Phosphofructokinase from Bumblebee Flight Muscle

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 33, Isaue of November 25, pp. 17527-17533,1988 Printed in U.S.A.

Phosphofructokinase from Bumblebee Flight Muscle MOLECULAR AND CATALYTIC PROPERTIES AND ROLE OF THE ENZYME IN REGULATION OF THE FRUCTOSE 6-PHOSPHATE/FRUCTOSE 1,6-BISPHOSPHATE CYCLE*

(Received for publication, October 7, 1987)

Adilson LeiteS, Jose Abrahiio Neto, Jaime F. LeytonQ, Omar Crivellaro, and Hamza A. El-Dorryll From the Department of Biochemistry, Institute of Chemistry, University of S& Paulo, Caixa Postal 20780, CEP 01498 S& Paulo, Brazil

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 26 "C or rest at 6 "C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 26 and 7.6 "C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bis- phoshate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.

Phosphofructokinase (ATP:D-fructose 6-phosphate l-phos- photransferase, EC 2.7.1.11) is a key regulatory enzyme of the glycolytic pathway in most living cells (for review see Ref. 1). In the flight muscle of the bumblebee, in addition to its glycolytic role, the enzyme is also involved in a substrate cycle between fructose 6-phosphate and fructose 1,6-bisphosphate (Fru-6-P/Fru-1,6-P2). The operation of a phosphofructokinase/fructose-l,6-bisphosphatase-mediated substrate cycle would produce continuous hydrolysis of ATP and generation of heat which could support bumblebee flight in cold weather (2). It has been reported that the rates of cycling of Fru-6-P

* This work was supported in part by FINEP Grant 43.84.0725.00/ 15 from the Financiadora de Estudos e Projetos. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Grant 81/1619-1 from the FundaCHo de Amparo a Pesquisa do Estado de SHo Paulo. Present address: Departamento de Genbtica e EvoluCHo, Universidade Estadual de Campinas, Campinas, SHo Paulo, Brazil.

5 Present address: Toxik6n Assessoria Toxicol6gica, Rua Salvador Correia 346, CEP 04109, SHo Paulo, Brazil.

7 To whom all correspondence should be addressed.

are 10, 0.48, and 0 mol/min/g of bumblebee flight muscle, respectively, resting at 5 "C, resting at 21 "C, or flying (3). The control mechanism for the regulation of the cycle during rest and flight was explained by a change in sarcoplasmic Ca2+ concentration associated with the contractile processes (3,4). Substrate cycling is turned off during flight as a result of fructose-1,6-bisphosphatase inhibition by physiological levels of Ca2+ which have no effect on phosphofructokinase activity (3). In addition, all fructose-1,6-bisphosphatases reported to date, with the exception of the enzymes isolated from chloroplasts (5,6) and bumblebee flight muscle (2), were found to be inhibited by AMP. It is this atypical characteristic that probably allows the simultaneous activity of fructose- 1,6-bisphosphatase andphosphofructokinase at rest (3). How- ever, regulation of the Fru-6-P/Fru-1,6-P~ cycle by Ca2+ does not explain the control mechanism underlying the observation that the rate of cycling is temperature-dependent. In order to examine the mechanisms which control Fru-6-P/Fru-1,6-P2 cycling, phosphofructokinase from the flight muscle of bumblebee was purified and its molecular and catalytic properties are presented. We also determined glycolytic intermediates, including Fru-2,6-P2, in the flight muscle of nonflying (resting) and flying insects exposed to low and normal temperatures. Since the activity of phosphofructokinase and fructose- 1,6-bisphosphatase is needed for the operation of the cycle, various allosteric properties were determined at 25 and 7.5 "C for both enzymes under conditions which approximate physiological concentrations of substrates and key effectors found in insects kept under stimulated and inhibited cycling conditions. The data presented in this study show the importance of Fru-2,6-P2 for the activity of bumblebee phosphofructokinase and also indicate a possible role for Fru-2,6-P2 in the regulation of the substrate cycling. In addition, it will be shown that the affinity of both enzymes for substrates and effectors is temperature-dependent, and this may be respon- sible for the increase in cycling activity at low temperature.

MATERIALS AND METHODS

Fructose 2,6-bisphosphate was prepared according to the procedure described by Van Schaftingen and Hers (7). Rat liver phosphofructokinase was purified according to the procedure described by Duna- way and Weber (8). Bumblebee fructose-1,6-bisphosphatase was purified as described by Leyton et al. (9). Cibacron Blue F-3-GA was obtained from Ciba Geigy do Brasil, SHo Paulo, Brazil, and was coupled to Sepharose 4B-200 as described by Heynz and DeMoor (10). All other enzymes and chemicals were reagent grade and obtained from commercial sources.

Bumblebees (Bombus atratus) were collected in the field from flowers and kept in the dark at 25 'C until required for enzyme extraction and metabolite determinations. Nonflying insects were kept in the dark at 25 or 5 "C for 5 min before immersion in liquid Nz. Flying insects were attached to thread, induced to fly for 2 min, and then immediately frozen in liquid Nz. Bumblebee thorax frozen

17527

Phosphofructokinase from Bumblebee Flight Muscle in liquid N:! was pulverized, and the tissue powder was weighed and extracted with 3 volumes of 6% perchloric acid at 0 "C. After centrifugation the supernatant solution was neutralized with 0.43 M triethanolamine containing 0.55 M KHCOa and centrifuged again. The supernatant was then used for all metabolite assays as described (11- 14). For the determination of Fru-2,6-P2 concentration, a hot (90 "C) solution of 10 mM HEPES' containing 2 mM EGTA and 10 mM KF at pH 9.3 was added to the tissue powder. After homogenization, the mixture was placed in a hot water bath (90 "C) for 10 min. The mixture was centrifuged in an Eppendorf centrifuge, and the supernatant was used for Fru-2,6-P2 analysis as described by Hue et al. (15). In order to insure that Fru-2,6-P2 was being measured, a test for acid lability was performed as described by Kuwajima and Uyeda (16).

Enzyme assays were carried out using a Zeiss PM6KS recording spectrophotometer equipped with a thermostatted cell holder whose temperature was controlled with a Lauda K2R refrigerated circulating bath and a Lauda R42/2 digital thermometer. The temperature of the cell holder was controlled within kO.1 "C.

Optimum phosphofructokinase activity was determined by following the rate of NADH oxidation at 340 nm, in a reaction mixture containing in a final volume of 1.0 ml: 50 mM triethanolamine/Cl (pH 8.0), 2 mM Fru-6-P, 1 mM ATP, 5 mM MgC12, 0.13 mM NADH, 4 mM 2-mercaptoethanol, 1 mM EDTA, 2 mM AMP, 50 mM KCl, aldolase (0.25 units), a-glycerophosphate dehydrogenase (1 unit), and triosephosphate isomerase (4.5 units). One unit of phosphofructokinase activity was defined as the amount of enzyme that catalyzes the phosphorylation of 1 pmol of Fru-g-P/min at 30 "C. The same reaction mixture was used in the study of catalytic properties at pH 8.0 with variation of the ATP and Fru-6-P concentrations.

Allosteric kinetic properties of phosphofructokinase were determined in a reaction mixture containing in a final volume of 1.0 ml: 50 mM HEPES (pH 7.4), 25 mM KC1, 0.2 mM EDTA, 4 mM 2- mercaptoethanol, 0.13 mM NADH, aldolase (0.25 units), a-glycerophosphate dehydrogenase (1 unit), and triosephosphate isomerase (4.5 units). The assays were performed at 30 "C, unless otherwise stated, and the reaction (pH 8.0 and 7.4) was initiated by the addition of ATP. The MgC12 concentration in all reactions was maintained at a 5 mM excess over the concentration of ATP, and all the coupling enzymes were dialyzed before use. When the enzyme activity is expressed as u/V, u is the activity at pH 7.4, and V is the optimum activity determined at pH 8.0.

Fructose-1,6-bisphosphatase activity was determined by following the rate of NADP reduction at 340 nm. The reaction mixture contained in a final volume of 1.0 ml: 40 mM triethanolamine/diethanol- amine buffer, pH 7.4, 5 mM MgCl:!, 0.2 mM NADP, 0.1 mM Fru-1,6- Pz, excess dialyzed phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase (2 gg each). The assays were performed at 30 "C, unless otherwise stated, and the reaction was initiated by the addition of the enzyme. One unit of fructose-1,6-bisphosphatase was defined as the amount of enzyme that catalyzes the hydrolysis of 1 pmol of Fru-l,B-Pz/min at 30 "C. When the activity of the enzymes was measured at 7.5 "C, the pH of the buffer was adjusted to 7.4 at 7.5 "C.

Water used in Fru-P2ase assay, in desalting of coupling enzymes, and in the preparation of stock solutions was purified in a Milli-R/Q water purifier system, obtained from Millipore do Brasil. Buffer used in the enzyme assay was treated with chelating resin purchased from Sigma as described by Nimmo and Tipton (17). Purified water and enzyme assay mixture were determined to contain less than 0.3 p~ Ca2+ using atomic absorption analysis.

The Hanes plots of the concentrations and velocities and the secondary plots were calculated with the weighed least square fit method (18). For all calculations, the general rate equation for a two substrates-two products reactions according to Cleland (19) was used. The Hill coefficients were calculated with a nonlinear least square fit method.

RESULTS

Purification of Phosphofructokinase-The purification procedure is described below and is summarized in Table I. All operations were performed at 0-4 "C. Frozen bumblebee muscle (5 g) was homogenized for 60 s in an Omni-Mixer with 40

The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylene- nitri1o)tetraacetic acid.

TABLE I Purification of phosphofructokinase from 5 g of

bumblebee flight muscle

Fraction Total Total Specific protein activity activity

ml rng units unitslmg % Crude extract 34 442 215 0.48 100

Precipitate 4 63 182 2.90 85 Blue-Sepharose Chromatography 0.3 1.0 65 65 30 Density gradient Centrifugation 0.5 0.17 21 123 10

45-65% (NH,)ZSO,

ml of 50 mM Tris/Cl, pH 8.0, containing 50 mM KF, 40 mM (NH4)2S04,0.1 mM EDTA, 2 mM t-aminocaproic acid, 0.5 mM ATP, and 10 mM dithiothreitol. The homogenate was centrifuged at 12,000 X g for 30 min. After passage through cheese- cloth, the crude extract was brought to 45% ammonium sulfate saturation and the pH was adjusted to 8.0 with 6 N NH4OH. After standing for 30 min, the solution was centrifuged at 12,000 X g for 30 min. The precipitate was discarded, the resulting supernatant was brought to 65% ammonium sulfate saturation, and the solution was allowed to stand for 30 min before centrifugation at 12,000 X g for 30 min. The 45-65% ammonium sulfate precipitate was dissolved in 50 mM sodium phosphate, pH 8.0, containing 50 mM KF, 0.1 mM EDTA, 50 mM Fru-1,6-Pz, 2 mM t-aminocaproic acid, and 10 mM dithiothreitol (buffer A). The solution was desalted over a Sephadex G-25 column equilibrated with the same buffer. The desalted 45-65% ammonium sulfate fraction was applied to a 1.4 X 22-cm blue-Sepharose column equilibrated with buffer A. The column was washed with buffer A and then with buffer A containing 0.15 mM ADP until the optical density of the effluent at 280 nm dropped below 0.05. Phos- phofructokinase was eluted with buffer A containing 2 mM Fru-6-P and 2 mM ATP. The peak fractions were combined and brought to 80% ammonium sulfate saturation. The 80% ammonium sulfate precipitate was dissolved in 0.3 ml of buffer A and dialyzed for 12 h against 2 liters of the same buffer. After dialysis, the enzyme solution was subjected to centrifugation for 18 h at 30,000 rpm at 4 "C through a 5-20% glycerol gradient (13 ml) in 10 mM Tris/Cl, pH 7.5, using a Beckman SW 41 rotor. The tube was then punctured at the bottom, and fractions (0.5 ml) were collected and monitored for enzyme activity. Fractions having a specific activity greater than 120 units/mg were combined, and the concentration was adjusted to 20% glycerol and stored at -20 "C with no loss of activity. The purified enzyme was homogeneous as demon- strated by polyacrylamide gel electrophoresis (Fig. 1). The specific activity of different preparations of phosphofructokinase varied from 110 to 130 units/mg.

Molecular Weight Determination-The purified enzyme ex- hibited a mobility corresponding to a molecular weight of 80,000 after gel electrophoresis in the reduced denatured state (Fig. 1). The molecular weight of the native enzyme was estimated by centrifugation on a glycerol gradient using the method of Martin and Aimes (20). A molecular weight of 360,000 was estimated for the native enzyme using catalase as standard.

Catalytic Properties at pH 8-The basic catalytic properties of bumblebee flight muscle enzyme are shown in Fig. 2. Primary plots of either Fru-6-Plu against Fru-6-P (Fig. 2 A ) or ATP/u against ATP (Fig. 2B) at various concentrations of ATP or Fru-6-P, respectively, show straight lines intersecting on the negative side and above the abscissa, and intercepts of the Fru-6-P or ATP axis with an apparent Michaelis constant

Phosphofructokinase from Bumblebee Flight Muscle 17529

A B C D E

FIG. 1. Slab gel electrophoresis of enzyme fractions. The reduced denatured proteins were subjected to slab gel electrophoresis through 5-1596 acrylamide gradient in the presence of 0.1% sodium dodecyl sulfate. Lanes A, R, C, D, and E were stained with Coomassie Blue and contained, respectively, 200 pg of crude extract, 200 pg of 45-65% (NH4)2S04 precipitate, 100 pg of blue-Sepharose eluate, 5 pg of the pooled density gradient centrifugation, and 50 pg of protein molecular weight markers included phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), and carbonic anhydrase (30,000).

for one substrate depending on the concentration of the other. The data in Fig. 2 indicate that the kinetic behavior of bumblebee phosphofructokinase is not a substituted-enzyme mechanism but rather random or compulsory-order ternary complex (21). Secondary plots of ATP/V against ATP (Fig. 2A, inset) or Fru-6-P/V against Fru-6-P (Fig. 2B, inset) are straight lines which intercept the KhTP or KEp on the ATP or Fru-6-P axis, respectively. The KEp and KhTP values calculated from the above mentioned plots are 39.2 f 3.8 and 39.0 f 2.2 pM, respectively.

Catalytic Properties a t p H 7.4-When bumblebee muscle phosphofructokinase is assayed at pH 7.4, the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward Fru-6-P (Fig. 3A), and inhibition by high concentrations of ATP (Fig. 3B). The effect of varying the concentration of Fru-6-P on enzyme activity in the presence of several fixed concentrations of ATP is shown in Fig. 3A. Increasing the concentration of ATP decreases the affinity of the enzyme for Fru-6-P and reduces the cooperativity as measured by the Hill coefficient (Fig. 3A; see also Table 11). The effect of AMP on the activation of the bumblebee muscle enzyme is shown in Fig. 4. In the presence of 5 mM ATP, the s0.5 for Fru-6-P in the absence of AMP appears to be 2.5 mM. The presence of 200 PM AMP decreased the So.5 for Fru-6-P to 0.6, indicating that AMP activates phosphofructokinase by increasing its Fru-6-P affinity.

Effect of Fru-2,6-P2 on Catalytic Properties-The effect of Fru-2,6-P2 on the activity of phosphofructokinase is shown in Fig. 3. Fructose-2,6-P2 at a concentration of 4 pM increases the enzyme affinity for Fru-6-P and decreases the cooperativity (Fig. 3A and Table 11). In addition, in the presence of 4

1 T P . l y Y I

I 1 I 1 -02 0 0.1 02

Fru 6-P (mM)

FIG. 2. Kinetic behavior at pH 8.0. Phosphofructokinase activity was assayed as described under "Materials and Methods." A, plot of Fru-B-P/u against Fru-6-P at various concentrations of ATP. Inset shows replots of ATP/V against ATP. B, plot of ATP/u against ATP at various concentrations of Fru-6-P. Inset show replots of Fru-6-P/ V against Fru-6-P.

p~ Fru-2,6-P2, an ATP concentration up to 3 mM has no inhibitory effect on the enzyme activity (Fig. 3B). The effect of varying the concentration of Fru-2,6-P2 at several fixed concentrations of ATP on bumblebee phosphofructokinase is shown in Fig. 5. Under these experimental conditions the K0.5

for Fru-2,6-P2 was 2 PM at 5.3 mM ATP. It is worth mentioning that the K0.5 for AMP in the absence of Fru-2,6-P2 is a t least 500 PM. The synergistic activation of bumblebee phosphofructokinase by Fru-2,6-P2 and AMP is shown in Fig. 4. Under these conditions and in the absence of both activators the s0.5 for Fru-6-P is 2.5 mM. The presence of 200 p M AMP or 4 p M Fru-2,6-P2 decreases the s0.5 to 0.6 and 0.28 mM, respectively, while the presence of both activators decreases the to 0.06 mM.

Effects of Environmental Temperature and Flight on Metab- olite Content of the Bumblebee Flight Muscle-To understand the mechanisms which control the Fru-6-P/Fru-1,6-P2 cycling in the flight muscle of the bumblebee, the concentrations of some glycolytic intermediates and metabolites were measured in the flight muscle of insects during rest and flight at 25 "C and rest at 5 "C. The results of these determinations are shown in Table 111. Statistically significant increases in the concentrations of AMP, Glu-6-P, and Fru-2,6-P2 were observed during flight a t 25 "C or rest at 5 "C when compared with insects at rest a t 25 "C. The values of glycolytic intermediates reported in this study are in good agreement with those previously published (2, 3, 22).

Catalytic Properties of Phosphofructokinase and Fructose- 1,6-Bisphosphatase under Physiological Conditions-Although

17530

A I .oc

0.7 e

: 0.JO >

0.22

a

B

Phosphofructokinase from Bumblebee Flight Muscle

0-0

0 0-~-0-0 1

0 I 2 3 4 5 ATP (mMJ

FIG. 3. Kinetic behavior at pH 7.4. Phosphofructokinase activity was assayed as described under "Materials and Methods." A, initial velocity as a function of Fru-6-P in the presence of: 5.3 mM ATP (O), 2.6 mM ATP (O), 1.3 mM ATP (X), and 5.3 mM ATP plus 4 pM Fru-2,6-P~ (A). B, initial velocity as a function of ATP in the presence of 0.45 mM Fru-6-P (O), 0.9 mM Fru-6-P (a), 1.8 mM Fru- 6-P (A), 0.45 mM Fru-6-P plus 4 pM Fru-2,6-P2 (A).

TABLE I1 Effect of A T P and Fru-2,6-P~ on phosphofructokinase affinity for

Fru-6-P and Hill coefficient (h)

ATP F~1-2,6-P2 h SO.5

(mM F-6-P) mM P M

0.53 3.30 k 0.85 1.25 2.66 2.03 k 0.61 1.50 5.31 1.42 k 0.57 2.27 5.31 4.0 1.00 & 0.43 0.29

\ W

0 1.0 2.0 3.0 4.0 Fru 6-P (mM)

FIG. 4. The influence of AMP and F r ~ 2 . 6 - P ~ on the activity of phosphofructokinase. The reaction mixture (pH 7.4) was the same as described under "Materials and Methods" except that 5 mM ATP was added. Initial velocity is shown as a function of Fru-6-P in the absence of AMP and Fru-2,6-P2 (A), and in the presence of: 200 p~ AMP (O), 3.8 pM Fru-2,6-Pz (X), and 200 PM AMP plus 3.8 pM Fru-2,6-P2 (0).

u O O 5 IO 15 2(

Fru 2.6-P2 ( p M l

FIG. 5. The effect of Fru-2,6-Pz on the inhibition of phosphofructokinase by ATP. Phosphofructokinase activity was assayed (pH 7.4) as described under "Materials and Methods" except that 0.5 mM Fru-6-P was added. Initial velocity is shown as a function of Fru-2,6-P~ in the presence of: 5.3 mM ATP (O), 2.7 mM ATP (O), and 1.3 mM ATP ( X ) .

the catalytic properties of phosphofructokinase reported above and those previously reported for fructose-1,6-bisphosphatase (4) were determined under varied concentrations of substrates and key effectors, they were redetermined under concentrations as close as possible to those found in the flight muscle of insects during rest and flight at 25 "C, and rest at 5 "C.

In the presence of 0.08 mM of Fru-6-P and 2.5 mM ATP, concentrations close to those found in muscle of insects flying at 25 "C and resting at 25 and 5 "C (see Table 111), phosphofructokinase was at least three times more active in the presence of physiological concentrations of Fru-2,6-P2 and AMP found in the muscle of nonflying insects at 5 "C or flying insects at 25 "C when compared with those concentrations found in the resting muscle of bumblebee at 25 "C (Fig. 6A).

The effect of varying the concentrations of Ca2+ at several fixed concentrations of Fru-2,6-Pz on bumblebee fructose-1,6-

Phosphofructokinase from Bumblebee Flight Muscle 1753 1

TABLE 111 Effect of environmental temDerature and fliPht on the metabolite content in the flieht muscle of bumblebee

~~

Condition of Content of metabolites

bumblebee Fm-2,6-p2 AMP Glu-6-P ATP ADP Fm-l,6-P2 FN-6-P Trioses-P

Nonflying 25 "C 2.77 f 0.50 0.16 f 0.04 0.07 f 0.01 3.02 f 0.35 0.64 & 0.09 0.05f 0.01 0.06 f 0.01 0.04 f 0.01 5 "C 7.26 f 0.86 0.37 f 0.05 0.15f 0.04 2.30f 0.27 0.60f 0.12 0.06 f 0.01 0.08 f 0.01 0.04 f 0.01

Flight 25 "C 7.13 f 0.58 0.39 f 0.09 0.16 f 0.03 2.56 f 0.33 0.70 f 0.18 0.05 f 0.01 0.06 f 0.01 0.04 f 0.01

L

b a

FIG. 6. Catalytic properties of phosphofructokinase and fructose- 1,6-bisphosphatase under in vivo conditions. The reaction mixture was the same as described under "Materials and Methods" except that phosphofructokinase was assayed in the presence of 0.08 mM Fru-6-P and 2.5 mM ATP, and fructose-1,6- bisphosphatase was assayed in the presence of 0.06 mM Fru-l,6-P~. A, effect of varying the concentrations of Fru- 2,6-Pz on the activity of phosphofructokinase in the presence oE 0.4 mM AMP (A), 0.16 mM AMP (O), and in the absence of AMP (0). a and b denote the activity of the phosphofructokinase in the presence of the in viuo concentrations of AMP and Fru-2,6-Pz found in muscle of insects, respectively, resting at 25 "C, and resting at 5 "C or flying at 25 "C. B, effect of varying the concentrations of Ca2+ on the activity of fructose-l,6-bisphosphatase in the presence of: 7.2 p M Fru-2,6-Pz (A), 2.5 pM Fru-2,6-Pz (O), and in the absence of Fru-2,6-P2 (0).

bisphosphatase activity is shown in Fig. 6B. These activities were measured using Fru-l,6-Pn concentration (0.06 mM) found in the muscle of bumblebee flying a t 25 "C and resting at 25 and 5 "C (see Table 111). The 10.5 of fructose-1,6-bisphosphatase in the absence of Fru-2,6-Pz appears to be 33 pM. The presence of 7.2 p~ Fru-2,6-P2 potentiates this inhibition and lowers the Io.6 for Can+ to 14 pM. It is important to note that phosphofructokinase is far less sensitive to Ca2+ inhibition (10.5 around 2 mM) than fructose 1,6-bisphosphatase (data not shown).

The Effect of Temperature on the Catalytic Properties of Phosphofructokinase and Fructose-1,6-bisphosphatase-In order to determine whether the catalytic properties of both enzymes as well as their sensitivities to effectors are temperature-dependent, the activity of the enzymes was measured at 25 and 7.5 "C at key concentrations of substrates and effectors.

In the presence of 2.5 mM ATP, 0.16 mM AMP, and 2.7 KM Fru-2,6-Pz, concentrations close to those found in muscle of insects at rest at 25 "C (see Table 111), the So.s for Fru-6-P for phosphofructokinase was found to be 101 p ~ . When the temperature of the reaction assay was lowered to 7.5 "C, the So.s for Fru-6-P decreased to 65 p ~ . The sO.5 values for phosphofructokinase determined at 25 and 7.5 "C a t concentrations of ATP and effectors close to those found in muscle of bumblebee resting at 5 "C or flying at 25 "C (2.5 mM ATP,

0.37 mM AMP, and 7.2 p~ Fru-2,6-P2) was 26 p~ at both temperatures. Finally, in the absence of both activators and in the presence of 2.5 mM ATP, the enzyme shows lower S0.5

(0.7 mM) for Fru-6-P at 7.5 "C than at 25 "c (1.5 mM). In the absence of Fru-2,6-P2, fructose-l,6-bisphosphatase

shows lower 10.5 (35 pM) for Can+ at 25 "c than at 7.5 "c (87 pM). In the presence of 7.2 pM Fru-2,6-Pz, the 10.5 for ca2+ measured at 7.5 "C was 80 p ~ . All the activities were measured using 60 p M Fru-l,6-P~. In the absence of Fru-2,6-P2, the 8 0 . 5

values for Fru-l,6-Pz at 25 and 7.5 "C were 18.6 and 6.6 pM, respectively.

DISCUSSION

The simultaneous operation of the enzymes involved in the interconversion of two substrates by irreversible reaction results in a "Futile Cycle" or the dispersing of energy without any metabolic gain (for review see Ref. 23). The operation of such a cycle, between glucose and glucose 6-phosphate, was first observed by Cahill et al. (24) and later confirmed by Clark et al. (25). Newsholme and co-workers (26-28) have proposed that the role of cycling in general, especially of the Fru-6-P/Fru-l,6-Pz cycle, is to increase the sensitivity of allosteric control. A second proposed function of cycling between Fru-6-P/Fru-1,6-P2 is to serve as an important heat source for the flight muscle of bumblebees in cold weather.

17532 Phosphofructokinase from Bumblebee Flight Muscle

This hypothesis was proved to be correct in experiments done by Clark et al. (3). They also found an increase in Fru-6-P/ Fru-l,6-Pn cycling in the muscle of pigs with halothane- induced malignant hyperthermia (29). In the case of the bumblebee, where the physiological function of the Fru-6-P/ Fru-l,6-P2 cycling is to produce energy through ATP hydrolysis, it has been proposed that it is inappropriate to call this cycle "futile" (30). Fructose-l,6-bisphosphatase from the flight muscle of bumblebee was purified and its role in the control of Fru-6-P/Fru-1,6-P2 substrate cycling was reported (4). In addition, the lack of susceptibility of bumblebee fructose-1,6-bisphosphatase to inhibition by AMP (2) is correlated with the absence of a structure corresponding to the nucleo- tide fold (9). Newsholme et al. (2) related this difference to the ability of the bumblebee to fly in cold weather when compared to other insects. On this basis, we have purified phosphofructokinase from the flight muscle of bumblebee to understand its role in the control of the Fru-6-P/Fru-1,6-P~ substrate cycle. Like phosphofructokinase from other sources (l), the bumblebee enzyme was found to be a tetramer with subunit molecular weight of 80,000. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory- order ternary complex and are in agreement with those results reported by Pettigrew and Frieden (21); the most probable is random-order ternary complex based on the product and dead-end inhibition studies reported by Hanson et al. (31) and Bar-Tana and Cleland (32).

Purified phosphofructokinase from the flight muscle of bumblebee was found to have a high s0.5 value (2.27 mM) for Fru-6-P as a result of inhibition by high concentration of ATP (5.31 mM). This result is in agreement with phosphofructokinase from other sources (1). Our results show that phosphofructokinase from the flight muscle of bumblebee is inactive when in vivo concentrations of substrates are used (Fig. 6A). Therefore, the in vivo concentration of Fru-2,6-Pz (2.8-7.3 nmol/g) seems to be essential for the activity of the bumblebee phosphofructokinase regardless of the insect's temperature and activity level. However, the physiological significance of Fru-2,6-P2 in skeletal muscle (33), but not heart muscle (34), is uncertain. In addition, our results clearly demonstrate synergistic activation of bumblebee flight muscle phosphofructokinase by both Fru-2,6-Pz and AMP. It is worth mentioning that the concentration of phosphofructokinase in the flight muscle of the bumblebee is approximately 4 pM (subunit concentration) based on a specific activity of 120 units/mg (Table I) and 40 units/g of flight muscle, which is comparable to the concentration of Fru-2,6-Pz (2.8-7.3 nmol/ g) found in that tissue.

In this study we show that fluctuations in Fru-2,6-Pz and AMP concentrations in flight muscle of bumblebee are temperature-dependent and that the concentrations of Fru-2,6- Pz and AMP are inversely related to the ambient temperature. In addition, our results show that at low concentration of Fru- 2,6-P2 (2.7 p ~ ) the affinity of phosphofructokinase for Fru-6- P increases with -decreasing temperature. It is important to note that the physiological concentration of Fru-6-P in the flight muscle is 0.06-0.08 mM (see Table 111), while the s0.5 values for Fru-6-P are 101 pM at 25 "C and 26 pM at 7.5 "c, when measured in the presence of the corresponding physiological concentrations of ATP and effectors. These results suggest that at rest the increase in cycling at 5 "C could be mediated by increases in the concentrations of Fru-2,6-P~ and AMP and increase the affinity of the enzyme for Fru-6-P. On the other hand, our results show that Fru-2,6-P2 is a potent inhibitor of bumblebee fructose-l,6-bisphosphatase. However, the data in the present study show that in the presence of

physiological concentration of substrate and Fru-2,6-P2 (7.26 @), and low concentration of Ca" M) found in resting muscle (35), fructose-l,6-bisphosphatase is sufficiently active to account for its activity in cycling at 5 "C. It is important to note that, in contrast to the enzyme from other tissues, fructose-1,6-bisphosphatase from the flight muscle of bumblebee is not inhibited by AMP (2). A rise in free Can+ concentrations from to M is required to initiate muscle contraction (35). Thus, the inhibitory effect of Fru-2,6-Pz on fructose-l,6-bisphosphatase at the concentration found in muscle of flying insects (7.13 MM) seem to be essential to lower the lo.s for Can+ (see Fig. 6B) within the range of Can+ levels required in contractile processes. The effect of temperature on fructose-1,6-bisphosphatase activity demonstrates that the affinity for substrate increases and the affinity for Ca2+ decreases with decreasing temperature, These changes in affinity would result in activation of fructose-l,6-bisphosphatase and a subsequent increase in cycling activity.

Although our results suggest a role for Fru-2,6-Pz in the cycling regulation during flight and rest at low temperature, they do not explain the inactivation of cycling in the resting bumblebee flight muscle a t 25 "C. However, it was recently reported that Glu-6-P regulates Can+ steady state in endo- plasmic reticulum and it was proposed that Glu-6-P could serve as "off" signal leading to a decrease in intracellular Ca2+ (36). It is of interest that we observed a 2-fold decrease in the concentration of Glu-6-P in the flight muscle of nonflying insects kept at 25 "C compared to those at 5 "C. Therefore, the fluctuation of Glu-6-P might have a role in regulation of cycling in resting bumblebee muscle at 25 "C.

In summary, our results demonstrate that: (a) Fru-2,6-P2 is essential for the activity of phosphofructokinase in the flight muscle of bumblebee; ( b ) cycling could be activated at low concentration of Ca2+ by increases in the affinities of phosphofructokinase and fructose-1,6-bisphosphatase for Fru-6-P and Fru-l,6-Pz, respectively, increases in the physiological concentrations of Fru-2,6-Pz and AMP, and a decrease in the affinity of fructose-1,6-bisphosphatase for CaZf; (c) during flight the increase in the intercellular concentration of Ca2+ in the presence of Fru-2,6-P2 should inhibit fructose- 1,6-bisphosphatase and turn off cycling activity.

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Phosphofructokinase from Bumblebee Flight Muscle

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