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Kinetic properties of phosphofructokinase from the flounder (Platichtys flesus L.)
Comp. Biochera.Physiol. Vol. 69B. pp, 435 to 443, 1981
0305-0491/81/070435.09502.00/0 Copyright © 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
KINETIC PROPERTIES OF PHOSPHOFRUCTOKINASE FROM THE FLOUNDER (PLATICHTYS FLESUS L.) OLE SAND Institute of Biochemistry, Odense Universitet Campusvej 55, 5230 Odense M, Denmark
(Received 25 September 1980) Abstract--1. Phosphofructokinase purified from flounder liver is characterized by sigmoidal kinetics with fructose 6-phosphate as substrate. 2, The enzyme is inhibited at higher ATP levels at both pH 6.9 and 8.2. The inhibition is more pronounced at pH 6.9. 3. This inhibition can be relieved by inorganic phosphate, fructose 6-phosphate, AMP and cyclic AMP. 4. In lower concentrations ADP acts as a deinhibitor, but in higher concentrations the effect is vanishing. 5. Citrate and phosphoenolpyruvate are inhibitors. 6. Fructose-l,6-diphosphate and creatinphosphate are without effect. 7. With increasing Mg 2+ level hyperbolic response curves are found with low ATP concentrations. With high ATP levels biphasic curves are demonstrated. 8. The enzyme behaves like mammalian phosphofructokinases but show some quantitative and qualitative differences.
INTRODUCTION
properties of phosphofructokinase purified from various sources, those of a fish phosphofructokinase have not hitherto been described. In a previous paper (Sand, 1981) dealing with the purification of phosphofructokinase from the flounder liver, its mol. wt was determined to 250,000, a value which falls between the minimum tool. wt of phosphofructokinase catalytically active from bacteria (mol. wt 140,000) and from mammalian species (tool. wt 320,000). With respect to pH response the flounder liver enzyme behaves like mammalian phosphofructokinase, showing maximal activity at pH 8.2. Addition of phosphate or sulphate stimulated the enzyme pronounced at pH 6.9, but only to some degree at pH 8.2 when the concentration of ATP was 1 mM and that of F-6-P was 2 raM. In the present paper further kinetic studies on the purified flounder liver enzyme are undertaken. These are performed at pH 6.9 and 8.2, and the results are compared with those of other phosphofructokinase enzymes that have been studied.
Phosphofructokinase (ATP: D-fructose 6-P-l-phosphotransferase, EC 2.7.1.11, PFK) is an important regulatory enzyme of carbohydrate metabolism and as such has been the subject of intensive investigation (for a recent review, see Uyeda, 1979). It is an allosteric enzyme subjected to a complex regulation by various metabolites, i.e. ATP, AMP, phosphate and citrate. The majority of the work has been done on phosphofructokinase isolated from primarily glycolytic cells, such as rabbit muscle. Phosphofructokinase isolated from livers has been found to have altered characteristics, which also was to be expected due to the gluconeogenesis in these cells. Phosphofructokinase isolated from livers include rabbit (Kemp, 1971; Ramaiah & Tejwani, 1973), pig (Massey & Deal, 1973), chicken (Kono & Uyeda, 1973), sheep (Brock, 1969), rat (Underwood & Newsholme, 1965; Brand & S61ing, 1974; Dunaway & Weber, 1974), monkey (Mattheyse, 1979) and mouse (Herzberg~ 1979). The number of effectors that influence phosphoMETHODS fructokinase and the extent of inhibition or activation vary, depending on the source of the enzyme. For Preparation of PFK example, only a few metabolites influence bacterial The enzyme is purified and diluted for measurements as phosphofructokinase, while the enzyme from mam- described by Sand (1980). The purified enzyme did not mals is affected by numerous effectors (Bloxham & contain the following enzymes, which otherwise might Lardy, 1973). interfere with the measurements: fructose-l,6-diphosphaIn respect to pH response, mammals and E. coli tase, adenylate kinase, NADH oxidases and ATPases. phosphofructokinase are oppositely influenced in their allosteric properties. In enzymes isolated from Assay of PFK-activity PFK was assayed at 30°C by following the appearance mammals an increase in pH from 7-8 causes a shift of the signoidal F-6-P velocity curve to the left and a of fructose-l,6-diphosphate (FDP) in a coupled assay system. All auxiliary enzymes are extensively dialyzed relief of the inhibitory action of ATP, whereas in the before use to get rid of ammoniumsulphate, which is an E. coil enzyme the opposite is found (Bloxham & activator for PFK. In the experiment where the influence Lardy, 1973; Uyeda, 1979). of FDP was investigated, PFK was, however, assayed by Despite the large number of reports of the kinetic monitoring the formation of ADP. 435
436
OLE SAND
In the former system the assay mixture contained a total vol of 810~1: 67mM potassium glycylglycine buffer, 0.14 mM NADH, 0.05 mg bovine serum albumin, 0.12 mM dithiothreitol, 0.6 U aldolase, 0.6 U glycerol 3-phosphate dehydrogenase, 1.0 U triosephosphate isomerase and ATP, F-6-P and MgCI2 as indicated for each experiment. The value of pH in the assay is also stated in each experiment. In the experiment where PFK activity is assayed by following the formation of ADP, the cuvette contained in a total vol. of 810#1: 67mM potassium glycylglycine buffer, pH 6.9, 0.14 mM NADH, 0.05 mg bovine serum albumin, 0.12 mM dithiothreitol, 1.0 U lactat dehydrogenase, 1.0 U pyruvate kinase, 2.0 mM F-6-P, 0.90 mM ATP, 1.30 mM MgC12 and 0.50 mM phosphoenolpyruvate (PEP). Most assays performed were non-linear with respect to time during the first minutes showing accelerating or decelerating rates, depending on the order of addition of various components to the assay, before a constant velocity was attained. This constant velocity, the steady-state rate, is used in the present paper. The steady-state rate obtained was not the same one, irrespectively of the addition of components, for the same assay mixture. It was decided to start the reaction by addition of 50 #1 containing a suitable amount of PFK preparation after allowing 4 min for temperature equilibration. An amount of PFK was added to each assay which would give a change in absorbance at 340 nm of 0.08 per rain in the "normal assay", which is the assay mixture monitoring the formation of FDP described above, but supplied with 10 mM ammoniumsulphate. The concentration of ATP, F-6-P and MgCI2 in this "normal assay" are respectively 1.30 mM, 1.90 mM and 1.30 mM and pH is 8.2. This velocity of reaction is defined as V,. The activities are expressed as viVa, where v represents the steady-state rate measured under the conditions of the experiment. This is done to allow for comparison of effects in preparations, which have different final specific activities. RESULTS
A variety of metabolites and inorganic ions has been shown to affect the activity of phosphofructokinase. Figure 1 shows the effect of increasing concentrations of sodium phosphate on the activity of flounder liver P F K at pH 6.9 and 8.2 in the presence of 0.5 m M F-6-P. The measurements are made at two fixed concentrations of ATP of 0.3 m M and 1.0 m M respectively. As seen from the figures, phosphate stimulates, but the stimulatory effect is very much dependent upon the concentration of A T P and on pH. At pH 6.9 and 0.3 m M ATP a hyperbolic curve is obtained with a concentration of phosphate giving half maximal activity at 0.4 mM. By increasing the concentration of A T P to 1 mM, which is an inhibitory
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Fig. 1. Phosphate dependence of phosphofructoklnase activity. An amount of enzyme was added to each assay that would give a change in absorbance at 340 nm of 0.08 per min in the "normal assay mixture". This is defined as Vt. The experimental assay is as described in Methods. The concentration of F-6-P is 0.50 mM. A, 0.3 mM ATP and A, 1.0mM ATP, pH6.9; O, 0.3mM ATP and O, 1.0raM ATP, pH 8.2. A T P concentration (Fig. 2A), a sigmoid curve is obtained with an apparent K,, for phosphate of 4 mM. Figure 1 also indicates that increasing phosphate concentrations counteract the inhibitory effect of 1 m M ATP. At pH 8.2 phosphate only has a slightly stimulatory effect at the low ATP concentration. Under these conditions 0.5 m M F-6-P has nearly abolished the inhibitory effect of 0.3 m M ATP (Fig. 3A). At an A T P concentration of 1 m M the enzyme is very susceptible to phosphate with an apparent K,, for phosphate of 0.2 mM. (Fig. 1) The stimulatory effect on the enzyme upon the addition of sodium phosphate is due to phosphate and not caused by N a ÷. Addition of NaC1 in the range 0-20 m M does not influence the enzyme activity. In addition to their function as substrates, F-6-P and ATP act under definite conditions as effectors on PFK, since F-6-P may relieve the ATP inhibition and ATP influences shape and position of the Fo6-P saturation curve. This is shown in Fig. 2 and Fig. 3. Figure 2A and 2B show the effect of varying the concentration of A T P at pH 6.9 at two fixed levels of F-6-P in the absence and presence of 12 m M sodium phosphate. This concentration of phosphate has been found to stimulate the enzyme maximally under the conditions indicated in Fig. 1. In the low region of ATP concentration, phosphate has no effect. Higher concentrations of A T P inhibits PFK, when phosphate is not present. Addition of 12 m M phosphate relieves
Table 1. The dependence of apparent K , and apparent K~ values for ATP on pH, F-6-P level and presence of phosphate
pH
mM phosphate added
6.9 6.9 8.2 8.2
0 12 0 12
ATP K,, (mM) 0.5 mM 2.0 mM F-6-P F-6-P 0.020 0.040 0.040 0.060
0.025 0.040 0.080 0.080
ATP K~ (mM) 0.5 mM 2.0 mM F-6-P F-6-P 0.22 1.45 0.45 >3
0.70 >3 1.3 >3
Kinetic properties of flounder's PFK
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Fig, 2. Effect of ATP concentration on phosphofructokinase activity at pH 6.9. Assay conditions as in Fig. 1. The concentration of F-6-P is 0.50 mM (A) and 2.0 mM (B). A, without phosphate added. O, 12 mM phosphate added.
the inhibitory effect of ATP at moderate ATP concentrations. At 0.5 mM F-6-P addition of 12 mM phosphate increases the apparent Ki for ATP from 0.22 to 1.45 mM (Table 1). In the absence of phosphate the apparent Kl for ATP is increased from 0.22 to 0.70 mM, when the F-6-P concentration is changed from 0.5 to 2.0 mM. Table 1 summarizes the values of the apparent K , for ATP measured at pH 6.9 derived from the curves in Fig. 2A and 2B. Changing the F-6-P concentration from 0.5 to 2.0 mM does not influence the value of K , . Addition of 12 mM phosphate increases Km with a factor of about 2 at both F-6-P concentrations. This
increase in K,, is a consequence of the fact that addition of phosphate increases the enzyme activity, but only when the concentration of ATP is inhibitory. Figures 3A and 3B show the effect of ATP at pH 8.2 at two fixed levels of F-6-P in the presence and absence of 12 mM sodium phosphate. In the lower range of ATP-concentration, phosphate has no effect. When phosphate is absent, the hyperbolic saturation curve is followed by a steep inhibition curve as the concentration of ATP is increased. The apparent Ki for ATP changes from 0.45 to 1.3 mM, when the F-6-P concentration changes from 0.5 to 2.0mM. (Table 1.) In the presence of phosphate the activity
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Fig. 3. Effect of ATP concentration on phosphofructokinase activity at pH 8.2. Assay conditions as in Fig. 1. The concentration of F-6-P is 0.50 mM (A) and 2.0 mM (B). A, without phosphate added. O, 12 mM phosphate added.
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Fig. 4. Effect of F-6-P concentration on phosphofructokinase activity at pH 6.9. At 0.3 mM ATP (A) and 1.0 mM (B), in the absence (A) and presence (O) of 12 mM phosphate. Assay conditions as in Fig. 1.
Fig. 5. Effect of F-6-P concentration of phosphofructokinase activity at pH 8.2 at 0.3 mM ATP (A) and 1.0 mM (B) in the absence (A) and presence (O) of 12 mM phosphate. Assay conditions as in Fig. 1.
curves reaches almost a plateau of maximal activity before inhibition is seen. Table I shows the values of K,, for ATP measured at pH 8.2 determined from the curves in Figs 3A and 3B. The addition of phosphate does not influence the apparent Km measured in the presence of 2 m M F-6-P, but at 0.5 m M F-6-P, K , is increased by 50%. When the F-6-P-concentration is increased from 0.5 to 2 m M in the presence or absence of phosphate the corresponding Km values for ATP are slightly increased. By comparing the curves in Figs 2 and 3, it is evident that the degree of inhibition by a fixed concentration of ATP, as well as the ATP concentration at which inhibition is first observed, is a function of pH, F-6-P concentration and phosphate concentration. Elevated pH, the presence of phosphate or increased F-6-P concentration increases the activity of P F K at
inhibitory concentrations of ATP, as well as delaying the onset of ATP inhibition. When the activity was determined at constant levels of ATP with varying levels of F-6-P in the absence of phosphate, sigmoid response curves typical of allosteric enzymes were observed at both pH 6.9 and 8.2. Addition of 12 m M phosphate abolished the sigrnoidicity leading to saturation curves steeper than a rectangular hyperbola. This is shown in Figs 4 and 5. Figures 4A and 4B show the results obtained at pH 6.9. Another effect of adding phosphate is an increase in the maximal activity above that obtainable with increasing F-6-P concentration in the absence of phosphate. Therefore F-6-P does not stimulate the enzyme to the same degree as phosphate does at pH 6.9. Table 2 shows the apparent K,, for F-6-P in the absence and presence of 12mM phosphate at
Table 2. The dependence of apparent K,, for F-6-P and Hill coefficient (nn) for F-6-P binding on pH, ATP level and presence of phosphate
pH
mM phosphate added
6.9 6.9 8.2 8.2
0 12 0 12
F-6-P K,n (mM) 0.3 mM 1.0 mM ATP ATP 0.95 0.080 0.36 0.085
3.80 0.32 1.24 0.16
nn
0.3 mM ATP
1.0 mM ATP
3.0 1.5 3.1 1.4
3.7 2.3 6.4 1.7
Kinetic properties of flounder's PFK fixed levels of 0.3 and 1 mM respectively, measured at pH 6.9 and 8.2 in the absence and presence of 12 mM phosphate. By increasing the ATP level from 0.3 to 1 mM, KM for F-6-P is increased by a factor of 4. Addition of 12 mM phosphate decreases KM for F-6-P by a factor of about 10 at both ATP concentrations tested. Figures 5A and 5B show the results obtained by varying the F-6-P concentration at constant levels of ATP at pH 8.2 in the absence and presence of 12 mM phosphate. In contrast to the measurements carried out at pH 6.9 no change in Vmax was observed when phosphate was added. Addition of phosphate or decreasing the ATP concentration decreases the values of Kin. (Table 2) When the curves in Figs 4A, 4B, 5A and 5B are repiotted in terms of the Hill equation (Hill, 1910): log - -
v
= nn log S - log K
Vma x --
where S is the substrate concentration (F-6-P), K is a constant and Vmaxis the maximal activity obtained-derived from each curve, straight lines are obtained from which could be calculated the interaction coefficient n , , which is presumed to be determined by the number of interacting sites for F-6-P and the strength of interaction among them. The values for nn are given in Table 2. It can be seen that addition of phosphate decreases nn, an effect which is also obtained by decreasing the concentration of ATP. Changing pH from 6.9 to 8.2 does not influence the value of nH at the low ATP concentration (0.3 raM), whereas at the high ATP-concentration (1 raM) nn is increased in the absence of phosphate, but decreased in the presence of phosphate. It should be noted that an unusual high value of nH of 6.4 is found at 1 mM A T E when phosphate is absent at pH 8.2. Mg 2+ has been shown to be essential for PFK, as is also the case for other kinases, which as one of their natural substrates uses ATP. The effect of Mg 2 + upon flounder liver P F K was examined by assaying the enzyme at pH 6.9 and 8.2 at two different concentrations of ATP in the presence of 2 mM F-6-P. The experiments were performed in the absence and pres-
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ence of 12 mM phosphate. The results are given in Figs 6A a n d 6B. When Mg 2÷ is absent, no P F K activity is found. The enzyme activity increases with increasing level of Mg 2+' up to a certain value of Mg 2+ concentration. Beyond this value increasing MgCl2 inhibits the enzyme. Figure 6A shows that at pH 6.9 in the presence of phosphate there is a more pronounced stimulation of the enzyme with increasing MgCl2 up to about 0.7 mM MgCl2 with 0.3 mM ATP present than is the case with I mM ATP present. The apparent K= for MgCI2 at 0.3mM ATP is 0.1raM, and the apparent K= at l m M ATP is 0.2 raM. When the experiment with 0.3 mM ATP is performed without phosphate added, a completely different type of curve is obtained. There is a little but distinct peak of activity at 0.1 mM MgCI2. Increasing the concentration of MgC12 above 0.15raM the enzyme activity increases. The apparent Km for MgC12 in the presence of 0.3 mM ATP without phosphate added is 0.35 raM. Phosphate, therefore, seems to increase the affinity of the enzyme for MgCl2. At I mM ATP without phosphate added, no activity is found by increasing MgCI2 (not shown), which is to be expected due to the inhibitory ATP concentration. Figure 6B shows that at pH 8.2 in the presence of 12 mM phosphate MgCl2 concentrations up to about 0.3 mM stimulate the enzyme to the same extent irrespective.of the ATP-concentration used. K~, for MgCl2 at both ATP-concentrations is 0.07raM. When 12 mM phosphate is ommitted in the reaction mixture, no change in the pattern of the curve representing 0.3mM ATP is observed. With I mM ATP, which at pH 8.2 is an optimal concentration (Fig. 3B), a completely different type of curve is obtained, which has an appearence similar to the curve in Fig. 6A, representing 0.3 mM ATP at pH 6.9, without phosphate present. A little peak of activity is noted at 0.07 mM MgCl2, then there is a decrease in activity with increasing MgC12 up to 0.3 mM MgCl2. At higher MgC12 a sigmoidal curve is found. The effect of phosphate at pH 8.2 with 1 mM ATP is therefore a decrease in the apparent K= from 0.95 to 0.07 mM for MgCl2. Due to the sigmoidicity of the curve it might be concluded that there is cooperativity among the sites for MgCl2 binding, when phosphate is absent in
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Fig, 6. Effect of MgC12 concentration on phosphofructokinase activity in the presence of 2.0 mM F-6-P, at pH6.9 (A) and 8.2 (B). Q, 0.3mM ATP; O, 0.3raM ATP + 12mM phosphate; &, 1.0raM ATP; A, 1.0 mM ATP + 12 mM phosphate. Assay conditions as in Fig. 1.
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the high ATP-concentration. By comparing the curves in Figs 6A and 6B representing the same conditions except for pH, it is evident that Ks for MgC12 is higher at pH 6.9 than at pH 8.2. The adenine nucleotides, AMP, cyclic AMP and ADP interfere with the activity of most P F K investigated. In the present study it was found that they stimulated the enzyme, and that the stimulatory effect was dependent on pH and on the degree of inhibition of ATP. At non-inhibitory ATP-concentration, there was a slight stimulatory effect of AMP and cyclic AMP (not shown), whereas in the presence of inhibitory concentration of ATP the stimulatory effect was pronounced. Figure 7A shows the effect of adding AMP and cyclic AMP (cAMP) respectively to the enzyme at pH 6.9, when the concentrations of F-6-P and ATP were 2 and 0.80 mM respectively. Without addition of the mononucleotides these conditions ensure that the enzyme activity is 35% of its maximal value due to the inhibition of ATP (see Fig. 2B). The figure shows that the maximal activity obtained with AMP and cAMP was the same regardless of the kind of effector used. This value is significantly higher than that obtained in the absence of ¢ffectors at optimal ATP concentration (0.30 mM, see Fig. 2B), and is also higher than those obtained in the presence of saturating levels of F-6-P. The concentrations of AMP and cAMP increasing the enzyme activity to half its maximal value are 6 and 50gM respectively. As seen from the curves in Fig. 7A, the same kinetic pattern is essentially applying to AMP as well as to cAMP.
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A completely different saturation curve is found for ADP, a product of the PFK-reaction, under the same assay conditions (Fig. 7B) ADP is also a stimulator, but the stimulatory effect does not simply increase with the concentration. When the level of ADP is increased from 0 to 0.25 mM the enzyme activity is increased 4-fold with a concentration of ADP giving half maximal velocity of 60 #M. When the concentration of ADP is increased above 0.25 mM the stimulatory effect is decreasing. It was not possible even at an ADP-concentration of 10 mM to obtain the same activity or a lower one as that obtained with no ADP present. With optimal ADP-concentration (0.25 raM) the stimulatory effect was smaller than that obtainable with AMP and cAMP respectively. FDP, which is also a product of the reaction, is reported as an activator for mammalian P F K (Bloxham & Lardy, 1973). The effect of F D P on flounder liver P F K was assayed at pH 6.9 by measuring the formation of ADP in the assay system described in "Methods". The concentration of PEP was kept as low as 0.5 mM to minimize the inhibition caused by this agent. With this choice of assay condition the activity of P F K without F D P added was 1S~o of that obtainable in the presence of an optimal ATP-concentration. It turned out that addition of F D P up to a concentration of 5 mM was without any effect on the enzyme activity. It might therefore be concluded, that F D P does not function as an activator or deinhibitor of the enzyme. P F K from many sources are inhibited by phosphoenolpyruvate (PEP), creatin phosphate and citrate. The effect of these compounds was tested at pH 6.9 in the presence of 2 mM F-6-P and 0.50 mM ATP. Without effectors present this choice of condition resulted in an enzyme activity of 909/o of that obtainable with an optimal ATP concentration. Figure 8 shows that phosphoenolpyruvate and citrate were inhibitors, whereas creatin phosphate was without effect in concentrations up to 4 raM. Citrate was a powerful inhibitor with a concentration decreasing the enzyme activity to S0~o of 0.23 mM. The corresponding value of PEP was 1.8 mM. Both effectors inhibit the enzyme 100~o when present in excess. Because the experiments were run in the presence of a fixed and relatively low MgCl2 concentration, and it is well known that citrate chelates Mg 2÷, the
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Fig. 7. A: Activation of ATP inhibited phosphofructokinase activity by AMP (O) and cyclic AMP (/\). B: Effect of ADP (D) on ATP inhibited phosphofructokinase activity. The expcrimems are performed at pH 6.9 at 2 mM F-6-P and 0.80 mM ATP. Assay conditions as in Fig. 1.
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Fig. 8. Inhibition of phosphofructokinase activity by citrate (r-l) and phosphoenolpyruvat {O). zX, Effect of phosphocreatin. The experiments are performed at pit 6.9 at 2 mM F-6-P and 0.50 mM ATP. Assay conditions as in Fig. 1.
Kinetic properties of flounder's PFK effect of 1 mM citrate upon the enzyme activity was also tested in the presence of increasing MgCI2 concentrations under the same conditions as those described for Fig. 8. It was found that the inhibitory action of 1 mM citrate could not be abolished by increasing the concentration of MgC12 (not shown), so it can be concluded that citrate inhibits the enzyme by interfering directly with it. DISCUSSION
In the present study, P F K from flounder liver was demonstrated to be regulated by a variety of effectors such as cAMP, AMP, ADP, citrate, PEP, inorganic phosphate, Mg 2÷ as well as by its substrates, F-6-P and ATP. Furthermore pH plays an important role in this regulation. Without Mg 2÷ present no activity was found consistent with the general proposal that M g ' A T P is substrate for PFK. When enzyme activity was measured as a function of the concentration of MgCIz it was found at both pH 6.9 and 8.2 that optimal enzyme activities were obtained when Mg2+/ATP was above 1 (Fig. 6). The simplest explanation for the rise in activity, observed by increasing Mg 2÷ above a Mg2+/ATP of 1 is that the enzyme is not fully saturated with substrate (Mg. ATP) until all of the ATP is effectively present as the Mg.ATP. There are two alternative explanations, namely: (1) that free ATP itself may be so highly inhibitory that a further rise in activity is not obtained before all ATP is bound by Mg 2+, or (2) that low concentrations of free Mg 2÷ may be necessary for enzyme activity (as with several kinases) so that full activity is not achieved until a saturating concentration of free metalion is present. More experiments should be done to distinguish between these alternatives. In the presence of inhibiting ATP concentrations without phosphate present biphasic curves were obtained (Fig. 6). Such responses have until now only been reported for P F K isolated from monkey liver (Mattheyse, 1979). The biphasic character was completely abolished by addition of phosphate. An explanation for this might be that Mg 2÷, besides being substrate, as Mg.ATP, furthermore favours the existence of a more active form of the enzyme, a form which is also favoured by the presence of phosphate. Sensitivity to inhibition of P F K at high levels of ATP differs significantly with the source of the enzyme. In general liver PFK is most strongly inhibited, muscle PFK is less sensitive and brain enzyme is least sensitive (Johnson et al., 1976; Tsai et al., 1974). PFK from a variety of bacteria are not inhibited by ATP (Ferdinandus et al., 1969; Doelle, 1972). P F K from flounder liver is found to be inhibited by ATP. The apparent inhibitor constants at pH 6.9 and 8.2 measured in the presence of 2 m M F-6-P without other effectors present are 0.7 and 1.3 mM respectively (Table 1). The corresponding values for sheep liver (Brock, 1969) measured under comparable circumstances are 1.7mM and 3.4mM respectively. From the paper of Mattheyse (1979) reporting the kinetics of P F K purified from monkey liver, an apparent inhibitor constant for ATP at pH 7.0 with 2 mM
441
F-6-P present without effector present can be derived from the figures to 0.8 raM. In the presence of a saturating level of the deinhibitor cAMP, the apparent Ki is increased to 2.4 raM. In the present paper a value above 3mM is found when 12mM inorganic phosphate is present. When assaying the activity in the presence of 0.5 mM and 2.0 mM F-6-P at pH 6.9 and 8.2 (Table 1), an apparent Michaelis constant (Kin) for ATP was found in the range 0.020-0.080 mM and this value was not greatly affected by 12mM phosphate. This effector did not affect the catalytical part of the ATP saturation curve, but only exerted its effect by relieving the inhibition at high levels of ATP. Massey & Deal (1975) found a Km for ATP at pH 7.0 of 0.025 mM when no effector was present. For monkey liver, Mattheyse (1979) reports K , for ATP at pH 7.0 and 8.0 in the same concentration range as that found for flounder liver. The kinetics of flounder liver P F K with respect to F-6-P were however found to be affected significantly by pH, ATP level and presence of inorganic phosphate. Plots of steady-state velocity vs F-6-P levels at different ATP concentrations at pH 6.9 and 8.2 gave S-formed curves indicating allosteric kinetics with respect to F-6-P (Figs 4 and 5). A change in pH from 6.9 to 8.2, presence of phosphate or decreased ATP level all caused a shift of the sigmoidal F-6-P activity curve to the left giving rise to smaller values of the apparent Kin. When pH is changed from 6.9 to 8.2, K~ for F-6-P with 0.3 mM ATP present is changed from 0.95 to 0.36 mM respectively. These values are close to those reported for monkey liver (Mattheyse, 1979), measured under comparable conditions, of 0.60 and 0.23raM respectively, but higher than those reported for pig liver (Massey & Deal, 1975) and rabbit liver (Kemp, 1971), which, however, were measured in the presence of ammonium sulphate known to increase the affinity of PFK to F-6-P. Values for nn, the Hill coefficient (Table 2), greater than 1 were found. In those cases, where phosphate was not present, unusual high values of nn were derived indicating a high degree of interaction. Presence of phosphate or decreasing ATP level resulted in decreasing nH values. This might be a consequence of a dissociation of the polymeric enzyme or conformational changes. In a recent paper concerning the kinetics of rat liver phosphofructokinase (Reinhart & Lardy, 1980) Hill coefficients as high as 5.2 were found, when steady-state activity, attained after completion of enzyme transient was taken as a function of the F-6-P level. Inorganic phosphate, F-6-P, AMP and cAMP were found to stimulate the flounder liver P F K when ATP was present in inhibitory concentrations (Figs 1, 4, 5 and 7). The stimulatory effect can be explained as a deinhibition of the effect of ATP. The most powerful deinhibitors demonstrated of the compounds tested was AMP (Fig. 7A), which at a concentration of 6 #M stimulated the enzyme to 50~o of that obtainable under conditions where the enzyme was inhibited 65Yo without AMP present. The corresponding values for AMP given for mouse liver (Herzberg, 1979), sheep liver (Kemp, 1971) and for hepatopancreas of the crayfish (Lesicki, 1980) are 35, 210 and 10-15 #M respectively. The concentration of cAMP giving half maximal ac-
442
OL~ SANO
tivity for flounder liver P F K (Fig. 7A) was found to be 50 #M, a value close to that of 75 #M found for rabbit liver P F K (Kemp, 1971). ADP, phosphate and F-6-P were not able at any level to deinhibit the enzyme to the same degree as did cAMP and AMP. It was demonstrated, that ADP (Fig. 7B) deinhibited the enzyme when present in a certain concentration range. In the presence of higher ADP levels the deinhibition was decreasing. This effect of ADP has also been published by Sumi (1972) and Kemerer (1975) for P F K from E. coll. An explanation might be, that ADP in lower concentrations binds to a "high-affinity ADP site", which might also be the site for AMP and cAMP, the effect of which is a deinhibition, and that ADP at higher concentrations effectively competes with ATP for the catalytical ATP-site. Fructose-l,6-diphosphate has been shown to be a very potent activator for mammalian P F K (Mansour, 1972; Kemerer, 1975; Uyeda, 1979), but without effect on P F K from E. coli and from many plant sources (Bloxham & Lardy, 1973). In the present paper it was found that F D P was without effect of the flounder liver enzyme. Also creatinphosphate (Fig. 8) was demonstrated to have no effect which is consistent with the general findings for other liver enzymes in contrast to the muscle enzymes, which are strongly inhibited by creatinphosphate (Tsai & Kemp, 1974; Hofmann, 1976). The inhibitory action of citrate was originally discovered by Garland et al. (1963) using rat heart PFK. Citrate is now accepted as a general potent inhibitor of mammalian PFK. Both citrate and PEP (Fig. 8) were found to be potent inhibitors of the flounder enzyme with concentrations giving half maximal inhibition of 0.23 and 1.8 mM respectively. In livers from mice (Herzberg, 1979) citrate inhibits 50% in a concentration of about 0.4mM, and Kemp (1971) reported that the concentration of citrate giving half maximal inhibition of P F K in muscle and liver from rabbit was 0.23 and 2 mM respectively. PEP inhibits 50% in rabbit muscle at a concentration of 2.5 mM, but is without effect on the liver enzyme. Citrate is neither an inhibitor for P F K in oyster adductor muscle (Storey, 1976) nor for P F K in other invertebrate tested (Walker & Bailey, 1969; Storey, unpublished data). The present paper shows that non sensitivity to citrate is not a general property for invertebrate PFK, as suggested by Storey (1976). In order to get a rough estimate as to the in vivo regulation of flounder liver P F K it is necessary to make some assumptions, which may not be valid. It is assumed that the enzyme in vivo has the same characteristics as those found for the purified enzyme, when assayed in high dilution at a temperature of 30°C. The concentrations of the nucleotides AMP, ADP and ATP are determined in the flounder liver (Sand, not published), according to the methods in Bergmeyer (1974) to 0.2, 0.4 and 1.5/tmoi/g liver respectively. These values are in good agreement with these given by Jorgensen (1980) for flounder liver. The values for AMP and ADP are close to these given for rat and mouse liver (Bergmeyer, 1974), but the concentration of ATP is only about 50% of that for rat and mouse liver. Beis (1975) has reported the concentration of different metabolites in muscles of invertebrates. The
AMP-level reported is 0.02-0.05 #mol/g a value about 10 times smaller than that found in flounder liver, and the concentration of F-6-P is 0.01-0.2/~mol/g, in which range also falls the concentration of F-6-P in mammals liver (Bergmeyer, 1974). No value for citrate is given, but in mammalian liver the concentration is about 0.20#mol/g (Bergmeyer, 1974). If it is assumed that 50% of the wet weight is intracellular water and that no compartmentation exists, then the concentration of ATP and F-6-P in the flounder liver is about 3 and 0.2 mM respectively. At pH 6.9, a value determined in flounder liver homogenized in 250mM sucrose (Sand, not published), P F K may be considered as completely inhibited, when no effector is present. In the presence of about 0.4 mM AMP and 0.4 mM citrate, effectors which for flounder liver PFK are supposed to be the most potent deinhibitor and inhibitor present at their respective concentrations, it is tempting to consider P F K as fairly active, due to the unusually high level of AMP present. But to be more conclusive, it is necessary to assay the enzyme by mixing the effectors in various combinations at levels, which represent values between the highest and lowest level likely to be encountered in the cell. Such studies have been performed on rat liver P F K by Reinhart (1980). He found that the enzyme activity at cellular concentrations of substrates and effectors was only a small percentage of that totally available--a situation opposite to that preliminary found for flounder liver. In conclusion it may be stated that flounder liver P F K behaves with regards to regulatory properties as P F K from mammalian sources. Especially noteworthy is, however, its lack of responsiveness to F D P and its high sensitivity to AMP, the latter of which is more characteristic for a muscle enzyme than for a liver enzyme. Acknowledgement--I should like to thank Jette Clemmensen for skilful technical assistance.
REFERENCES BEISI. & NEWSHOLMEE. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscle from vertebrates and invertebrates. Biochem. J. 152, 23-32. BERGMEYERH. V. & BERNTE. (1974) Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.). Verlag Chemie GmbH. BLOXHAM D. P. & LARDY H. A. (1973) Phosphofructokinase. In The Enzymes, Vol. VIII (Edited by BORER P.), 3rd edn, pp. 239-278. Academic Press, New York. BRAND I. A. & SOLING H. D. (1974) Rat liver phosphofructokinase. Purification and characterisation of its reaction mechanism. J. biol. Chem. 249, 7824-7831. BROCKD. J. H. (1969) Purification and properties of sheep liver phosphofructokinase. Biochem. J. 113, 235-242. DOELLE H. W. (1972) Kinetic characteristics of phosphofructokinase from Lactobacillus Casei Vat Rhamnosus ATCC 7469 and Lactobacillus Plantarum ATCC 14917. Biochim. biophys. Acta 258, 404-410. DUNAWAV(3. A. & WEBER(3. (1974) Rat liver phosphofructokinase isozymes. Archs Biochem. Biophys. 162, 620-628.
Kinetic properties of flounder's PFK FERDINANDUSJ. & CLARKJ. B. (1969) The phosphofructokinase from Arthrobacter crystallopoites. Biochem. J. 113, 735-736. GARLAND P. B. et al. (1963) Citrate as an intermediate in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate diabetes and starvation. Nature 200, 169-170. HERZnERG G. R. (1979) Purification and properties of liver phosphofructokinase from normal and obese mice. Int. J. Biochem. 10, 803-806. HILL A. V. (1910) The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol. 40, 4p. HOFMANE. (1976) The significance of pbosphofructokinase to the regulation of carbohydrate metabolism. In Reviews of Physiology, Biochemistry and Pharmacology, Vol. 75, pp. 1-68. Springer, Berlin. JOHNSONC. S. et al. (1976) Viscometric comparison of the asymmetry properties of phosphofructokinases from pig kidney and rabbit muscle. Biochem. biophys. Res. Commun. 73, 391-395. JORGENSENJ. B. & MUSTAFAT. (1980) The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys Flesus L.). High energy phosphate compounds and their role of glycolytic and gluconeogenetic enzymes. Comp. Biochem. Physiol. 67, 243-256. KEMERER V. F. et al. (1975) Phosphofructokinase from E. coll. In Methods in Enzymology, Vol. XLII (Edited by WOOD W. A.), pp. 91-98. Academic Press, New York. KEMP R. G. (1971) Rabbit liver phosphofructokinase. Comparison of some properties with those of muscle phosphofructokinase. J. biol. Chem. 246, 245-252. KONO N. & UYEDAK. (1973) Chicken liver phosphofructokinase, d. biol. Chem. 248, 8603-8609. LESlCKI A. (1980) The regulation of phosphofructokinase activity in hepatopancreas and abdominal muscle of the crayfish Orconectes Limosces Raf. (crustacea: decapoda). Comp. Biochem. Physiol. 66B, 291-296. MANSOUR T. E. (1972) Phosphofructokinase. In Current Topics in Cellular Regulation, Vol. V (Edited by HORECKER B. L. & STADTMANE. R.), pp. 146. Academic Press, New York.
c.8.P. 69/3B
l
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MASSEYT, H. & DEAL W. C. Jr (1973) Unusual, metabolite-dependent solubility properties of phosphofructokinase. J. biol. Chem. 248, 56--62. MASSEY T. H. & DEAL W. C. Jr (1975) Phosphofructokinases from porcine liver and kidney and from other mammalian tissues. In Methods in Enzymology, Vol. XLII (Edited by WOOD W. A.), pp. 99-110. Academic Press, New York. MATrnEYSE M. E. et al. (1979) Purification and properties of monkey liver phosphofructokinase. Int. J. Biochem. 10, 295-302. RAMAIAH A. & TEJWANI G. A. (1973) Interconvertible forms of phosphofructokinase from rabbit liver. The role of effectors on the interconversion. Eur. J. Biochem. 39, 183-192. REINHARTG. D. & LARDYH. A. (1980) Rat liver phosphofructokinase. Kinetic activity under near-physiological conditions. Biochemistry 19, 1477-1484. SAND O. (1980) Purification and properties of phosphofructokinase from liver of the flounder (Platichtys Flesus L.). Comp. Biochem. Physiol. 68, 77-81. STOREYK. B. (1976) Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. Eur. J. Biochem. 70, 331-337. SUMIT. & UI M. (1972) Kinetic studies on phosphofructokinase from Erlich Ascites Tumor cells. Biochim. biophys. Acta 276, 19-30. TSAI M. Y. & KEMP R. G. (1974) Rabbit brain phosphofructokinase. Comparison of regulatory properties with these of other phosphofructokinase isozymes. J. biol. Chem. 249, 6590-6596. UNDERWOODA. H. & NEWSHOLMEE. A. (1965) Properties of phosphofructokinase from rabbit liver and their relation to the control of glycolysis and gluconeogenesis. Biochem. J. 95, 868-875. UYEDA K. (1979) Phosphofructokinase. In Advances in Enzymology, Vol. 48 (Edited by MEiSTER A.), pp. 193-244. Wiley, New York. WALKER P. g.- BAILEY I. (1969) A comparison of the properties of the phosphofructokinase of the fat body and flight muscle of the adult male desert locust. Biochem. J. 111, 365-371.
0305-0491/81/070435.09502.00/0 Copyright © 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
KINETIC PROPERTIES OF PHOSPHOFRUCTOKINASE FROM THE FLOUNDER (PLATICHTYS FLESUS L.) OLE SAND Institute of Biochemistry, Odense Universitet Campusvej 55, 5230 Odense M, Denmark
(Received 25 September 1980) Abstract--1. Phosphofructokinase purified from flounder liver is characterized by sigmoidal kinetics with fructose 6-phosphate as substrate. 2, The enzyme is inhibited at higher ATP levels at both pH 6.9 and 8.2. The inhibition is more pronounced at pH 6.9. 3. This inhibition can be relieved by inorganic phosphate, fructose 6-phosphate, AMP and cyclic AMP. 4. In lower concentrations ADP acts as a deinhibitor, but in higher concentrations the effect is vanishing. 5. Citrate and phosphoenolpyruvate are inhibitors. 6. Fructose-l,6-diphosphate and creatinphosphate are without effect. 7. With increasing Mg 2+ level hyperbolic response curves are found with low ATP concentrations. With high ATP levels biphasic curves are demonstrated. 8. The enzyme behaves like mammalian phosphofructokinases but show some quantitative and qualitative differences.
INTRODUCTION
properties of phosphofructokinase purified from various sources, those of a fish phosphofructokinase have not hitherto been described. In a previous paper (Sand, 1981) dealing with the purification of phosphofructokinase from the flounder liver, its mol. wt was determined to 250,000, a value which falls between the minimum tool. wt of phosphofructokinase catalytically active from bacteria (mol. wt 140,000) and from mammalian species (tool. wt 320,000). With respect to pH response the flounder liver enzyme behaves like mammalian phosphofructokinase, showing maximal activity at pH 8.2. Addition of phosphate or sulphate stimulated the enzyme pronounced at pH 6.9, but only to some degree at pH 8.2 when the concentration of ATP was 1 mM and that of F-6-P was 2 raM. In the present paper further kinetic studies on the purified flounder liver enzyme are undertaken. These are performed at pH 6.9 and 8.2, and the results are compared with those of other phosphofructokinase enzymes that have been studied.
Phosphofructokinase (ATP: D-fructose 6-P-l-phosphotransferase, EC 2.7.1.11, PFK) is an important regulatory enzyme of carbohydrate metabolism and as such has been the subject of intensive investigation (for a recent review, see Uyeda, 1979). It is an allosteric enzyme subjected to a complex regulation by various metabolites, i.e. ATP, AMP, phosphate and citrate. The majority of the work has been done on phosphofructokinase isolated from primarily glycolytic cells, such as rabbit muscle. Phosphofructokinase isolated from livers has been found to have altered characteristics, which also was to be expected due to the gluconeogenesis in these cells. Phosphofructokinase isolated from livers include rabbit (Kemp, 1971; Ramaiah & Tejwani, 1973), pig (Massey & Deal, 1973), chicken (Kono & Uyeda, 1973), sheep (Brock, 1969), rat (Underwood & Newsholme, 1965; Brand & S61ing, 1974; Dunaway & Weber, 1974), monkey (Mattheyse, 1979) and mouse (Herzberg~ 1979). The number of effectors that influence phosphoMETHODS fructokinase and the extent of inhibition or activation vary, depending on the source of the enzyme. For Preparation of PFK example, only a few metabolites influence bacterial The enzyme is purified and diluted for measurements as phosphofructokinase, while the enzyme from mam- described by Sand (1980). The purified enzyme did not mals is affected by numerous effectors (Bloxham & contain the following enzymes, which otherwise might Lardy, 1973). interfere with the measurements: fructose-l,6-diphosphaIn respect to pH response, mammals and E. coli tase, adenylate kinase, NADH oxidases and ATPases. phosphofructokinase are oppositely influenced in their allosteric properties. In enzymes isolated from Assay of PFK-activity PFK was assayed at 30°C by following the appearance mammals an increase in pH from 7-8 causes a shift of the signoidal F-6-P velocity curve to the left and a of fructose-l,6-diphosphate (FDP) in a coupled assay system. All auxiliary enzymes are extensively dialyzed relief of the inhibitory action of ATP, whereas in the before use to get rid of ammoniumsulphate, which is an E. coil enzyme the opposite is found (Bloxham & activator for PFK. In the experiment where the influence Lardy, 1973; Uyeda, 1979). of FDP was investigated, PFK was, however, assayed by Despite the large number of reports of the kinetic monitoring the formation of ADP. 435
436
OLE SAND
In the former system the assay mixture contained a total vol of 810~1: 67mM potassium glycylglycine buffer, 0.14 mM NADH, 0.05 mg bovine serum albumin, 0.12 mM dithiothreitol, 0.6 U aldolase, 0.6 U glycerol 3-phosphate dehydrogenase, 1.0 U triosephosphate isomerase and ATP, F-6-P and MgCI2 as indicated for each experiment. The value of pH in the assay is also stated in each experiment. In the experiment where PFK activity is assayed by following the formation of ADP, the cuvette contained in a total vol. of 810#1: 67mM potassium glycylglycine buffer, pH 6.9, 0.14 mM NADH, 0.05 mg bovine serum albumin, 0.12 mM dithiothreitol, 1.0 U lactat dehydrogenase, 1.0 U pyruvate kinase, 2.0 mM F-6-P, 0.90 mM ATP, 1.30 mM MgC12 and 0.50 mM phosphoenolpyruvate (PEP). Most assays performed were non-linear with respect to time during the first minutes showing accelerating or decelerating rates, depending on the order of addition of various components to the assay, before a constant velocity was attained. This constant velocity, the steady-state rate, is used in the present paper. The steady-state rate obtained was not the same one, irrespectively of the addition of components, for the same assay mixture. It was decided to start the reaction by addition of 50 #1 containing a suitable amount of PFK preparation after allowing 4 min for temperature equilibration. An amount of PFK was added to each assay which would give a change in absorbance at 340 nm of 0.08 per rain in the "normal assay", which is the assay mixture monitoring the formation of FDP described above, but supplied with 10 mM ammoniumsulphate. The concentration of ATP, F-6-P and MgCI2 in this "normal assay" are respectively 1.30 mM, 1.90 mM and 1.30 mM and pH is 8.2. This velocity of reaction is defined as V,. The activities are expressed as viVa, where v represents the steady-state rate measured under the conditions of the experiment. This is done to allow for comparison of effects in preparations, which have different final specific activities. RESULTS
A variety of metabolites and inorganic ions has been shown to affect the activity of phosphofructokinase. Figure 1 shows the effect of increasing concentrations of sodium phosphate on the activity of flounder liver P F K at pH 6.9 and 8.2 in the presence of 0.5 m M F-6-P. The measurements are made at two fixed concentrations of ATP of 0.3 m M and 1.0 m M respectively. As seen from the figures, phosphate stimulates, but the stimulatory effect is very much dependent upon the concentration of A T P and on pH. At pH 6.9 and 0.3 m M ATP a hyperbolic curve is obtained with a concentration of phosphate giving half maximal activity at 0.4 mM. By increasing the concentration of A T P to 1 mM, which is an inhibitory
1.0 oO/0~0 -o-
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Fig. 1. Phosphate dependence of phosphofructoklnase activity. An amount of enzyme was added to each assay that would give a change in absorbance at 340 nm of 0.08 per min in the "normal assay mixture". This is defined as Vt. The experimental assay is as described in Methods. The concentration of F-6-P is 0.50 mM. A, 0.3 mM ATP and A, 1.0mM ATP, pH6.9; O, 0.3mM ATP and O, 1.0raM ATP, pH 8.2. A T P concentration (Fig. 2A), a sigmoid curve is obtained with an apparent K,, for phosphate of 4 mM. Figure 1 also indicates that increasing phosphate concentrations counteract the inhibitory effect of 1 m M ATP. At pH 8.2 phosphate only has a slightly stimulatory effect at the low ATP concentration. Under these conditions 0.5 m M F-6-P has nearly abolished the inhibitory effect of 0.3 m M ATP (Fig. 3A). At an A T P concentration of 1 m M the enzyme is very susceptible to phosphate with an apparent K,, for phosphate of 0.2 mM. (Fig. 1) The stimulatory effect on the enzyme upon the addition of sodium phosphate is due to phosphate and not caused by N a ÷. Addition of NaC1 in the range 0-20 m M does not influence the enzyme activity. In addition to their function as substrates, F-6-P and ATP act under definite conditions as effectors on PFK, since F-6-P may relieve the ATP inhibition and ATP influences shape and position of the Fo6-P saturation curve. This is shown in Fig. 2 and Fig. 3. Figure 2A and 2B show the effect of varying the concentration of A T P at pH 6.9 at two fixed levels of F-6-P in the absence and presence of 12 m M sodium phosphate. This concentration of phosphate has been found to stimulate the enzyme maximally under the conditions indicated in Fig. 1. In the low region of ATP concentration, phosphate has no effect. Higher concentrations of A T P inhibits PFK, when phosphate is not present. Addition of 12 m M phosphate relieves
Table 1. The dependence of apparent K , and apparent K~ values for ATP on pH, F-6-P level and presence of phosphate
pH
mM phosphate added
6.9 6.9 8.2 8.2
0 12 0 12
ATP K,, (mM) 0.5 mM 2.0 mM F-6-P F-6-P 0.020 0.040 0.040 0.060
0.025 0.040 0.080 0.080
ATP K~ (mM) 0.5 mM 2.0 mM F-6-P F-6-P 0.22 1.45 0.45 >3
0.70 >3 1.3 >3
Kinetic properties of flounder's PFK
0.8
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Fig, 2. Effect of ATP concentration on phosphofructokinase activity at pH 6.9. Assay conditions as in Fig. 1. The concentration of F-6-P is 0.50 mM (A) and 2.0 mM (B). A, without phosphate added. O, 12 mM phosphate added.
the inhibitory effect of ATP at moderate ATP concentrations. At 0.5 mM F-6-P addition of 12 mM phosphate increases the apparent Ki for ATP from 0.22 to 1.45 mM (Table 1). In the absence of phosphate the apparent Kl for ATP is increased from 0.22 to 0.70 mM, when the F-6-P concentration is changed from 0.5 to 2.0 mM. Table 1 summarizes the values of the apparent K , for ATP measured at pH 6.9 derived from the curves in Fig. 2A and 2B. Changing the F-6-P concentration from 0.5 to 2.0 mM does not influence the value of K , . Addition of 12 mM phosphate increases Km with a factor of about 2 at both F-6-P concentrations. This
increase in K,, is a consequence of the fact that addition of phosphate increases the enzyme activity, but only when the concentration of ATP is inhibitory. Figures 3A and 3B show the effect of ATP at pH 8.2 at two fixed levels of F-6-P in the presence and absence of 12 mM sodium phosphate. In the lower range of ATP-concentration, phosphate has no effect. When phosphate is absent, the hyperbolic saturation curve is followed by a steep inhibition curve as the concentration of ATP is increased. The apparent Ki for ATP changes from 0.45 to 1.3 mM, when the F-6-P concentration changes from 0.5 to 2.0mM. (Table 1.) In the presence of phosphate the activity
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Fig. 3. Effect of ATP concentration on phosphofructokinase activity at pH 8.2. Assay conditions as in Fig. 1. The concentration of F-6-P is 0.50 mM (A) and 2.0 mM (B). A, without phosphate added. O, 12 mM phosphate added.
438
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Fig. 4. Effect of F-6-P concentration on phosphofructokinase activity at pH 6.9. At 0.3 mM ATP (A) and 1.0 mM (B), in the absence (A) and presence (O) of 12 mM phosphate. Assay conditions as in Fig. 1.
Fig. 5. Effect of F-6-P concentration of phosphofructokinase activity at pH 8.2 at 0.3 mM ATP (A) and 1.0 mM (B) in the absence (A) and presence (O) of 12 mM phosphate. Assay conditions as in Fig. 1.
curves reaches almost a plateau of maximal activity before inhibition is seen. Table I shows the values of K,, for ATP measured at pH 8.2 determined from the curves in Figs 3A and 3B. The addition of phosphate does not influence the apparent Km measured in the presence of 2 m M F-6-P, but at 0.5 m M F-6-P, K , is increased by 50%. When the F-6-P-concentration is increased from 0.5 to 2 m M in the presence or absence of phosphate the corresponding Km values for ATP are slightly increased. By comparing the curves in Figs 2 and 3, it is evident that the degree of inhibition by a fixed concentration of ATP, as well as the ATP concentration at which inhibition is first observed, is a function of pH, F-6-P concentration and phosphate concentration. Elevated pH, the presence of phosphate or increased F-6-P concentration increases the activity of P F K at
inhibitory concentrations of ATP, as well as delaying the onset of ATP inhibition. When the activity was determined at constant levels of ATP with varying levels of F-6-P in the absence of phosphate, sigmoid response curves typical of allosteric enzymes were observed at both pH 6.9 and 8.2. Addition of 12 m M phosphate abolished the sigrnoidicity leading to saturation curves steeper than a rectangular hyperbola. This is shown in Figs 4 and 5. Figures 4A and 4B show the results obtained at pH 6.9. Another effect of adding phosphate is an increase in the maximal activity above that obtainable with increasing F-6-P concentration in the absence of phosphate. Therefore F-6-P does not stimulate the enzyme to the same degree as phosphate does at pH 6.9. Table 2 shows the apparent K,, for F-6-P in the absence and presence of 12mM phosphate at
Table 2. The dependence of apparent K,, for F-6-P and Hill coefficient (nn) for F-6-P binding on pH, ATP level and presence of phosphate
pH
mM phosphate added
6.9 6.9 8.2 8.2
0 12 0 12
F-6-P K,n (mM) 0.3 mM 1.0 mM ATP ATP 0.95 0.080 0.36 0.085
3.80 0.32 1.24 0.16
nn
0.3 mM ATP
1.0 mM ATP
3.0 1.5 3.1 1.4
3.7 2.3 6.4 1.7
Kinetic properties of flounder's PFK fixed levels of 0.3 and 1 mM respectively, measured at pH 6.9 and 8.2 in the absence and presence of 12 mM phosphate. By increasing the ATP level from 0.3 to 1 mM, KM for F-6-P is increased by a factor of 4. Addition of 12 mM phosphate decreases KM for F-6-P by a factor of about 10 at both ATP concentrations tested. Figures 5A and 5B show the results obtained by varying the F-6-P concentration at constant levels of ATP at pH 8.2 in the absence and presence of 12 mM phosphate. In contrast to the measurements carried out at pH 6.9 no change in Vmax was observed when phosphate was added. Addition of phosphate or decreasing the ATP concentration decreases the values of Kin. (Table 2) When the curves in Figs 4A, 4B, 5A and 5B are repiotted in terms of the Hill equation (Hill, 1910): log - -
v
= nn log S - log K
Vma x --
where S is the substrate concentration (F-6-P), K is a constant and Vmaxis the maximal activity obtained-derived from each curve, straight lines are obtained from which could be calculated the interaction coefficient n , , which is presumed to be determined by the number of interacting sites for F-6-P and the strength of interaction among them. The values for nn are given in Table 2. It can be seen that addition of phosphate decreases nn, an effect which is also obtained by decreasing the concentration of ATP. Changing pH from 6.9 to 8.2 does not influence the value of nH at the low ATP concentration (0.3 raM), whereas at the high ATP-concentration (1 raM) nn is increased in the absence of phosphate, but decreased in the presence of phosphate. It should be noted that an unusual high value of nH of 6.4 is found at 1 mM A T E when phosphate is absent at pH 8.2. Mg 2+ has been shown to be essential for PFK, as is also the case for other kinases, which as one of their natural substrates uses ATP. The effect of Mg 2 + upon flounder liver P F K was examined by assaying the enzyme at pH 6.9 and 8.2 at two different concentrations of ATP in the presence of 2 mM F-6-P. The experiments were performed in the absence and pres-
I.o
&
o2
B
o.
o/
O.e
~v,
ence of 12 mM phosphate. The results are given in Figs 6A a n d 6B. When Mg 2÷ is absent, no P F K activity is found. The enzyme activity increases with increasing level of Mg 2+' up to a certain value of Mg 2+ concentration. Beyond this value increasing MgCl2 inhibits the enzyme. Figure 6A shows that at pH 6.9 in the presence of phosphate there is a more pronounced stimulation of the enzyme with increasing MgCl2 up to about 0.7 mM MgCl2 with 0.3 mM ATP present than is the case with I mM ATP present. The apparent K= for MgCI2 at 0.3mM ATP is 0.1raM, and the apparent K= at l m M ATP is 0.2 raM. When the experiment with 0.3 mM ATP is performed without phosphate added, a completely different type of curve is obtained. There is a little but distinct peak of activity at 0.1 mM MgCI2. Increasing the concentration of MgC12 above 0.15raM the enzyme activity increases. The apparent Km for MgC12 in the presence of 0.3 mM ATP without phosphate added is 0.35 raM. Phosphate, therefore, seems to increase the affinity of the enzyme for MgCl2. At I mM ATP without phosphate added, no activity is found by increasing MgCI2 (not shown), which is to be expected due to the inhibitory ATP concentration. Figure 6B shows that at pH 8.2 in the presence of 12 mM phosphate MgCl2 concentrations up to about 0.3 mM stimulate the enzyme to the same extent irrespective.of the ATP-concentration used. K~, for MgCl2 at both ATP-concentrations is 0.07raM. When 12 mM phosphate is ommitted in the reaction mixture, no change in the pattern of the curve representing 0.3mM ATP is observed. With I mM ATP, which at pH 8.2 is an optimal concentration (Fig. 3B), a completely different type of curve is obtained, which has an appearence similar to the curve in Fig. 6A, representing 0.3 mM ATP at pH 6.9, without phosphate present. A little peak of activity is noted at 0.07 mM MgCl2, then there is a decrease in activity with increasing MgC12 up to 0.3 mM MgCl2. At higher MgC12 a sigmoidal curve is found. The effect of phosphate at pH 8.2 with 1 mM ATP is therefore a decrease in the apparent K= from 0.95 to 0.07 mM for MgCl2. Due to the sigmoidicity of the curve it might be concluded that there is cooperativity among the sites for MgCl2 binding, when phosphate is absent in
to
o8
°/~/
//
? ~/
OS
./
/
.~.
,~v,
/
~ /
o2
bT
/
/
'
mM M~I2
439
,/
~
MIICIa
Fig, 6. Effect of MgC12 concentration on phosphofructokinase activity in the presence of 2.0 mM F-6-P, at pH6.9 (A) and 8.2 (B). Q, 0.3mM ATP; O, 0.3raM ATP + 12mM phosphate; &, 1.0raM ATP; A, 1.0 mM ATP + 12 mM phosphate. Assay conditions as in Fig. 1.
440
OLE SAND
the high ATP-concentration. By comparing the curves in Figs 6A and 6B representing the same conditions except for pH, it is evident that Ks for MgC12 is higher at pH 6.9 than at pH 8.2. The adenine nucleotides, AMP, cyclic AMP and ADP interfere with the activity of most P F K investigated. In the present study it was found that they stimulated the enzyme, and that the stimulatory effect was dependent on pH and on the degree of inhibition of ATP. At non-inhibitory ATP-concentration, there was a slight stimulatory effect of AMP and cyclic AMP (not shown), whereas in the presence of inhibitory concentration of ATP the stimulatory effect was pronounced. Figure 7A shows the effect of adding AMP and cyclic AMP (cAMP) respectively to the enzyme at pH 6.9, when the concentrations of F-6-P and ATP were 2 and 0.80 mM respectively. Without addition of the mononucleotides these conditions ensure that the enzyme activity is 35% of its maximal value due to the inhibition of ATP (see Fig. 2B). The figure shows that the maximal activity obtained with AMP and cAMP was the same regardless of the kind of effector used. This value is significantly higher than that obtained in the absence of ¢ffectors at optimal ATP concentration (0.30 mM, see Fig. 2B), and is also higher than those obtained in the presence of saturating levels of F-6-P. The concentrations of AMP and cAMP increasing the enzyme activity to half its maximal value are 6 and 50gM respectively. As seen from the curves in Fig. 7A, the same kinetic pattern is essentially applying to AMP as well as to cAMP.
1.0
0~- -~
Q8
/
./
02
mM
A completely different saturation curve is found for ADP, a product of the PFK-reaction, under the same assay conditions (Fig. 7B) ADP is also a stimulator, but the stimulatory effect does not simply increase with the concentration. When the level of ADP is increased from 0 to 0.25 mM the enzyme activity is increased 4-fold with a concentration of ADP giving half maximal velocity of 60 #M. When the concentration of ADP is increased above 0.25 mM the stimulatory effect is decreasing. It was not possible even at an ADP-concentration of 10 mM to obtain the same activity or a lower one as that obtained with no ADP present. With optimal ADP-concentration (0.25 raM) the stimulatory effect was smaller than that obtainable with AMP and cAMP respectively. FDP, which is also a product of the reaction, is reported as an activator for mammalian P F K (Bloxham & Lardy, 1973). The effect of F D P on flounder liver P F K was assayed at pH 6.9 by measuring the formation of ADP in the assay system described in "Methods". The concentration of PEP was kept as low as 0.5 mM to minimize the inhibition caused by this agent. With this choice of assay condition the activity of P F K without F D P added was 1S~o of that obtainable in the presence of an optimal ATP-concentration. It turned out that addition of F D P up to a concentration of 5 mM was without any effect on the enzyme activity. It might therefore be concluded, that F D P does not function as an activator or deinhibitor of the enzyme. P F K from many sources are inhibited by phosphoenolpyruvate (PEP), creatin phosphate and citrate. The effect of these compounds was tested at pH 6.9 in the presence of 2 mM F-6-P and 0.50 mM ATP. Without effectors present this choice of condition resulted in an enzyme activity of 909/o of that obtainable with an optimal ATP concentration. Figure 8 shows that phosphoenolpyruvate and citrate were inhibitors, whereas creatin phosphate was without effect in concentrations up to 4 raM. Citrate was a powerful inhibitor with a concentration decreasing the enzyme activity to S0~o of 0.23 mM. The corresponding value of PEP was 1.8 mM. Both effectors inhibit the enzyme 100~o when present in excess. Because the experiments were run in the presence of a fixed and relatively low MgCl2 concentration, and it is well known that citrate chelates Mg 2÷, the
1.0 (IS O8
\
~
0.4
°~o
wVt
O,4
v/Vt
o
0.1 Oq mM
Fig. 7. A: Activation of ATP inhibited phosphofructokinase activity by AMP (O) and cyclic AMP (/\). B: Effect of ADP (D) on ATP inhibited phosphofructokinase activity. The expcrimems are performed at pH 6.9 at 2 mM F-6-P and 0.80 mM ATP. Assay conditions as in Fig. 1.
0
1
2
3
4
mM
Fig. 8. Inhibition of phosphofructokinase activity by citrate (r-l) and phosphoenolpyruvat {O). zX, Effect of phosphocreatin. The experiments are performed at pit 6.9 at 2 mM F-6-P and 0.50 mM ATP. Assay conditions as in Fig. 1.
Kinetic properties of flounder's PFK effect of 1 mM citrate upon the enzyme activity was also tested in the presence of increasing MgCI2 concentrations under the same conditions as those described for Fig. 8. It was found that the inhibitory action of 1 mM citrate could not be abolished by increasing the concentration of MgC12 (not shown), so it can be concluded that citrate inhibits the enzyme by interfering directly with it. DISCUSSION
In the present study, P F K from flounder liver was demonstrated to be regulated by a variety of effectors such as cAMP, AMP, ADP, citrate, PEP, inorganic phosphate, Mg 2÷ as well as by its substrates, F-6-P and ATP. Furthermore pH plays an important role in this regulation. Without Mg 2÷ present no activity was found consistent with the general proposal that M g ' A T P is substrate for PFK. When enzyme activity was measured as a function of the concentration of MgCIz it was found at both pH 6.9 and 8.2 that optimal enzyme activities were obtained when Mg2+/ATP was above 1 (Fig. 6). The simplest explanation for the rise in activity, observed by increasing Mg 2÷ above a Mg2+/ATP of 1 is that the enzyme is not fully saturated with substrate (Mg. ATP) until all of the ATP is effectively present as the Mg.ATP. There are two alternative explanations, namely: (1) that free ATP itself may be so highly inhibitory that a further rise in activity is not obtained before all ATP is bound by Mg 2+, or (2) that low concentrations of free Mg 2÷ may be necessary for enzyme activity (as with several kinases) so that full activity is not achieved until a saturating concentration of free metalion is present. More experiments should be done to distinguish between these alternatives. In the presence of inhibiting ATP concentrations without phosphate present biphasic curves were obtained (Fig. 6). Such responses have until now only been reported for P F K isolated from monkey liver (Mattheyse, 1979). The biphasic character was completely abolished by addition of phosphate. An explanation for this might be that Mg 2÷, besides being substrate, as Mg.ATP, furthermore favours the existence of a more active form of the enzyme, a form which is also favoured by the presence of phosphate. Sensitivity to inhibition of P F K at high levels of ATP differs significantly with the source of the enzyme. In general liver PFK is most strongly inhibited, muscle PFK is less sensitive and brain enzyme is least sensitive (Johnson et al., 1976; Tsai et al., 1974). PFK from a variety of bacteria are not inhibited by ATP (Ferdinandus et al., 1969; Doelle, 1972). P F K from flounder liver is found to be inhibited by ATP. The apparent inhibitor constants at pH 6.9 and 8.2 measured in the presence of 2 m M F-6-P without other effectors present are 0.7 and 1.3 mM respectively (Table 1). The corresponding values for sheep liver (Brock, 1969) measured under comparable circumstances are 1.7mM and 3.4mM respectively. From the paper of Mattheyse (1979) reporting the kinetics of P F K purified from monkey liver, an apparent inhibitor constant for ATP at pH 7.0 with 2 mM
441
F-6-P present without effector present can be derived from the figures to 0.8 raM. In the presence of a saturating level of the deinhibitor cAMP, the apparent Ki is increased to 2.4 raM. In the present paper a value above 3mM is found when 12mM inorganic phosphate is present. When assaying the activity in the presence of 0.5 mM and 2.0 mM F-6-P at pH 6.9 and 8.2 (Table 1), an apparent Michaelis constant (Kin) for ATP was found in the range 0.020-0.080 mM and this value was not greatly affected by 12mM phosphate. This effector did not affect the catalytical part of the ATP saturation curve, but only exerted its effect by relieving the inhibition at high levels of ATP. Massey & Deal (1975) found a Km for ATP at pH 7.0 of 0.025 mM when no effector was present. For monkey liver, Mattheyse (1979) reports K , for ATP at pH 7.0 and 8.0 in the same concentration range as that found for flounder liver. The kinetics of flounder liver P F K with respect to F-6-P were however found to be affected significantly by pH, ATP level and presence of inorganic phosphate. Plots of steady-state velocity vs F-6-P levels at different ATP concentrations at pH 6.9 and 8.2 gave S-formed curves indicating allosteric kinetics with respect to F-6-P (Figs 4 and 5). A change in pH from 6.9 to 8.2, presence of phosphate or decreased ATP level all caused a shift of the sigmoidal F-6-P activity curve to the left giving rise to smaller values of the apparent Kin. When pH is changed from 6.9 to 8.2, K~ for F-6-P with 0.3 mM ATP present is changed from 0.95 to 0.36 mM respectively. These values are close to those reported for monkey liver (Mattheyse, 1979), measured under comparable conditions, of 0.60 and 0.23raM respectively, but higher than those reported for pig liver (Massey & Deal, 1975) and rabbit liver (Kemp, 1971), which, however, were measured in the presence of ammonium sulphate known to increase the affinity of PFK to F-6-P. Values for nn, the Hill coefficient (Table 2), greater than 1 were found. In those cases, where phosphate was not present, unusual high values of nn were derived indicating a high degree of interaction. Presence of phosphate or decreasing ATP level resulted in decreasing nH values. This might be a consequence of a dissociation of the polymeric enzyme or conformational changes. In a recent paper concerning the kinetics of rat liver phosphofructokinase (Reinhart & Lardy, 1980) Hill coefficients as high as 5.2 were found, when steady-state activity, attained after completion of enzyme transient was taken as a function of the F-6-P level. Inorganic phosphate, F-6-P, AMP and cAMP were found to stimulate the flounder liver P F K when ATP was present in inhibitory concentrations (Figs 1, 4, 5 and 7). The stimulatory effect can be explained as a deinhibition of the effect of ATP. The most powerful deinhibitors demonstrated of the compounds tested was AMP (Fig. 7A), which at a concentration of 6 #M stimulated the enzyme to 50~o of that obtainable under conditions where the enzyme was inhibited 65Yo without AMP present. The corresponding values for AMP given for mouse liver (Herzberg, 1979), sheep liver (Kemp, 1971) and for hepatopancreas of the crayfish (Lesicki, 1980) are 35, 210 and 10-15 #M respectively. The concentration of cAMP giving half maximal ac-
442
OL~ SANO
tivity for flounder liver P F K (Fig. 7A) was found to be 50 #M, a value close to that of 75 #M found for rabbit liver P F K (Kemp, 1971). ADP, phosphate and F-6-P were not able at any level to deinhibit the enzyme to the same degree as did cAMP and AMP. It was demonstrated, that ADP (Fig. 7B) deinhibited the enzyme when present in a certain concentration range. In the presence of higher ADP levels the deinhibition was decreasing. This effect of ADP has also been published by Sumi (1972) and Kemerer (1975) for P F K from E. coll. An explanation might be, that ADP in lower concentrations binds to a "high-affinity ADP site", which might also be the site for AMP and cAMP, the effect of which is a deinhibition, and that ADP at higher concentrations effectively competes with ATP for the catalytical ATP-site. Fructose-l,6-diphosphate has been shown to be a very potent activator for mammalian P F K (Mansour, 1972; Kemerer, 1975; Uyeda, 1979), but without effect on P F K from E. coli and from many plant sources (Bloxham & Lardy, 1973). In the present paper it was found that F D P was without effect of the flounder liver enzyme. Also creatinphosphate (Fig. 8) was demonstrated to have no effect which is consistent with the general findings for other liver enzymes in contrast to the muscle enzymes, which are strongly inhibited by creatinphosphate (Tsai & Kemp, 1974; Hofmann, 1976). The inhibitory action of citrate was originally discovered by Garland et al. (1963) using rat heart PFK. Citrate is now accepted as a general potent inhibitor of mammalian PFK. Both citrate and PEP (Fig. 8) were found to be potent inhibitors of the flounder enzyme with concentrations giving half maximal inhibition of 0.23 and 1.8 mM respectively. In livers from mice (Herzberg, 1979) citrate inhibits 50% in a concentration of about 0.4mM, and Kemp (1971) reported that the concentration of citrate giving half maximal inhibition of P F K in muscle and liver from rabbit was 0.23 and 2 mM respectively. PEP inhibits 50% in rabbit muscle at a concentration of 2.5 mM, but is without effect on the liver enzyme. Citrate is neither an inhibitor for P F K in oyster adductor muscle (Storey, 1976) nor for P F K in other invertebrate tested (Walker & Bailey, 1969; Storey, unpublished data). The present paper shows that non sensitivity to citrate is not a general property for invertebrate PFK, as suggested by Storey (1976). In order to get a rough estimate as to the in vivo regulation of flounder liver P F K it is necessary to make some assumptions, which may not be valid. It is assumed that the enzyme in vivo has the same characteristics as those found for the purified enzyme, when assayed in high dilution at a temperature of 30°C. The concentrations of the nucleotides AMP, ADP and ATP are determined in the flounder liver (Sand, not published), according to the methods in Bergmeyer (1974) to 0.2, 0.4 and 1.5/tmoi/g liver respectively. These values are in good agreement with these given by Jorgensen (1980) for flounder liver. The values for AMP and ADP are close to these given for rat and mouse liver (Bergmeyer, 1974), but the concentration of ATP is only about 50% of that for rat and mouse liver. Beis (1975) has reported the concentration of different metabolites in muscles of invertebrates. The
AMP-level reported is 0.02-0.05 #mol/g a value about 10 times smaller than that found in flounder liver, and the concentration of F-6-P is 0.01-0.2/~mol/g, in which range also falls the concentration of F-6-P in mammals liver (Bergmeyer, 1974). No value for citrate is given, but in mammalian liver the concentration is about 0.20#mol/g (Bergmeyer, 1974). If it is assumed that 50% of the wet weight is intracellular water and that no compartmentation exists, then the concentration of ATP and F-6-P in the flounder liver is about 3 and 0.2 mM respectively. At pH 6.9, a value determined in flounder liver homogenized in 250mM sucrose (Sand, not published), P F K may be considered as completely inhibited, when no effector is present. In the presence of about 0.4 mM AMP and 0.4 mM citrate, effectors which for flounder liver PFK are supposed to be the most potent deinhibitor and inhibitor present at their respective concentrations, it is tempting to consider P F K as fairly active, due to the unusually high level of AMP present. But to be more conclusive, it is necessary to assay the enzyme by mixing the effectors in various combinations at levels, which represent values between the highest and lowest level likely to be encountered in the cell. Such studies have been performed on rat liver P F K by Reinhart (1980). He found that the enzyme activity at cellular concentrations of substrates and effectors was only a small percentage of that totally available--a situation opposite to that preliminary found for flounder liver. In conclusion it may be stated that flounder liver P F K behaves with regards to regulatory properties as P F K from mammalian sources. Especially noteworthy is, however, its lack of responsiveness to F D P and its high sensitivity to AMP, the latter of which is more characteristic for a muscle enzyme than for a liver enzyme. Acknowledgement--I should like to thank Jette Clemmensen for skilful technical assistance.
REFERENCES BEISI. & NEWSHOLMEE. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscle from vertebrates and invertebrates. Biochem. J. 152, 23-32. BERGMEYERH. V. & BERNTE. (1974) Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.). Verlag Chemie GmbH. BLOXHAM D. P. & LARDY H. A. (1973) Phosphofructokinase. In The Enzymes, Vol. VIII (Edited by BORER P.), 3rd edn, pp. 239-278. Academic Press, New York. BRAND I. A. & SOLING H. D. (1974) Rat liver phosphofructokinase. Purification and characterisation of its reaction mechanism. J. biol. Chem. 249, 7824-7831. BROCKD. J. H. (1969) Purification and properties of sheep liver phosphofructokinase. Biochem. J. 113, 235-242. DOELLE H. W. (1972) Kinetic characteristics of phosphofructokinase from Lactobacillus Casei Vat Rhamnosus ATCC 7469 and Lactobacillus Plantarum ATCC 14917. Biochim. biophys. Acta 258, 404-410. DUNAWAV(3. A. & WEBER(3. (1974) Rat liver phosphofructokinase isozymes. Archs Biochem. Biophys. 162, 620-628.
Kinetic properties of flounder's PFK FERDINANDUSJ. & CLARKJ. B. (1969) The phosphofructokinase from Arthrobacter crystallopoites. Biochem. J. 113, 735-736. GARLAND P. B. et al. (1963) Citrate as an intermediate in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate diabetes and starvation. Nature 200, 169-170. HERZnERG G. R. (1979) Purification and properties of liver phosphofructokinase from normal and obese mice. Int. J. Biochem. 10, 803-806. HILL A. V. (1910) The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol. 40, 4p. HOFMANE. (1976) The significance of pbosphofructokinase to the regulation of carbohydrate metabolism. In Reviews of Physiology, Biochemistry and Pharmacology, Vol. 75, pp. 1-68. Springer, Berlin. JOHNSONC. S. et al. (1976) Viscometric comparison of the asymmetry properties of phosphofructokinases from pig kidney and rabbit muscle. Biochem. biophys. Res. Commun. 73, 391-395. JORGENSENJ. B. & MUSTAFAT. (1980) The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys Flesus L.). High energy phosphate compounds and their role of glycolytic and gluconeogenetic enzymes. Comp. Biochem. Physiol. 67, 243-256. KEMERER V. F. et al. (1975) Phosphofructokinase from E. coll. In Methods in Enzymology, Vol. XLII (Edited by WOOD W. A.), pp. 91-98. Academic Press, New York. KEMP R. G. (1971) Rabbit liver phosphofructokinase. Comparison of some properties with those of muscle phosphofructokinase. J. biol. Chem. 246, 245-252. KONO N. & UYEDAK. (1973) Chicken liver phosphofructokinase, d. biol. Chem. 248, 8603-8609. LESlCKI A. (1980) The regulation of phosphofructokinase activity in hepatopancreas and abdominal muscle of the crayfish Orconectes Limosces Raf. (crustacea: decapoda). Comp. Biochem. Physiol. 66B, 291-296. MANSOUR T. E. (1972) Phosphofructokinase. In Current Topics in Cellular Regulation, Vol. V (Edited by HORECKER B. L. & STADTMANE. R.), pp. 146. Academic Press, New York.
c.8.P. 69/3B
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MASSEYT, H. & DEAL W. C. Jr (1973) Unusual, metabolite-dependent solubility properties of phosphofructokinase. J. biol. Chem. 248, 56--62. MASSEY T. H. & DEAL W. C. Jr (1975) Phosphofructokinases from porcine liver and kidney and from other mammalian tissues. In Methods in Enzymology, Vol. XLII (Edited by WOOD W. A.), pp. 99-110. Academic Press, New York. MATrnEYSE M. E. et al. (1979) Purification and properties of monkey liver phosphofructokinase. Int. J. Biochem. 10, 295-302. RAMAIAH A. & TEJWANI G. A. (1973) Interconvertible forms of phosphofructokinase from rabbit liver. The role of effectors on the interconversion. Eur. J. Biochem. 39, 183-192. REINHARTG. D. & LARDYH. A. (1980) Rat liver phosphofructokinase. Kinetic activity under near-physiological conditions. Biochemistry 19, 1477-1484. SAND O. (1980) Purification and properties of phosphofructokinase from liver of the flounder (Platichtys Flesus L.). Comp. Biochem. Physiol. 68, 77-81. STOREYK. B. (1976) Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. Eur. J. Biochem. 70, 331-337. SUMIT. & UI M. (1972) Kinetic studies on phosphofructokinase from Erlich Ascites Tumor cells. Biochim. biophys. Acta 276, 19-30. TSAI M. Y. & KEMP R. G. (1974) Rabbit brain phosphofructokinase. Comparison of regulatory properties with these of other phosphofructokinase isozymes. J. biol. Chem. 249, 6590-6596. UNDERWOODA. H. & NEWSHOLMEE. A. (1965) Properties of phosphofructokinase from rabbit liver and their relation to the control of glycolysis and gluconeogenesis. Biochem. J. 95, 868-875. UYEDA K. (1979) Phosphofructokinase. In Advances in Enzymology, Vol. 48 (Edited by MEiSTER A.), pp. 193-244. Wiley, New York. WALKER P. g.- BAILEY I. (1969) A comparison of the properties of the phosphofructokinase of the fat body and flight muscle of the adult male desert locust. Biochem. J. 111, 365-371.