Acad. Sci. USA 71, 2166-2168. 19. SCATCHARD, G. (1949)Ann. N. Y. Acad. Sci. 51,
660-672.
Evidence
for an Interaction between Fructose 1,6-Bisphosphatase and Fructose 1,6-Bisphosphate Aldolase
S. PONTREMOLI,” E. MELLONI,” F. SALAMINO,” B. SPARATORE,* M. MICHETTI,” V. N. SINGH,? AND B. L. HORECKERt *Institute of Biological Chemistry, University of Genoa, Genoa, Italy, and tRoche Institute of Molecular Biology, Nutley, New Jersey 07110
Received April 11, 19’79;revised May 25, 1979 Three distinct lines of evidence suggest interaction and possible complex formation between fructose l&biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn *+ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.
There has been considerable discussion of the possibility that in the cell the “soluble” cytoplasmic enzymes may be bound to cellular matrices or organized into multienzyme complexes (for reviews and discussion of the problem see Refs. (l-3)). A multienzyme complex containing all of the glycolytic enzyme activities has been detected in extracts of Escherichia coli (41, and both kinetic and physicochemical evidence for an interaction of fructose 1,6-biphosphate aldolase and glyceraldehyde 3-phosphate dehydrogenase isolated from rabbit muscle has been reported (5, 6). Direct evidence for an interaction of glycolytic or gluconeogenie enzymes involving weak noncovalent forces is difficult to obtain by the methods usually employed. Recently Fahien and Smith (‘7) have used a technique based on the extent of penetration of the enzyme proteins into Sephadex G-200 to demonstrate an association between glutamateoxaloacetate transaminase and glutamic dehydrogenase. 0003-9861/79/l10356-08$02.00/O Copyright 0 1979by AcademicPress, Inc. AII rights of reproductionin any form reserved.
Our interest in a possible interaction between rabbit liver fructose l,gbisphosphate aldolase and rabbit liver fructose 1,6bisphosphatase (Fru-P,ase)‘, which catalyze successive reaction in gluconeogenesis, arose from the observation that rabbit liver aldolase tended to copurify with rabbit liver Fru-Pzase when the livers were obtained from fed, but not from fasted rabbits (8). In the present paper we report evidence for an interaction between the two enzymes based on changes in physicochemical properties of each enzyme caused by the presence of the other enzyme protein. We have also observed that the ability of liver Fru-Pzase and liver aldolase to penetrate into Ultrogel AcA34 was decreased when both enzymes were present. These effects were observed only with mixtures of enzymes isolated from the same tissue (i.e., I Abbreviations used: Fru-Po, fructose 1,6-bisphosphate; Fru-P*ase, fructose 1,6-bisphosphatase; PEP, phosphoenol pyruvate.
356
INTERACTION
BETWEEN
Fru-P,ase AND Fru-P,ase ALDOLASE
liver), but not when one of the proteins was replaced by the corresponding enzyme purified from muscle, MATERIALS Fru-P,ase was purified from rabbit liver as previously described (9). Muscle Fru-P,ase was purified from skeletal muscle of commercial field-grown brown rabbits by the procedure described by Black et al. (lo), as modified by Pontremoli et al. (11) except that a column of Ultrogel AcA44 was employed instead of Sephadex G-100 for the final filtration. The purified enzyme was stored at 2°C as a precipitate in 80% saturated ammonium sulfate solution. The activity assayed according to the procedure of Black et al. (10) was 15.6 pmol Fru-PP hydrolyzed/min/mg of protein. In polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, the preparation showed a single band with electrophoretic mobility identical to that of purified rabbit liver FruP,ase with an approximate subunit molecular weight of 36,000. Fructose 1,6-biphosphate aldolase (EC 4.1.2.13) was purified from rabbit liver by the following procedure carried out at 0-4°C: 100 g of fresh liver was homogenized in 10-g batches with 45-ml quantities of 0.25 M sucrose containing 1 mM EDTA, pH 7.0, using a Potter-Elvehjem homogenizer with a Teflon pestle and a clearance of 0.2 mm. The combined homogenates were centrifuged for 30 min at 15,000g and the supernatant solution (500 ml) was treated with 10 g of moist phosphocellulose Pll (Whatman Biochemicals Ltd., Springfield Mill, England) which had been prepared and washed as previously described (12). During the addition the pH was maintained at 7.0 with 2 N NaOH. The suspension was filtered and the clear filtrate was treated with additional phosphocellulose, maintaining the pH at 6.4 with 2 N NaOH, until 80-96% of the aldolase activity was absorbed. The resin was then collected by vacuum filtration, washed with 1 liter of 0.15 M Na-acetate buffer, pH 6.0, containing 0.1 mM EDTA, and transferred to a column (approximately 3 x 15 cm) where washing was continued with 0.2 M Na-acetate buffer in 0.1 mM EDTA, pH 6.1, until the absorbance at 280 nm was less than 0.04. Aldolase was then eluted with the last washing buffer containing 2 mM Fru-P,. The fractions containing aldolase were combined and precipitated with 4 vol of saturated (NH&SO,. The specific activity was 2 units/mgof protein and the recovery was 60-80% of the activity present in the first supernatant solution. Before use, enzyme solutions were filtered through a column of Sephadex G-100 equilibrated with 0.01 M Na-acetate, 0.1 mM EDTA, pH 6.5, to remove Fru-P,, and triose phosphates. Rabbit muscle fructose 1,6-biphosphate aldolase, pyruvate kinase, myokinase, glycerophosphate dehy-
357
drogenase and triose-phosphate isomerase, ATP, AMP, and PEP were purchased from BoehringerMannheim, Mannheim, German Federal Republic. FIX-P, and NADP were obtained from Sigma Chemical Company, St. Louis, Missouri. Ultrogel AcA44 and Ultrogel AcA34 were obtained from LKB, Bromma 1, Sweden. All other chemicals were reagent grade. Radioactive 65ZnCl, (carrier free, 100 mCi/mg) was obtained from Radiochemical Centre Ltd., Amersham, England. Antiserum against rabbit liver aldolase, prepared in the guinea pig, or serum from nonimmunized guinea pigs were coupled (13) to BrCN-activated Sepharose (Pharmacia Fine Chemicals). The resin was then washed with 0.1 M NaHCO,, pH 9.0, and 20 mM Naphosphate, pH 7.0, containing 0.15 M NaCl and finally equilibrated with 20 mM diethanolamine-20 mM triethanolamine, pH 8.5. METHODS Fru-P,ase activity was measured in a spectrophotometric assay system based on reduction of NA.DP at pH 7.5 in the presence of 2 mM MgCl, (19, or by the release of inorganic phosphate, using the method of Tashima and Yoshimura (14) modified as previously described (9). Fructose 1,6-bisphosphate aldolase was assayed as described by Gracy et al. (15). Measurement of the binding of 65Zn*+to Fru-P,ase was carried out by the gel-filtration technique previously described (9), using Sephadex G-50 columns (1.2 x 54 cm) equilibrated with 20 mM diethanolamine-20 mM triethanolamine at the indicated pH and a flow rate of 0.8 ml/min. Fractions of 1 ml were collected in plastic tubes and radioactivity content was measured in a Packard auto-counter Model 5110. The protein concentration was determined by the absorbance at 280 nm using a value of 0.73 for the absorbance of a solution containing 1 mg/ml. When mixtures of Fru-P2ase and aldolase were used, the aldolase content of each fraction was calculated from the aldolase activity, using a specific activity of 2 units/mg for the liver enzyme and 10 units/mg for the muscle enzyme. The molecular weights of liver and muscle Fru-P,ase were taken as 144,000(12) and 142,000 (10) and those of liver and muscle aldolase as 158,000 (15) and 160,000 (16), respectively. Unless indicated, incubation of Fru-P,ase with aldolase was carried out at room temperature (202PC). Fluorescence measurements were carried out in an Aminoc Bowman spectrophofluorometer with a 10 x lo-mm cell and right angle optical geometry. Fluorescence spectra were recorded using an excitation wavelength of 280 nm. All experiments were carried out at 20°C in 20 mM diethanolamine-20 mM triethanolamine, pH 8.5. Gel penetration experiments. These experiments were carried out according to the procedure of Ackers
358
PONTREMOLI
ET AL.
(17) as described by Fahien and Smith (7). Two milliliters of packed Ultrogel AcA34, washed three times in plastic tubes with 5 mM diethanolamine-5 mM triethanolamine, pH 3.5, was mixed with 2 ml of the same buffer solution containing either Fru-P,ase alone or aldolase alone or mixtures of the two enzymes. The suspensions were stirred for 2 h at 4°C. At the end of this time the suspensions were centrifuged (5009) and aliquots of the clear supernatant were removed for determination of FruP,ase activity. The distribution coefficient (K,) was defined as: K = Vf - va D v, -v, where VI is the penetration volume of Fru-P,ase calculated from the total units of Fru-P,ase added, divided by the units per milliliter of Fru-Ppase recovered in the clear supernatant, V, is the total volume of the system. V,, the volume of the aqueous phase, was calculated from a parallel experiment with the same quantity of Ultrogel AcA34 and the same volume of buffer containing blue Dextran 2000 as a marker, by dividing the total amount of blue Dextran added by the concentration of blue Dextran recovered in the clear supernatant. RESULTS
Effect of Rabbit Liver Fru-P,ase on the Fluorescence Emission Spectrum of Rabbit Liver Aldolase The addition of rabbit liver Fru-P,ase to a solution of rabbit liver aldolase caused the fluorescence emission spectrum of the latter to increase in intensity by nearly twofold, with a shift in the emission maximum from 336 to 330 nm (Fig. 1, solid curves). FruP,ase also largely prevented the red shift that is observed when the emission spectrum of aldolase is measured in the presence of nondissociating concentrations of urea (Fig. 1, broken curves). The blue shift in the fluorescence emission spectrum of aldolase was dependent on the concentration of FruPease and reached a maximum when the molar ratio of the Fru-P,ase to aldolase was 2:l (Fig. 2). The effects of 2 eq of liver Fru-P,ase on the location of the emission maximum of liver aldolase are summarized in Table I. The addition of liver Fru-Pzase to a solution of liver aldolase caused a blue shift of 6 nm in the absence of urea, and of 9 nm in the presence of 3 M urea. The red shift observed when the emission spectrum of liver al-
300
320
340
WAVELENGTH
380
380
(nm)
FIG. 1. Effect of rabbit liver Fru-P,ase on the fluorescence emission spectrum of rabbit liver aldolase in the presence or absence of 3 M urea. The solutions contained 0.6 nmol of rabbit liver aldolase in 1.5 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5, or a mixture of 0.6 nmol of rabbit liver aldolaae and 1.2 nmol of rabbit liver Fru-Pzase. They were incubated for 10 min at 20°C and the fluorescence spectra were recorded as reported under Methods. A solution (0.65 ml) containing 10 M urea dissolved in the same buffer was then added to each enzyme solution and the fluorescence spectra were again recorded. The broken lines, representing the spectra recorded after the addition of urea, were corrected for dilution by the urea solution. A solution containing 1.2 nmol of rabbit liver Fru-P,ase alone showed only a small emission peak at 322 nm, with relative fluorescence intensity at that wavelength of approximately 1.3 (not plotted).
dolase was measured in 3 M urea was reduced from 4 to 1 nm by the addition of 2 eq of liver Fru-P,ase. No change in the intensity of fluorescence (data not shown) or in the position of the emission maxima (Table I) was observed when rabbit muscle FruP,ase was added instead of rabbit liver FruP,ase. The effects were therefore specific for the enzymes isolated from the same tissue, and were not due to the catalytic activity of Fru-P2ase. Similar, but smaller, effects on the fluorescence spectrum of rabbit muscle aldolase were observed when the spectra were measured in the presence of rabbit muscle Fru-Pzase; but the emission spectrum was not affected by Fru-P,ase from liver (Table I).
INTERACTION
BETWEEN
336 I
nmol
I / 1 2 Fru-P2asepNette
I 3
359
Fru-P,ase AND Fru-P,ase ALDOLASE
z
J
FIG. 2. Changes in the fluorescence emission spectrum of rabbit liver aldolase as a function of the quantity of rabbit liver Fru-P,ase added. The spectra were recorded as described in the legend to Fig. 1, with the indicated amounts of purified rabbit liver Fru-P,ase added to 0.6 nmol of rabbit liver aldolase, in a total volume of 1.5 ml.
Effect of Rabbit Liver and Muscle Aldolases on the Binding of Zn2+ by Rabbit Liver Fm-P,ase We have previously reported (9) that rabbit liver Fru-P*ase binds 3 eq of Zn2+
per subunit; 2 with high affinity in the absence of the substrate, fructose 1,6-bisphosphate, and the third, with lower affinity, whose binding requires the presence of the substrate. Binding of Zn2+ to the first site inhibits the activity of the enzyme. The binding of Zn2+ to this site, measured in the presence of 0.05 )(LM Zn2+, was found to be decreased by nearly one-half when rabbit liver aldolase was present (Fig. 3). No decrease in the binding of Zn2+ to Fru-P,ase was detected when rabbit muscle aldolase was added in place of rabbit liver aldolase. At this concentration of Zn2+, no binding to either liver or muscle aldolase alone was detected. Similar binding experiments were carried out with varying concentrations of Zn2+ in the column buffer ranging from 40 InM to 10 ,XM and the data plotted according to Scatchard (Fig. 4). These experiments confirmed that the binding of Zn2+ to the first and second sets of binding sites of rabbit liver Fru-P,ase was decreased when liver aldolase was present. The concentrations of Zn2+ required to fill half of the first set of four binding sites were calculated to be 0.018 and 0.09 PM, in the absence or presence of aldolase, respectively. The value of
TABLE I EFFECTOFFru-P,ase ONTHEFLUORESCENCEEMISSIONSPECTRUMOFRABBITLIVERANDMUSCLEALDOLASES Emission maxima and blue shift (-1 in enzyme mixture” No urea
+3 M urea
Red shift produced by 3 M urea’
(nm)
(nm)
(nm)
Liver aldolase +Liver Fru-P,ase +Muscle Fru-Pzase
336 330 (-6) 336 (0)
340 331 (-9) 340 (0)
+4 +1 +4
Muscle aldolase + Muscle Fru-Pzase + Liver Fru-P,ase
331 329 (-2) 331 (0)
336 331 c-51 336 (0)
+5 +2 +5
Additions”
B Purified rabbit liver aldolase (0.6 nmol) or purified rabbit muscle aldolase (0.6 nmol) was incubated with or without 1.2 nmol of purified rabbit liver Fru-P2ase or purified rabbit muscle Fru-P,ase for 10 min at 20°C in 1.5 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5. b The fluorescence spectra were recorded as described in the legend to Fig. 1 before and after the addition to the enzyme solution of 0.65 ml of 10 M urea dissolved in the same buffer. The numbers in parentheses represent the blue shift observed in the presence of Fru-P2ase. c The red shift was calculated by subtracting the maximum emission wavelength of the sample without urea from the maximum emission wavelength of the sample in 3 M urea.
360
PONTREMOLI
ET AL.
KD for the second set of sites was similarly increased from 0.3 PM in the absence of aldolase to 0.8 PM in its presence (Fig. 43). Binding of Zn*+ to the third set of sites, which depends on the presence of FruPz, was not affected and the value of K, was estimated to be 3 PM (Fig. 4B) in agreement with the value reported previously (9). Does Aldolase Affect the Binding of Mg2+ to the First Two Sets of Binding Sites? We have previously reported that the addition of Mg2+ reduces the apparent affinity of the first two sets of binding sites for Zn2+ by approximately one order of magnitude (9) and we employed this property to evaluate the effect of aldolase on the binding of Mg 2+ to these sites. The concentration of Zn2+ required to fill nearly half of the first set of sites was increased lo-fold in the presence of 2 MM Mg and the same ratio was observed when aldolase was present, al-
10
20
30 FRACTION
40
so
00
70
NUMBERS
FIG. 3. Binding of Zn*+ to rabbit liver Fru-P,ase and the effect of rabbit liver aldolase or rabbit muscle aldolase. The binding experiments were carried out as described under Methods. Before filtration, 1.7 nmol of purified rabbit liver Fru-P,ase was incubated for 10 min at 20°C in 0.2 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5, containing 7 nmol of 65Zn2+,either alone or together with 0.85 nmol of purified rabbit liver aldolase or 0.35 nmol of purified rabbit muscle aldolase. In other experiments, as indicated, 1.7 nmol of either rabbit liver aldolase or rabbit muscle aldolase was incubated with Zn*+ in the same conditions. Each sample was filtered through a Sephadex G-50 column equilibrated with the same buffer solution containing 0.05 pM s5Zn2+.The speciAc radioactivity of ssZne+was 12 cpm/pmol.
FIG. 4. Scatchard (19) plots for the binding of 6sZn2+to rabbit liver Fru-P,ase in the presence or in the absence of rabbit liver aldolase. The binding experiments were carried out as described under Methods and in the legend to Fig. 3. The quantity of 65Zn2+in the enzyme mixtures before filtration was 7 nmol in A and 20 nmol in B. The concentrations of 6sZn2+used to equilibrate and develop the columns ranged from 0.04 to 0.25 @Min A and from 0.4 to 10 pM in B. In B 0.1 mM Fru-P, was also added to the incubation mixtures and to the buffers used to equilibrate the columns. The specific radioactivity of 6sZnz+ varied from 12 cpm/pmol at the lower concentrations to 2.5 cpmlpmol at the higher concentrations. The amount of 65Zn*+bound to Fru-P,ase was calculated from the excess radioactivity in the protein peak after subtracting the radioactivity in the same volume of equilibrating buffer. The values (V) are expressed as mol of 65Zn2+boundlmol of Fru-P,ase. When mixtures of Fru-P,ase and aldolase were used the amount of aldolase in the protein peak was estimated from its catalytic activity, using a specific activity of 2 unitslmg for the liver enzyme, and 10 units/mg for the muscle enzyme. The amount of Fru-P,aae was established by the absorbance at 280 nm and by measurement of catalytic activity of the enzyme in each fraction of the protein peak by the spectrophotometric assay system in the presence of 0.1 mM EDTA as reported under Methods, using a specific activity of 14.6 units/mg.
though in each case the concentration of Zn2+ required was increased by 2-fold (Table II, compare lines 1 and 2). Similarly when the concentration of Zn2+ was increased and that of Mg2+ decreased, to permit measurements of binding to the second set of sites, the ratio of Zn2+ concentrations required was increased by 1.5fold both in the presence and absence of aldolase. Although the experiments were indirect the results suggest that the binding of Mg*+ to the first two sets of sites is modified to the same extent as is the binding of Zn2+.
INTERACTION
BETWEEN
361
Fru-P,ase AND Fru-P,ase ALDOLASE TABLE II
EFFECTOFALDOLASE ONTHE RATIOOF%?+ BOUNDIN THE PRESENCEANDABSENCEOF Mg*+ Equilibrium Zn*+ concentration (PM)' Experiment 1 2 3 4
Conditions”
Equivalents of Zn2+ bound”
No Mg*+ (A)
+Mg2+ (B)
Ratio WA)
No Aldolase +Aldolase No Aldolase +Aldolase
1.57 1.55 6.6 6.6
0.02 0.04 1.6 3.5
0.2 0.44 2.5 5.0
10 11 1.56 1.51
a The binding experiments were carried out as described under Methods, using 1.7 nmol of Fru-P*ase mixed, where indicated, with 0.85 nmol of aldolase. Before filtration 7 nmol of 65Zn2Cwas added in experiments 1 and 2 and 14 nmol in experiments 3 and 4. b Determined as described under Methods and in the legend to Fig. 4. c The concentrations of ZnZ+ required in the equilibrating buffers to achieve the amount of binding shown in the second column. The equilibrating buffers also contained 2 mM Mg*+ in experiments 1 and 2; 0.1 mM in experiments 3 and 4.
Correlation of Zn2+ Binding and Inhibition by Zn2+ of Fru-Ppse Activity at Low Concentrations of Zn2f
conditions (Table III). The results obtained indicated that the decreased binding of Znzf to rabbit liver Fru-P*ase in the presence of rabbit liver aldolase correlates with the deThe binding of 65Zn2+was measured in creased inhibition of Fru-P,ase activity. the presence of 2 mM MgClz and the activity The plot of these data revealed that the of the enzyme measured under the same experimental values fall on a straight line TABLE III EFFECT OF RABBIT LIVER ALDOLASE ON THE BINDING AND INHIBITION BY asZn2+OF RABBIT LIVER Fru-Ppase IN THE PRESENCE0~2 mM MgCl,
Experiment” 1 2 3 4 5
Equivalents of 6sZnZ+bound (mol/mol of Fru-P,ase)D
Inhibition of Fru-P,ase activity (%Y
6sZnZ+added to the equilibrating buffer (PM)
No addition
+Aldolase
No addition
+Aldolase
0.2 0.4 0.6 0.8 1.2
0.88 2.16 2.4 3.0 3.62
0.31 1.04 1.28 1.81 2.57
21 55 63 78 92
6 22 32 49 70
n The binding experiments were carried out as described under Methods and in the legends to Figs. 3 and 4. Before filtration 2.6 nmol of Fru-Pzase was incubated for 10 min at 20°C with 1.3 nmol of aldolase in 0.25 ml of 20 mM diethanolamine-20 mM triethanolamine, pH 8.5, containing 2 mM MgCl, and 11 nmol of 65Zn2+. The enzyme mixtures were applied to a Sephadex C-50 column equilibrated with the same buffer solution containing the indicated amounts of ssZn2+and 2 mM MgCl,. The specific radioactivity of 6sZn2+was 8.6 cpm/pmol. b The amount of 6sZn2+bound was calculated as described under Methods and in the legend to Fig. 4. c Fru-P,ase activity was measured on aliquots (0.05 ml) of the fractions containing the enzyme protein (from 0.09 to 0.06 mg/ml) diluted loo-fold with the buffer that had eluted from the column before the protein peak. To 0.25 ml of the diluted enzyme solution, 1~1 of 25 mM EDTA and 1~1 of 25 mM Fru-P, or 1~1 of 25 mM Fru-P, alone were added. After 10 min the inorganic phosphate released was determined as described under Methods. The extent of inhibition was calculated from the aetivity determined in the presence of Fru-P, alone and the activity determined in the presence of Fru-P, and EDTA, with the latter taken as 100%.
362
PONTREMOLI
ET AL.
TABLE
IV
DISTRIBUTION COEFFICIENTS(K,) OF RABBIT LIVER Fru-P,ase INTO ULTROGEL AcA34”
KLI
Addition and concentration Liver Fru-P,ase (nmol)
Liver aldolase (nmol)
Liver Fru-P,ase
Liver aldolase
1.5 0 1.5 1.5
0 1.5 0.75 1.5
0.84 0.50 0.50
-b 0.81 0.52
Muscle Fru-P,ase (nmol)
Liver aldolase (nmol)
1.5 1.5 1.5
0 0.75 1.5
Muscle Fru-P,ase 0.83
-
0.83 0.83
-
a The experimental procedure and the calculations are described under Methods. 6 Not measured.
that extrapolated to 100% inhibition with 4 eq of Zn2+ bound; this supports the previously proposed model in which the inhibitory effects of Zn*+ were related to its binding to the first set of sites (9). In the presence of rabbit liver aldolase the decreased binding of Zn2+ is correlated with the decreased inhibition of catalytic activity. Reversibility
of the Effect of Aldolase
When the mixture containing rabbit liver Fru-P2ase and rabbit liver aldolase was incubated for 10 min and the aldolase removed by passage through a column containing anti-rabbit liver aldolase serum coupled to Sepharose the affinity of the FruPzase emerging from the column for Zn2+ was the same as that of the native enzyme (data not shown). Thus the effect of aldolase on the affinity of Fru-Pzase for Zn2+ is fully reversible. Gel Filtration Equilibration of Fru-P,ase in the Presence and in the Absence of Rabbit Liver Aldolase The penetration of rabbit liver Fru-P2ase into Ultrogel AcA34 was examined in the absence and in the presence of rabbit liver aldolase (Table IV). The distribution coefficients of rabbit liver Fru-P2ase and rabbit liver aldolase were both significantly decreased in a mixture of the two enzymes, but the distribution coefficient for rabbit
muscle Fru-Pzase was not altered by the addition of rabbit liver aldolase. In similar experiments, muscle aldolase was shown to have no effect on the distribution coefficient of liver Fru-P,ase (data not shown). DISCUSSION
The present results provide evidence for an interaction between liver Fru-Pzase and liver aldolase that is specific for the enzymes isolated from the same tissue. The maximum interaction appears to occur in mixtures containing 2 eq of Fru-P2ase tetramer per equivalent of aldolase tetramer. The reciprocal effects do not appear to be due to the catalytic activities of the enzymes, for example to the removal of traces of Fru-P, that might be present in the enzyme preparations. Thus an equal concentration of muscle aldolase had no effect on the binding of Zn2+ to rabbit liver Fru-P,ase, although its intrinsic specific activity is eightfold greater. Futhermore the effect of aldolase was reversed when it was removed by absorption to specific antiserum coupled to Sepharose. Direct evidence for the formation of a complex could not be obtained. In sucrose gradient sedimentation experiments with a mixture of rabbit liver Fru-P2ase and rabbit liver aldolase each sedimented at the same rate as in the absence of the other proteins. No differences could be observed when the individual enzymes or mixtures of
INTERACTION
BETWEEN
Fru-P,ase AND Fru-P,ase ALDOLASE
the two were filtered through Sephadex G-200 columns. If a complex of these two enzymes were to exist in the cell it might be expected to favor the flux in the direction of gluconeogenesis, since the product of the reaction catalyzed by aldolase, Fru-P,, is the substrate for Fru-Pzase. In addition, the interaction of aldolase and Fru-P*ase might contribute to the regulation of Fru-P2ase activity by reducing the binding of Zn*+ to the sites of high or intermediate affinity. We have previously reported (9) for the rabbit liver enzyme that binding of Zn2+ to the high affinity sites results in decreased activity, whereas binding of Zn2+ to the second set of sites results in enhanced activity. Thus, depending on the concentration of Zn2+ ions, which could be influenced by the levels of endogenous chelating agents, such as histidine (18), the interaction between FruP,ase and aldolase may contribute to enhanced or diminished Fru-P2ase activity. The effect of liver Fru-Pzase on the tryptophan emission spectrum of liver aldolase suggests that the conformation of the latter enzyme is also changed. Thus far, however, we have been unable to detect any effect on the catalytic properties of aldolase in the presumed complex. ACKNOWLEDGMENT The Institute of Biological Chemistry, University of Genoa, acknowledges support from the Italian CNR. REFERENCES 1. MASTERS, C. J. (1977) in Current Topics in Cellular Regulation (Horeeker, B. L., and Stadtman, E. R., eds.), Vol. 12, pp. 75-105, Academic Press, New York. 2. OTTAWAY,J. H., AND MOWBRAY,J. (1977)in Cur-
3.
4. 5. 6.
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