Inhibition of rabbit liver fructose 1,6-bisphosphatase by AMP: Effect of temperature and physiological concentrations of cations and anions

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 209, No. 2, July, pp. 598-695, 1981

Inhibition of Rabbit Liver Fructose 1,6-Bisphosphatase by AMP: Effect

of Temperature and Physiological Concentrations of

Cations and Anions’

ALBERT0 VITA,**’ HIROSHI KIDO, *s3 S. PONTREMOLI,? AND B. L. HORECKER*

*Rode Institute of Molecular Biology, Nuti, New Jersm 07110,’ and fkvtitute of Biolo&al Chemistry, University of Genoa, Gema, Italy

Received December 9. 1980

The sensitivity of rabbit liver fructose l,&bisphosphatase (EC 3.1.3.11) to inhibition by AMP is highly dependent on the temperature, the ionic composition of the assay mixture, and whether Me or Mn*+ . 1s present as the activating cation. The sensitivity to AMP is increased on addition of K+ and decreased by the addition of Na+. At 38’C, in the presenee of physiological concentrations of K+, Na’, HPC$, and histidine, and with saturating levels of Mn2’, the concentration of AMP required for 50% inhibition approaches 100 pM, which is 5-10 times greater than is required with the usual assay conditions. In the presence of both M2+ and Mn *+ the AMP inhibition curve is identical , to that observed with Mns+ alone. In agreement with these effects, the affinity of the enzyme for AMP was found to be decreased in the presence of Mn*+, which also causes the binding curve to become sigmoidal. The preference for Mna+ over Mga+ was confirmed by measurement of Mn *+ binding. The enzyme tetramer was found to bind 8 eq of MnZ+, 4 with high and 4 with lower affinity. In the presence of the substrate analog (01 + o)methyl-D-fructofuranoside the number of binding sites in each set is increased to eight. Mg+ competes for the low-affinity binding sites, but not for the sites with high affinity. The results suggest that the enzyme contains eight structural sites for Mn”+, plus four additional sites that bind the M&-substrate (or Mga-substrate) complex.

The allosteric inhibition of fructose 1,6- bisphosphatase (Fru-Pzase,4 EC 3.1.3.11) by AMP was reported in 1963 by Taketa and Pogell (1) and Mendicino and Va- sarhely (2) and has since been confirmed in a number of laboratories with both crude and purified preparations from a variety of vertebrate species and tissues (3-S), as well as from Saccharomyces cer- eviseae (9), Candida utilus (lo), and Esch- erichia coli (11). In the early experiments

i This paper is dedicated to Professor David Shemin on the occasion of his 70th birthday.

* Present address: Lab. Biochimica Applicata. Universita Camerino, 62032 Camerino (MC), Italy.

’ Present address: Department of Enzyme Chem- istry, Institute for Enzyme F&search, Tokushima University School of Medicine, Tokushima 7’70, Ja- pan.

’ Abbreviations used: Fru-Passe, fructose 1,6-bis- phosphstase; Fru-Pa, fructose 1,6-bisphosphate.

using crude extracts of mammalian liver or kidney, the concentrations of AMP required for 50% inhibition were relatively high, 0.1-0.3 mM, and within the concentration range reported for AMP in rat liver (12). Later workers, however, using purified Fru-Pzase preparations from mammalian liver or kidney, found that much lower concentrations of AMP were required, particularly when the enzyme was assayed with MS+ as the activating cation (8, 13-17).

In an effort to assess the role of AMP as a physiological regulator of Fru-Pzase activity, we undertook a study of the fac- tors that might influence the inhibition of rabbit liver Fru-Pease by this ligand. Our attention was drawn particularly to two earlier observations: (i) The inhibition of partially purified rat liver Fru-Pzase was reported by Taketa and Pogell (5) to be

598 OOS3-9861/81/080598-08$02.OS/O Copyrkht 0 1981 by Academic Preea, Inc. Al1 rights of reproduction in any form reserved.

AMP INHIBITION OF RABBIT LIVER FRUCTOSE 1,6-BISPHOSPHATASE 599

very sensitive to temperature and a similar effect of temperature was observed with more highly purified enzyme preparations from rabbit (13) and mouse (18) liver. (ii) The sensitivity of liver Fru-Pzase to inhibition by AMP was also markedly decreased when Mn2+ replaced Me as the activating cation (8,14,15). The decreased sensitivity to AMP in the presence of Mn2+ was also reported for muscle Fru-P2ase (7, 19, 20). On the basis of this and other observations, Van To1 et ccl. (20) concluded that the activity of Fru-P2ase in muscle might be controlled by the intracellular levels of Ca2+ and Mn2+. Of particular interest was the observation of Opie and Newsholme (7) that the inhibition of muscle Fru-Pzase by AMP measured in the presence of both M$+ and Mn2+ resembled that observed with Mn2+ alone. We have reported the same observation with purified rabbit liver Fru-Pease and also found that the inhibition of this enzyme by AMP is more readily reversed by pyridoxal phosphate when the activity is assayed with Mn2+ or a mixture of Mn2+ and Me, as compared with Mg2f alone (21).

Monovalent cations have also been reported to affect both the activity and the sensitivity to AMP of vertebrate Fru- Pzases. Hubert et al. (22) observed activation of pig kidney and other vertebrate Fru-P2ases by NH4+ and K+ and to a lesser extent by Na+. The sensitivity of rabbit muscle Fru-P2ase to inhibition by AMP was also reported to be increased by K+, but this effect was not observed with the pig kidney or rabbit liver enzymes. These effects of monovalent cations on rabbit muscle Fru-P2ase were confirmed with the purified enzyme by Black et al. (23). More recently Nakashima and Tuboi (24) have carried out a detailed study of the effects of monovalent cations on the activity of neutral rabbit liver Fru-P2ase. They confirmed the activation by NH,+ and K+ and also reported that the sensitivity to inhibition by AMP was enhanced by cations larger than K+ and decreased by smaller cations (e.g., Na+ and Li+); K+ had little or no effect on this parameter.

In the present paper we have reexam- ined the changes in sensitivity of purified

rabbit liver Fru-Pzase to inhibition by AMP that result from the addition of physiological concentrations of cations and anions, including Me, Mn2', K+, Na+, and HPOf. We have also examined the effect of temperature on binding of AMP to Fru-P2ase and on the inhibition by AMP of Fru-Pease activity. The results demon- strate that when the assay is carried out at 3’7°C in the presence of physiological concentrations of monovalent cations and HPOf, AMP becomes an effective modu- lator within its physiological concentration range. We have also found that the enzyme possesses preferred binding sites for Mn2+; the results suggest that Mn2+ and Mgz+ may cooperate in the in vivo regulation of its activity.

EXPERIMENTAL PROCEDURES

Materials. Fru-Prase was purified from rabbit liver

(25) and stored in 80% saturated (NH&SOI. Before use, the enzyme suspensions were centrifuged, the

precipitate dissolved in the appropriate buffer for each experiment and dialyzed overnight at 4’C

against the same buffer containing 1 MM EDTA.

Adenosine 5’-monophosphate (Na salt) and [U-

“CJadenosine 5-monophosphate, diammonium salt (479 Ci/mol) were purchased from the Sigma Chem-

ical Company and from New England Nuclear Cor- poration, respectively. 54MnC1s was from Amersham,

histidine from Sigma Chemical Company, and Seph- adex G56 coarse and blue dextran from Pharmacia

Fine Chemicals. Other materials were from the sources previously identified (26).

(a + @Methyl+-fructofuranoside 1,6-bisphosphate

was a gift from Dr. S. Benkovic of Pennsylvania State University.

Methods. Fru-Pease was assayed as previously described (16) except as otherwise indicated in the leg-

ends to the figures. Protein concentrations were cal-

culated from the absorbance at 296 nm, assuming a value of 0.63 for a solution containing 1 mg/ml.

For measurements of binding of AMP and Mn” an equilibrium solute partitioning method was em-

ployed (27) using a total fluid volume of 0.5 ml and

40 mg of Sephadex G56 coarse powder for each de- termination and protein concentrations as indicated in the figure legends. The equilibrations were carried out with gentle rocking in 50 X 15-mm plastic vials

for 5 h at the temperature indicated. The excluded volume was determined in a similar mixture containing Blue Dextran 2666 (Pharmacia Fine Chemi-

cals).

600 VITA ET AL.

RESULTS

wfect of b2&Gers and monovalent cations on the inhibition of Fru-P2ase by AMP. We have previously reported (21) that in the presence of Mn2+ rabbit liver Fru- P2ase is significantly less sensitive to inhibition by AMP in the presence of Me, and also that the inhibition curve gener- ated in the presence of both Mn2+ and Mg2 is identical to that observed with Mn2+ alone. We therefore employed a mixture of the two cations to evaluate the effect of buffers and other monovalent cations. In these experiments 40 mM (NHJ2S04, which abolishes the inhibition by excess substrate (28), was also added to the assay mixtures. At pH 7.5, with either Tris.HCl or a diethanolamine-triethanolamine . HCl buffer mixture, identical results were obtained for both catalytic activity and inhibition by increasing concentrations of AMP (Fig. 1, curve 2,3). When these buffers were replaced by a K2HP04-KH2P04 buffer mixture at the same pH, we found that the catalytic activity was somewhat

[AMPI YM

FIG. 1. Effect of buffers on the inhibition of Fru- Ppase by AMP. The reaction mixtures (1.0 ml) con-

tained 2.0 mM MgCla, 0.15 mM MnCla, 0.1 mM EDTA, 40 mM (NH4)aSOI, 0.1 mM Fru-Ps, 0.2 mM NADP, and 5 pg each of glucose-g-phosphate dehydrogenase and glucose-g-phosphate isomerase in the following buff-

ers, all at pH 7.5 and 25°C: (1) 20 mM NaPO,, 0; (2) 20 mM diethananolamine/20 mM triethanolamine. HCl, 0; (3) 20 mM Tris.HCl, 0; (4) 20 mM KPO,, A;

(5) 50 mM KPOI, A. In the inset the results are plotted as percentage of activity measured in the absence of AMP. No correction was made for binding of AMP

by M$+; this correction would reduce the concentration of AMP by approximately 15%.

[AMP] PM

FIG. 2. Effect of Na salts on the inhibition of Fru-

Pgse by AMP. The assays were carried out in the

presence of 50 mM KPOl buffer, pH 7.5, as described in the legend to Fig. 1, with the addition of the fol-

lowing Na salts: (1) 36 mM NaCOa, pH 7.5, 0; (2) 20 mM NaPOd, A; (3) 18 mM NaaSOI, V; (4) 36 mM NaCl,

0; (5) none, 0. The inset shows the results plotted as percentage of activity measured in the absence of AMP.

enhanced, but also more sensitive to inhibition by AMP (Fig. 1, curves 4 and 5). When the K2HP04-KH2P04 buffer mixture was replaced by an Na2HP04- NaH2P04 buffer mixture the effects were reversed, the catalytic activity was decreased as was the sensitivity to inhibition by AMP (Fig. 1, curve 1).

From the results reported in Fig. 1 it may be concluded that K+ increases while Na+ decreases the sensitivity of rabbit liver Fru-P2ase to inhibition by AMP. A decrease in the Ki for AMP on addition of Na+ was also reported by Nakashima and Tuboi (24), but in contrast to the present results they, and also Hubert et al. (22), reported that K+ had little or no effect on the AMP inhibition curve. In order to evaluate the effect of a physiological mixture of K+ and Na+, and also the effect of anions, we tested a variety of Na salts added to an assay mixture containing 50 InM KPOl buffer (pH 7.5, 92 mM K+). Un- der these conditions 36 mM Na+ caused a 50% inhibition of the activity measured in the absence of AMP, but the inhibition by AMP was also decreased (Fig. 2). At concentrations of AMP greater than 25 InM, the effect of Na+ was to increase the catalytic activity. There were small dif- ferences due to the anions, but the Na salts

AMP INHIBITION OF RABBIT LIVER FRUCTOSE 1,CBISPHOSPHATASE 601

of divalent and trivalent anions were al- most equally effective in reversing the inhibition by AMP.

In subsequent experiments the buffer employed was a mixture of Na and K phos- phates, pH 7.5, yielding final concentrations of Na+ = 36 mM, K+ = 92 mM, and HPOZ- + HzPO; = 70 mM, to approximate the concentrations of these ions reported for mammalian cells (29). Ammonium ions were omitted since they were found to have no further effect in the presence of 92 mM K+. We also found that replacing EDTA by 1 RIM histidine (30) did not significantly alter the shape of the AMP-inhibition curve.

Effect of Mg2+ and Mn2’ concentration. The concentrations of MgC12 (2 InM) and MnC12 (0.15 mM) employed in the preceding experiments were those found to be optimal in the usual assay mixtures buff- ered with the diethanolamine-triethanolamine mixture (16). In the presence of 70 mM phosphate buffer, containing 92 mM

K+ and 36 mM Na+, optimum activity was observed with lo-15 mM Mg”‘, which is close to the reported intracellular concentration (29); the optimum concentration for Mn2+ was still 0.15 mM (data not shown).

With the new assay conditions, including concentrations of K+, Na+, and phosphate within the physiological range, the activities observed with Mgz+ and Mn2+ were comparable (see Fig. 3) but, as reported earlier (21), much higher concentrations of AMP were required for comparable inhibition when Mn2+ was the activating cation. The effect of Mn2+ was not altered by the addition of O-20 mM MgSO, (Fig. 3).

Effect of temperature on the inhibition of Fru-P,ase by AMP. Using the more physiological concentrations of anions and cations, which significantly decreased the sensitivity to inhibition by AMP, it was of interest to reexamine the effect of temperature. In confirmation of the earlier reports (5, 13, 18) we found that raising the temperature from 25 to 38°C caused a threefold increase in the concentration of AMP required for 50% inhibition of the

FIG. 3. Effect of Mgr+ and Mn*+ on the inhibition of Fru-Psase by AMP. The assays were carried out

in the presence of 59 mM KPOJ26 mM NaPO, buffer,

pH 7.5 (70 mM P@, 92 mM K+, and 36 mM Na+), as described in the legends to Figs. 1 and 2. EDTA was

replaced by 1 mM histidine. The concentrations of

MgSOl were as indicated. The lower curve represents results obtained in the absence of Mn2+; the upper

curve in the presence of 0.15 mM Mn&. The concentration of free AMP was calculated assuming asso-

ciation constants of 85 M-’ and 299 M-' for the Mg- AMP and Mn-AMP complexes, respectively (8). In

the absence of AMP the specific activity with 0.15 rnM

Mn2+ alone was 11.3 amol/min/mg protein. In the presence of 0.15 mM Mn2+ plus 20 mM MgSO, this

value increased to 13.1 gmol/min/mg protein. With 10 or 29 mM MgSO,, in the absence of Mn2+, the spe-

cific activity was 14.6/rmol/min/mg protein.

activity of Fru-Pzase (Fig. 4). At the physiological temperature, in the presence of approximately physiological concentrations of the major anions and cations, the apparent Ki for free AMP was 90 PM; the total AMP concentration required to achieve this concentration of free AMP in the presence of 18 mM Me would be 227 FM.

Effect of Mn2’ and temperature on the binding of AMP to Fru-P2ase. In order to determine whether the decreased inhibition was correlated with a decreased affinity of the enzyme for AMP, we examined the effect of both Mn2+ and temperature on AMP binding. The presence of Mn2+ was found to have a profound effect, increasing the apparent KD by more than lo-fold and inducing a marked positive cooperativity (Fig. 5). In the absence of Mn2+ the binding curve was hyperbolic and yielded a linear Scatchard plot. The number of binding sites (one per subunit) was not altered by the presence of Mn2+.

602 VITA ET AL.

Binding of AMP to rabbit liver Fru- Pzase was also decreased by raising the temperature from 4 to 38°C (Fig. 6). The apparent KD increased from 0.7 PM at 4°C to 10.5 PM at 38°C and at the latter temperature the binding curve became somewhat sigmoid in shape.

The binding experiments were carried out in the absence of the cations and anions that were reported in the preceding sections to affect the inhibition by AMP. The purpose of these measurements, however, was to compare the binding affinity with or without Mn2+, and at the lower and higher temperatures. To measure dissociation constants greater than 10m5 M would have required the use of very high concentrations of protein.

Effect of M#+ on the binding of Mn2+ to rabbit liver Fru-P2ase. The measurements of AMP inhibition carried out with Mn2+ alone or with a mixture of Mn2+ and MS (see Fig. 3) suggested that at least some of the binding sites for these divalent cations remained occupied by Mn2+ even in the presence of much higher concentrations of Me. In order to determine the number of

Free[AMFj pt.4

FIG. 4. Effect of temperature on the inhibition of Fru-Pzase by AMP. The assay was carried out with the K,NaPO, buffer mixture as described in the legend to Fig. 2, with the following additions: 4 mM MgCls, 14 mM MgSOI, 0.15 mM MnCls, 1 mM histidine. Fru-Ps, NADP, and coupling enzyme were as described in the legend to Fig. 1. The total concentration of AMP ranged from 0 to 1 mM and the concentration of free AMP was calculated as described in the legend to Fig. 3. The activities measured at 25, 30, and 38°C in the absence of AMP were 15, 19.6, and 29 pmol Fru-6-P formed/min/mg of protein, respectively.

[AMP] “M -3

FIG. 5. Effect of Mn2’ on the binding of AMP to Fru-Ppase. The binding experiments were carried out as described under Materials and Methods. The solutions (0.5 ml) contained 20 mM diethanolamine-20 mM triethanolemine buffer, pH 7.5, 16 pg of Fru- Psase, 0.1 mM Fru-P2, and concentrations of [U- ‘%]AMP (specific activity = 5700 cpm/nmol) with or without MnC12 as indicated. Fru-P2 was added in these experiments because it is known to be required for optimal binding of AMP (8, 13, 31). After equilibration for 5 min the solutions were transferred to 50 X 15-mm plastic vials containing 40 mg of Seph- adex G50 coarse and several small glass beads. After equilibration for 5 h at 4°C on a rocking platform, aliquots of the supernatant solutions were transferred to scintillation vials containing 10 ml of Aquasol (New England Nuclear) and counted on a Beckman Model LS-250 scintillation counter. (A) Equivalents of AMP bound to Fru-P2ase as a function of the total AMP concentration. (B) Scatchard plot (32) of the data in A, with the free AMP concentrations corrected for binding to protein (free AMP). The values of KD were calculated to be 0.7 X 10e6 M in the absence of Mn2+ and 8.6 X 10e6 M in the presence of 0.2 mM MnC12. The concentration of protein was calculated assuming a value of M, = 143,000 (16).

binding sites occupied by Mn2+ and the effect of Me on its binding to these sites, we carried out binding measurements with “Mn as the ligand (Fig. 7).

In the absence of the substrate analog, ((Y + @)methyl-D-fructofuranoside l,6-P2, the Scatchard plot indicated the presence of eight binding sites, one set of four with affinity too high to measure and a second set with lower affinity (KD = 3.5 X lo-’ M) (Fig. 7, curve 1). In the presence of the substrate analog the number of high-affinity binding sites increased to eight, and eight additional binding sites were detected with an apparent KD of 2.2 X 10e5 M (Fig. ‘7, curve 3). On addition of 10 mM

M$+, which was the concentration re-

AMP INHIBITION OF RABBIT LIVER FRUCTOSE 1,6-BISPHOSPHATASE 603

FIG. 6. Effect of temperature on the binding of

AMP to Fru-Pgse. The conditions were as described

in the legend to Fig. 5. The solutions were equili- brated with Sephadex G50 coarse for 5 h at 4 and

25°C and for 3 h at 38%. The total concentration of

AMP was as indicated. No Mgs+ or Mn2+ was added. (A) Equivalents of AMP bound to Fru-Pgse as a

function of the total AMP concentration. (B) Scat- chard plot of the data in A, using the corrected free

AMP concentrations. The values of KD were calcu-

lated to be 0.7 X 10m6 M at 4”C, 2.37 X 10m6 M at 25”C, and 10.5 X 10e6 M at 38°C.

quired for maximum activity (see above), the total number of equivalents of Mn2+ bound did not change, but a large decrease was observed in its apparent affinity (& = 8 X 10F5 M) for the low-affinity site.

DISCUSSION

The experiments reported here were de- signed to evaluate the influence of physiological concentrations of various anions and cations on the activity of fructose 1,6- bisphosphatase and its inhibition by AMP. With the assay mixtures usually employed, containing Tris . HCl or diethanolamine-triethanolamine buffer, and with Mg2+ as the activating cation, the enzyme is sensitive to low concentrations (lo-50 mM) of AMP (8,14,16). We have confirmed previous reports indicating that this sensitivity is decreased when the temperature of the assay mixture is raised to 38°C (5, 13,18), and when Mn2+, rather than Me, is the activating cation (8, 15, 16). In addition we have found specific effects of the monovalent cations K+ and Na+. The sensitivity to inhibition by AMP is increased by the addition of physiological concentrations of K+, and decreased in the presence of physiological . concentrations of Na+. Particularly noteworthy is the fact that Na+ causes a marked decrease in the

sensitivity to AMP inhibition even in the presence of a physiological concentration of K+. The effect of Na+ appears to be enhanced by a physiological concentration of phosphate ions.

Also of interest is the effect of Mn2+ added to a reaction mixture that already contains a saturating concentration of Me. Under these conditions the inhibition by AMP is identical to that observed with Mn2+ alone.

Under conditions that approximate those in the intracellular environment, namely at 38°C in the presence of 36 mM Na+, 92 mM K+, 70 mM Pi, 14 mM SOd2-, and 1 mM

histidine to remove inhibiting Zn2+ ions, the Kn for free AMP is approximately 100 PM, equivalent to a total AMP concentration of 227 PM and approaching concentrations reported (12) for mammalian liver (cf. Fig. 4).

3

FIG. 7. Binding of Mn*+ to Fru-Psse and effect of

Me. The binding measurements were carried out in 20 mM diethanolamine-20 mM triethanolamine

buffer, pH 7.5, as described in the legend to Fig. 5

except that no Fru-Pr was added, the total aqueous volume was 1.0 ml, the amount of protein was 2 mg/

tube, and the equilibration was carried out with 80 mg of Sephadex G50. Each tube contained 10.8 X 10’

cpm of =MnC& plus the following: Curue 1: Nonra- dioactive MnCle to bring the final concentrations to

0.05-0.03 mM. Curve 3: 0.25 mM ((Y + @methyl-D- fructofuranoside 1,6-Pe and MnCla to bring the final

concentrations to 0.08 and 0.6 mM. Curve 2: As Curve

3 with the addition of 10 mM MgSO,. For calculation of the free Mn’+, the total concentration was corrected for binding by (a + @)methyl-D-fructofuranoside 1,6-P, using the value of 1.8 X 10s M-’ for the

association constant for the complex (33). Radioac- tivity was measured with a Packard Auto-Gamma Scintillation Spectrometer Model 5130.

604 VITA ET AL.

In order to provide information on the physical basis for the decreased inhibition by AMP observed with Mn2+ as the activating cation, we examined the effect of Mn2+ on the binding of AMP by rabbit liver Fru-P2ase. Mn2+ decreased the apparent KD for AMP by a factor of 10 and changed the shape of the binding curve from hyperbolic to sigmoidal. A similar decrease in affinity for AMP and induction of positive cooperativity in its binding on addition of Mn2+ was reported earlier by Pontremoli et al. (8).

Earlier workers (14,30,33) had reported that rabbit liver Fru-P2ase binds four equivalents of Mn2+ in the absence of substrate and 8 eq in the presence of substrate or the substrate analog (a + @)methyl-D- fructofuranoside. The addition of the substrate analog not only increased the number of binding sites, but also abolished the negative cooperativity and increased the affinity of binding to the first set of sites (33). These observations with Mn2+ were in contrast to those reported with Zn2+, which binds to 12 sites on rabbit liver Fru- P2ase, 8 in the absence of substrate, and 4 more in its presence (25).

In the present experiments we detected a total of 8 binding sites for Mn2+ per mole of Fru-P2ase in the absence of substrate, in agreement with the number reported for Zn2+. However, the number of additional equivalents of Mn2+ bound in the presence of the substrate analog was 8, rather than the 4 observed with Zn2+ (25) or reported by Libby et al. for Mn2+ (33). More recently, Benkovic et al. (31) have reported new data on the binding of Mn2+ to rabbit liver Fru-P2ase in the presence of (C-X + @)methyl-D-fructofuranoside. The results suggest that at higher concentrations of Mn2+ the total number of binding sites might extrapolate to a value somewhat larger than 8, but not to the value of 16 reported here. The basis for the discrepancy between their results and our remains unknown. However, if the complex formed between Mn2+ and (cy + fl)methyl-D-fructofuranoside 1,6-bisphosphate contains two equivalents of Mn2+ per mole, as reported by Libby et aI. (33), this could account for the fact that

2 additional equivalents of Mn2+ bind with the substrate to each Fru-P,ase subunit. Except for the difference in number of binding sites, our results fit the model pro- posed by Libby et a2. (33) in which a structural Mn2+-enzyme complex is formed, which then binds the substrate-metal ion complex.

The original goal of these binding experiments was to determine the effect of Me on the binding of Mn2+. We find that Mg2+ affects the binding of Mn2+ only to the eight sites that are dependent on the presence of the substrate analog; the value for the apparent dissociation constant for these sites increased from 2.2 X 10e5 M in the absence of Me to 8.0 X 10S5 M in the presence of 10 mM MgS04. At this concentration, Me had little or no effect on the binding of Mn2+ to the high-affinity binding sites.

The sensitivity of rabbit liver Fru-P2ase to inhibition by AMP appears to be determined by the presence of Mn2+ at the high- affinity sites. It should be noted that in the assays for AMP inhibition EDTA or histidine was added to remove any Zn2’ that might be present. We have previously reported that binding of Zn2+ to the high- affinity sites increases the inhibition of the activity by AMP (34). Under physiological conditions the enzyme may exist either as a Mn2+ (active) or Zn2+ (inhib- ited) complex, to which the substrate would bind as the Mg2-Fru-P2 or Mn2-Fru- P2 chelate. The intracellular concentration of histidine, which is 0.2 M in livers of fed rabbits and increases to 1.0 M in livers of fasted rabbits (30), may determine whether the structural site(s) are occupied by Zn2+ or by Mn2+.

ACKNOWLEDGMENTS

The Institute of Biological Chemistry, University

of Genoa, acknowledges support from the Italian CNR. The authors are indebted to Dr. S. Benkovic of Pennsylvania State University for the generous gift

of (a + fl)methyl-D-fructofuranoside 1,6-bisphosphate.

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