Regulation of phosphofructokinase during haemopoiesis of rainbow trout (Salmo gairdneri R.): Kinetic studies

Comp. Biochem. Physiol. Vol.95B, No. 3, pp. 641~46, 1990 Printed in Great Britain

0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc

REGULATION OF PHOSPHOFRUCTOKINASE DURING HAEMOPOIESIS OF RAINBOW TROUT (SALMO GAIRDNERI R.): KINETIC STUDIES SOLEDADGAIT.~.N,ELVIRACUENLLAS,MANUELRUIZ-AMIL and CONCEPCI6NTEJERO* Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain (Received 8 August 1989)

Phosphofructokinase of haemopoietic cells and erythrocytes exhibit biphasic kinetics at intracellular concentrations of both substrates. 2. Fru-l,(~P2 acts as a positive allosteric effector in haemopietic cells, and an activator effect may be proposed in erythrocytes. 3. AMP acts as an activator in haemopoietic cells and as an inhibitor in erythrocytes, while ADP acts as an inhibitor in both cellular populations. 4. These results show a different regulation of PFK during trout haemopoiesis. Abstract--1.

INTRODUCTION

In fish erythrocytes (Leray and Bachand, 1975) as in other species (Rapoport, 1968), glycolysis is the main metabolic pathway, although in other fish tissues proteins and lipids are the main source of energy (Walton and Cowey, 1982). Phosphofructokinase (PFK) (EC 2,7.1.11) is a key catalyst of glycolysis in most organisms. The regulation of this enzyme is complex and controlled by several metabolites. The number of effectors that influence phosphofructokinase activity and the extent of inhibition or activation vary, depending on the source of the enzyme. For example, only a few metabolites influence bacterial PFK, while the enzyme from mammals is influenced by numerous effectors (Bloxham and Lardy, 1973; Uyeda, 1979). Studies of the enzyme in normal and malignant tissues of the rat, first demonstrated the presence of several chromatographically and immunologically distinguishable forms of PFK in this species (Tanaka et al., 1972). Actually, mammals are known to have three distinct subunit types; denominated M or A, L or B and P or C. These subunits are distinctly expressed by different organs and undergo random tetramerization to produce various homo- and heterotetrameric isoenzymes, with specific physicochemical, immunochemical and kinetic-regulatory properties; cooperativity with respect to fructose 6-phosphate and ATP inhibition in the order P4 > L4 > M4- The most sensitive to fructose 1,6-bisphosphate being M4 (Foe et al., 1983). However, as kinetic properties of heterotetramers, or hybrid forms, do not coincide with the arithmetic mean of homotetramer properties, they might demonstrate kinetic properties per se (Gonzalez and Kemp, 1978). It has been described that in neoplastic and cellular differentiation processes, phosphofructokinase activity decreases according to an increase in the differen*Author to whom correspondence should be addressed.

tiation grade. This difference in the enzymatic activity is related to changes in enzymatic levels, kinetic regulatory properties and/or isoenzymatic patterns (Pinilla et al., 1982; Dunaway et al., 1981; Nijhof et al., 1984; Oskam et al., 1985; Vora et al., 1985a; Bristow et al., 1987). In this research we have carried out a comparative kinetic study of phosphofructokinase at intracellular concentrations of substrates and effectors in trout haemopoietic cells and erythrocytes. The role of adenine nucleotides and fructose 1,6-bisphosphate in the regulation of PFK during trout haemopoiesis has also been established. MATERIALS AND METHODS

Materials Adult rainbow trout (Salmo gairdneri R.) weighing 150 g were obtained from a fish farm in Segovia (Spain). All biochemicals were acquired from Boehringer Mannheim (FRG). Other reagents of analytical grade were purchased from Merck, Darmstadt. Methods Exact preparations. The kidney cephalic part, (the main haemopoietic organ in trout), was removed from the animals to obtain haemopoietic cells. Two grams of tissue was diluted in 3 ml of 100 mM Tris-HCl buffer (pH 8.5) and homogenized for 5 rain. Blood was obtained by puncturing the trout at the venous sinous, collecting it in heparinized tubes and then centrifuging (ll00g, 10rain) in order to separate the plasma. Red cells were washed three times with 1% NaC1, freed of leucocytes and platelets by centrifugation, and lysed with an equal volume of deionized water. After stirring for 10rain the hemolysate was frozen and thawed twice. Both preparations, homogenate and hemolysate, were centrifuged at 15,000g for 30 min. The supernatants were filtered by Sephadex G-25 column (I x 12cm) and employed for enzymatic determinations. All procedures were carried out at a temperature between 0 and 4°C. Phosphofructokinase assay. Phosphofructokinase activity was estimated according to Layzer (1975) by measuring the

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Fig. 1. Kinetic behaviour of PFK activity vs fructose 6-phosphate concentration at intracellular concentrations of Mg ATP. Mean values )7 + SD, N =4. Hill plots are inserted. (A) Haemopoietic cells (13.25 mM Mg ATP). (B) Erythrocytes (5.7 mM Mg ATP).

rate of NADH oxidation in a coupled system with aldolase, triosephosphate isomerase and phosphoglycerol dehydrogenase. Assays were carried out at pH 6.8 and 15°C. The reaction mixture for haemopoietic cells contained 90 mM Tris-Maleate; 90 mM KC1; 13.25mM MgC12;5 mM dithiotreitol; 0.3 mM NADH; 0.3 IU/ml aldolase; 8.2 IU/ml triosephosphate isomerase; 2.8 IU/ml phosphoglycerol dehydrogenase and extract containing 0.5 mg/ml protein. The assay mixture for erythrocytes contained 90mM Tris-maleate; 90mM KC1; 5,7mM MgC12; 10mM dithiotreitol; 0.15mM NADH; 0.6IU/ml aldolase; 16 IU/ml triosephosphate isomerase; 5.7IU/ml phosphoglycerol dehydrogenase and extract containing 1 mg/ml protein. Fru-6-P and ATP were employed at the concentration required in each assay. GIc~-P was added to Fru-6-P at a concentration of 3:1. Proteins were determined by the method of Lowry et al. (1951).

Substrates Fructose 6-phosphate. Assays are carried out at intracellular concentrations of ATP (13.25 mM in haemopoietic cells and 5.20 mM in erythrocytes) with an ATP: Mg 2+ ratio of 1:1. In both cellular populations PFK exhibits biphasic kinetics (Fig. 1) with a transition of negative cooperativity to a positive one, depending on the increase of the substrate (Table 1). The affinity for PFK-Fru-6-P is lower at high concentrations in haemopoietic, however, in erythrocytes the affinity is the same in both concentration ranges. As can be observed in Table 1, in haemopoietic cells, at intracellular concentrations of Mg ATP, the enzyme demonstrates a higher affinity for Fru-6-P than at non-intracellular concentrations of Mg ATP. The behaviour is opposite to that found in erythrocytes. Mg A TP. Experiments are carried out at intracellular concentrations of Fru-6-P (30 #M in haemopoietic cells and 50 # M in erythrocytes). The results (Fig. 2) demonstrate that in both cellular populations the enzyme exhibits biphasic kinetics, and there is no inhibition by Mg ATP. In haemopoietic cells the enzyme exhibits a similar affinity for the substrate at both concentration ranges. However, in erythrocytes the affinity is slightly higher at high concentrations of the substrate. In Table 2 the data obtained at the saturated concentration of Fru-6-P (6 mM in haemopoietic cells and 4 mM in erythrocytes) can be observed. The enzyme affinity for Mg ATP is higher at intracellular concentrations of Fru-6-P in erythrocytes. This effect is observed only at the higher end of the range of Mg ATP concentrations in haemopoietic cells. Effectors Fructose 1,6-bisphosphate. Assays have been carried out at intracellular concentrations of Fru-l,6-P2 (0.16mM in haemopoietic cells and 0.04raM in erythrocytes) and at two concentrations of Mg ATP. In haemopoietic cells it was seen (Fig. 3A) that biphasic kinetics are maintained, and Fru-l,6-P2 acts as a positive allosteric effector. There is an increase in the PFK activity, as well as in the affinity of

Table 1. Influence of fructose 1,6-bisphosphate at intracellular concentrations on PFK kinetic parameters vs fructose 6-phoshate at two concentrations of Mg ATP Control h

S0.smM

0.01-1.50 1.50~.00

0.8 3.5

1.01 1.33

0.01-1.00 1.0~6.00

0.4 2.5

0.30 0.85

13.25

0.01-1.50 1.5&6.00

0.5 1.8

0.63 1.14

0.01-1.00 1.0(~6.00

0.2 1.2

0.22 0.86

1.50

0.01~).80 0.80~..00

0.3 2.7

0.10 0.66

0.01-1.00 1.00-4.00

0.3 1.5

0.12 0.58

5.70

0.01~).80 0.80~,.00

0.4 2.8

0.77 0.80

0.01~).60 0.60~,.00

0.4 2.5

0.33 0.58

mM M g A T P mM range 3.00

Fru-l,6-P 2 mM range h S0.smM

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PFK-Fru-6-P, and a decrease in the value of the Hill coefficient (Table 1). The activator effect of Fru-l,6P2 is superior when assays are carried out at intracellular levels of Mg ATP. However, the differences of the values of h and S0.5 (in the presence and absence of Fru-l,6-P2) are proportional at both Mg ATP concentrations. Biphasic kinetics are maintained, and a clear activation by Fru-1,6-P2 at both Mg ATP concentrations is detected in erythrocytes (Fig. 3B). Table 1 displays the influence of bisphosphorylated hexose on kinetic parameters and that the effect is relevant for S0.s only when assays are carried out at 5.7raM Mg ATP (intracellular). The increase in P F K activity produced by Fru-l,6P2 is similar at both Mg ATP concentrations. A M P . These assays are performed at intracellular concentrations of A M P (20 p M in haemopoietic cells

and 10/~M in erythrocytes) and at two different concentrations of Fru-6-P. Results are shown in Fig. 4; the biphasic kinetic behaviour is maintained at all conditions and in the two cellular populations. A M P acts as an activator for haemopoietic cells PFK. This effect is superior when Fru-6-P concentration is higher, This nucleotide acts as an inhibitor in erythrocytes. This effect is more potent at 4 mM Fru-6-P. A M P increases the affinity of the enzyme for Mg ATP in haemopoietic cells (Table 2), while in erythrocytes the effect is opposite. With respect to the cooperative status of the enzyme, negligible changes in h values are observed, except for the higher range values of Mg ATP, when assays are carried out in 4 mM Fru-6-P in erythrocytes. A D P . Assays are performed at intracellular concentrations of A D P (360#M in haemopoietic cells and 30/~M in erythrocytes), and two concentrations of Fru-6-P. The biphasic kinetics are maintained in the presence of this nucleotide (Fig. 5). A D P acts as an inhibitor of P F K in both cellular populations, decreasing the enzymatic activity and the Mg ATP enzyme affinity (Table 2). Changes in Hill coefficients are negligible, except for erythrocytes in the higher value range of Mg ATP and at 4 mM Fru-6-P, similar to that of AMP. The effect of A D P is independent of the Fru-6-P concentration in haemopoietic cells, while in erythrocytes the effect is higher at intracellular concentrations of this hexose phosphate (50 ~tM).

DISCUSSION

The present results prove that phosphofructokinase exhibits a biphasic behaviour at intracellular concentrations of both substrates (Fru-6-P and Mg ATP) in haemopoietic cells and in erythrocytes (Figs 1 and 2). We also detected this behaviour in crude extracts at saturated concentrations of the substrates. The reason for this type of kinetics is the presence of two isoenzymatic phosphofructokinase forms in each cellular population. We have designated them PFK1 and PFK2 (Gaitfin et al., 1987). Biphasic kinetics have also been described for P F K of other species (Kelly and Turner, 1971; Lee et al., 1973; Fideu

Table 2. Effect of AMP and ADP at intracellular concentrations of PFK kinetic parameters vs Mg ATP at two concentrations of fructose 6-phosphate mM Fru-6-P

Control mM range h

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mM range

AMP h

S0smM

mM range

ADP h

S0smM

0.03

0.054).80 0.80-13.25

0.4 1.5

0.89 0.83

0.054).80 0.80-13.25

0.3 1.3

0.20 0.56

0.05-1.00 1.00-13.25

0.4 1.4

2,05 1.20

6.00

0.01-1.50 3.00-13.25

0.6 1.7

0.26 1.50

0.05-1.00 1.00-13.25

0.7 1.6

0.10 0.74

0.05-1.50 3.00-13.25

0.6 1.8

0.59 2.17

0.05

0.10-0.80 0.80-7.20

0.6 1.5

0.72 0.60

0.10~).80 0.80-7.20

0.4 1.5

3.27 0.85

0.10-1.00 1.50-7.20

0.7 1.2

2.00 2.30

4,00

0.014).80 0.80-7.20

0.3 1.3

1.70 0.88

0.10-1,10 1.10-7,20

0.8 2.5

2.50 1.43

0.1ff4).80 0.80-7.20

0,5 2,1

2.20 1.10

HC

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HC--haemopoietic cells (20/~ M AMP, 360 p M ADP). E--erythrocytes (10 gtM AMP, 30 # M ADP). mM ranges are Mg ATP concentrations which correspond to those of the first and second sections of the biphasic curve.

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Fig. 4. Influence of AMP at intracellular concentrations on the kinetic behavior of PFK vs Mg ATP at two concentrations of fructose 6-phosphate. Mean values 2 + SD, N = 4. (A) Haemopoietic cells: at 30#M Fru-6-P; • control; • 20 #M AMP; at 6 mM Fru-6-P: • control; q~-20 #M AMP. (B) Erythrocytes, at 50 #M Fru-6-P; • control; • 10 #M AMP; at 4raM Fru-6-P: • control; "k 10ttM AMP.

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Fig. 5. Effect of intracellular concentrations of ADP on the kinetic behaviour of PFK vs Mg ATP at two concentrations of fructose 6-phosphate. Mean values ~ + SD, N = 4. (A) Haemopoietic cells at 30/~M Fru-6-P: • control; • 360/~M A.DP; at 6mM Fru-6P: • control; "k" 360#M ADP; (B) Erythrocytes; at 50 #M Fru-6-P: • control; • 30fzM ADP; at 4 mM Fru-6-P: • control; "k" 30/~M ADP.

PFK in trout haemopoiesis Alonso et al., 1985) although this behaviour is attributed to several mechanisms. The fact that S0.5 values are higher than the intracellular concentration of Fru-6-P (Table 1) in both cellular populations implies an important feature, i.e. the low in vivo P F K activity. Inhibition of phosphofructokinase by Mg ATP (Bloxham and Lardy, 1973; Uyeda, 1979) is a common feature of phosphofructokinase from different origins. However, in P F K of trout haemopoietic cells and erythrocytes this nucleotide displays no inhibition, although the assays were performed at pH 6.8 and at low Fru-6-P concentrations (intracellulars) when the inhibition effect is thought to be more potent (Bloxham and Lardy, 1973; Uyeda, 1979). Jensen (1981) found negligible inhibition of P F K by this substrate in trout myocardial. In mammals, M-type P F K is the least sensitive to inhibition by Mg ATP (Vora et al., 1985b). So, similar subunits could be present in trout haemopoietic cells and erythrocytes. The affinity of the enzyme for both substrates is different in each cellular population (Table I). This might imply that different intracellular levels of substrates may be responsible for the regulation of PFK. We have found in previous assays that Fru-l,6-P2 is the most potent activator of phosphofructokinase in both cellular populations, even superior to Fru2,6-P 2 (Gait~m et al., 1989). This fact may be related to the position of the trout in the phylogenetic scale, since Fru-2,6-P2 plays a relevant role in the regulation of PFK, mainly in mammals (Torres et al., 1984). Data obtained with respect to the influence of Fru-l,6-P2 (Figs 3A and B, and Table 1) exhibits differences between P F K of haemopoietic cells and erythrocytes. Therefore, Fru-l,6-P2 acts as a positive allosteric modulator in haemopoietic cells, nevertheless, only an activator effect can be attributed in erythrocytes. These results allow us to emphazise the presence of M-type P F K subunits in haemopoietic cells (Foe et al., 1983). Comparing the results obtained with the effect of Fru-l,6-P 2 at two Mg ATP concentrations (Figs 3A and B, and Table 1) we can conclude that in haemopoietic cells a synergistic effect between Fru-1,6-P 2 and Mg ATP could be proposed. Increases in Mg ATP concentration would therefore facilitate the binding of Fru-l,6-P 2 to PFK. An opposite effect of A M P in both cellular populations is deduced from data in Fig. 4 and Table 2. A M P acts as an activator in haemopoietic cells, and as an inhibitor in erythrocyes at all Mg ATP and Fru-6-P concentrations studied. The activation of A M P has been described for muscle and liver of several species using the phylogenetic scale (Leaver and Burt, 1981; Sakakibara and Uyeda, 1983; Torres et al., 1984; Tornheim, 1985). We can therefore suggest that in haemopoietic cells M-type and L-type subunits are present. The different effects of A M P on both cellular populations could imply a distinct isoenzymatic pattern. A synergistic effect of A M P and Fru-6-P is observed during activation as well as during inhibition. ADP, according to different authors, exhibits different effects on phosphofructokinase activity in several species (Uyeda, 1979; Leaver and Burt, 1981; Dunaway, 1983; Isaac and Rhodes, 1986) sometimes

645

acting as an activator and sometimes as an inhibitor. A D P acts as an inhibitor in both types of cells studied (Fig. 5 and Table 2). This effect is independent of the Fru-6-P concentration in haemopoietic cells, however, in erythrocytes the inhibition increases at low Fru-6-P concentrations. A general pattern is deduced from the data: P F K presents a higher activity in haemopoietic cells than in erythrocytes. These results agree with those found in neoplasias and cellular differentiation processes (Pinilla et aL, 1982; Nijhof et aL, 1984; Oskam et al., 1985; Vora et al., 1985a; Bristow et al., 1987). We can conclude from this investigation that there is a different regulation of phosphofructokinase in haemopoietic cells and erythrocytes. Fru-l,6-P 2 and A M P in haemopoietic cells seem to be responsible for the maintenance of activity levels related with the higher energetic requirements of these cells.

SUMMARY

Some regulatory properties of phosphofructokinase (PFK) (ATP; D-fructose 6-phosphate-l-phosphotransferase EC 2.7.1.11) have been investigated in two cellular populations, representative of trout haemopoiesis: haemopoietic cells (undifferentiated and capable of replication and differentiation) and erythrocytes (highly specialized cells). The kinetic behaviour has been studied at intracellular concentrations of substrates, as well as the influence of fructose 1,6-bisphosphate, A M P and ADP. Phosphofructokinase, of both cellular populations, exhibits biphasic kinetics at intracellular concentrations of both substrates. Fructose 1,6-bisphosphate acts as a positive allosteric effector in haemopoietic cells, and an activator effect may be proposed in erythrocytes. A synergistic effect of fructose 1,6-biphosphate and Mg ATP could be only observed in haemopoietic cells PFK. With respect to A M P this nucleotide acts as an activator in haemopoietic cells and as an inhibitor in erythrocytes. On the other hand, A D P acts as an inhibitor in both cellular populations. These results show a different regulation of phosphofructokinase in haemopoietic cells and erythrocytes, which could be related to the physiological role of each cellular population. Acknowledgements--The authors wish to thank Drs J. M. J. Herranz and M. L. Pdrez for their valuable advice and thorough review of the manuscript. Mr Tomfis Urrialde for supplying the trout for this investigation. REFERENCES

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Nijhof W., Wierenga P. K., Staal G. E. J. and Jansen G. (1984) Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro. Blood. 64, 607-613. Oskam R., Rijksen G., Staal G. E. J. and Vora S. (1985) Isozymic composition and regulatory properties of phosphofructokinase from well-differentiated and anaplastic medullary thyroid carcinomas of rat. Cancer Res. 45, 135-142. Pinilla M., Perez-Artes E., Roncales P., Tejero C. and Luque J. (1982) Estudio de la enzimas reguladoras glicoliticas en eritrocitos, reticu|ocitos y c61ulas inmaduras de m~dula 6sea de rata. Rev. Esp. Fisiol. 38, 97-100. Rapoport S. (1968) The regulation of glycolysis in mammalian erythrocyte. In Assays in Biochemistry. (Edited by Cambell P. and Creville G. D.), Vol. 4, pp. 69-103. Academic Press, New York. Sakakibara R. and Uyeda K. (1983) Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver. J. biol. Chem. 258, 8656-8662. Tanaka T., Imamura K., Ann T. and Taninchi K. (1972) Multimolecular forms of pyruvate kinase and phosphofructokinase in normal and cancer tissues. GANN Monogr. Cancer Res. 13, 219-234. Tornheim K. (1985) Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites. J. biol. Chem. 260, 7985-7989. Torres N. V., Sanchez L. D., Perez J. A. and MelendezHevia E. (1984) Regulation of glycolysis in lizards: kinetic studies on liver pyruvate kinase and phosphofructokinase from Lacerta galloti. Comp. Biochem. Physiol. 77B, 289-294. Uyeda K. (1979) Phosphofructokinase. In Advances in Enzymology (Edited by Meister A.), Vol. 48, pp. 193-244. J. Wiley, New York. Vora S., Halper J. P. and Knowles D. M. (1985a) Alterations in the activity and isozymic profile of human phosphofructokinase during malignant transformation in vivo and in vitro. Transformation and progression linked discriminant of malignancy. Cancer Res. 45, 2953-3001. Vora S., Oskam R. and Staal G. E. (1985b) Isozymes of phosphofructokinase in the rat. Demonstration of the three nonidentical subunits by chemical, immunochemical and kinetic studies. Biochem. J. 219, 333-341. Walton M. J. and Cowey C. B. (1982) Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B, 59-79.