30 T I B S - February 1978

tions relating to myosin phosphorylation. Among the most prominent ones are:

(13 How are the kinase and phosphatase regulated? This is of particular relevance to cells containing a Ca2+-independent kinase. Is there a mechanism for convert- ing a Ca 2 +-insensitive kinase into a Ca 2 +- sensitive one? Cyclic nucleotides represent another potential control. Although cAMP does not appear to alter kinase activity [9,13] a possible effect of cyclic G M P on the kinase or phosphatase has not been ruled out.

(2) How does phosphorylat ion alter the kinetics of actin-myosin interaction? Ex- periments using enzymatically active pro- teolytic fragments of myosin in the phos- phorylated and unphosphorylated state should permit a detailed kinetic analysis of this interaction. These fragments, which can be prepared with and without the P- light chain should help resolve another question: does this light chain act as an inhibitor ofact in-myosin interaction in the unphosphorylated state and does phos- phorylation work by relieving this inhibi- tion. If this were so, an enzymatically active fragment of myosin which contained no P-light chain should be activated by aetin even though phosphorylat ion was not possible.

(3) Can myosin phosphorylat ion be. directly related to an in vivo process in- volving actin-myosin interaction in non- muscle cells? Such an experiment would enhance our understanding of how myosin phosphorylat ion might act to regulate cel- lular functions.

(4) Finally, what about skeletal and car- diac muscles which contain the necessary enzymes and substrates [23], but where phosphorylat ion has not been shown to alter the actin-activated ATPase activities of the myosins? Will future experiments show that myosin phosphorylat ion plays a key role not only in the exciting realms of cell motility and vas deferens contrac- tion, but also in the mundane processes of pumping blood and flexing biceps?

Acknowledgments

I am grateful to Mary Anne Conti and Dr. Stylianos Scordilis for critically read- ing the manuscript , and to Exa Murray for typing it.

References

1 Clarke, M. and Spudich, J.A. (19773 in Annual Review of Biocbemistry (Snell, E. E., ed.) Vol. 46, pp. 797~822, Annual Reviews, Inc., Palo Alto

2 Hitchcock, S.E.(1977)J. CelIBiol. 74, 1-15 3 Adelstein, R.S. and Conti, M.A. (1975) Nature

(London) 256, 597-598 4 Small J.V. and Sobieszek A. (19773 Eur. J.

Biochem. 76, 521-530 5 Gorecka, A., Aksoy, M.O. and Hartshornc, D.J.

(19763 Biochem. Biophys. Res. Commun. 71, 325- 331

6 Chacko, S., Conti, M.A. and Adelstein, R.S. (1977) Proe. NatL Acad. Sci. U.S.A. 74, 129-133

7 Scordilis, S. P. and Adelstein, R.S. (19773 Nature ( LondmO 268,558-560

8 Maruta, H. and Korn, E.D., J. Biol. Chem. in press •

9 Perry, S.V., Cole, H.A., Fearson, N., Moir, A.J.G., Morgan. M. and Pires, E. 119761 in Molecular Basis of Motility (Heilmeyer, L.M.G., J r., Ruegg, J. C. and Wieland, T., eds) pp. 107-121, Springer-Verlag, Berlin

10 Adelstein, R.S. and Conti, M.A. 11976) CoM Spring Harbor Conference on Cell Proliferation, Vol. 3, pp. 725-738, Cold Spring Harbor Labora- tory, Cold Spring Harbor, NY

11 Scordilis, S.P., Anderson, J.L., Pollack, R. and Adelstein, R.S. (1977) 3". Cell. Biol. 74, 940-949

12 Jakes, R., Northrop, F. and Kendrick-Jones. J. (19763 FEBS Lett. 70. 229-234

13 Daniel, J.L. and Adelstein, R.S. (19763 Biochem- istry 15, 2370-2377

14 Perry, S.V. (1976) in Contractile Systems in Non- Muscle Tissues (Perry, S.V.. Margreth, A. and Adelstein. R. S.. eds) pp. 141-!51, North- Holland, Amsterdam

15 Adelstein, R.S., Chacko, S., Scordilis. S.P., Barylko, B., Conti, M.A. and Trotter, J. A. ( 19771 in Calcium-binding Proteins and Calcium Function

(Wasserman, R. H., Corradino, R.A., Carafoli, E., Kretsinger, R.H., MacLennan, D.H. and Siegel, F.L., eds) pp. 251-261, North-Holland. Amster- dam

16 Morgan, M.. Perry, S.V. and Ottaway, J. (1976) Biochem. J. 157, 687-697

17 Lebowitz, E.A. and Cooke, R. (19773 circulation (Abstr.) in press

18 Sobieszek, A. (19773 in Tbe Biochemistry of Smooth Muscle (Stephens, N. L., ed.) pp. 413-433. University Park Press. Baltimore

19 Daniel, J.L., Holmscn, H. and Adelstein, R.S. (1977) Thrombosis and Haemostasis in press

20 Haslam, R.J. and Lynham, J.A. (1977) Biochem. Biophys. Res. Commun. 77, 714--722

21 Barany, K. and Barany, M. (1977) ,I. Biol. Chem. 252.4752-4754

22 Stul[, J.T. and High, C.W. (1977) Biochem. Bio- phys. Res. Commun. 77, 1078-1083

23 Frearson, N. and Perry, S.V. (19753 Biochem. J. 151,99-107

The role of phosphofructokinase in

the Pasteur effect Gopi A. Tejwani

The inhibition of glycolysis in the presence o f oxygen is mediated through the changes in the concentrations of metabolites which regulate the activity of phosphofructokinase. Cer- tain tissues which lack this control of glycolysis by respiration, exhibit high aerobic glycol- ),sis which is needed for the very survival of these tissues.

In 1861, Louis Pasteur observed that the rate of fermentation of sugar by yeast was higher in the absence of oxygen as com- pared to when oxygen was present [1]. Sixty years later, Pasteur 's original obser- vation was confirmed by Warburg and Meyerhof, who independently observed that the production of lactic acid from glu- cose in various animal tissues was in- creased in the absence of oxygen. The term 'Pas teur effect' was introduced by Warburg and since then this phenomenon has been observed in a number of systems including skeletal muscle, heart, brain, kid- ney cortex, liver, Novikoff hepatoma, ade- nocarcinoma, yeast and bacteria (see refer- ence 2 for details).

The biochemical basis of the Pasteur effect is still unclear, a l though a number

G.A.T. is at the Departments of Pharmacology and Radiology, College of Medicine, The Ohio State Uni- versity, Columbus, OH 43210, U.S.A.

of possible explanat ions have been pre- sented [3-6]. Some of the earlier theories were advanced by pioneer scientists like Warburg, Meyerhof, Burk, Lynen, and Lipmann, when little was known of com- plexity of allosteric regulation, and yet these theories are still valid [3]. For ex- ample, in 1941, Lynen [7] and Johnson [8] independently suggested that P~ was re- sponsible for the enhanced glycolytic rate in the absence of oxygen. They observed that when oxygen is present, P~ is preferen- tially utilized for respiration and is not available for glycolysis. The involvement of P~ in the Pasteur effect was also sup- ported by Loomis and Lipmann [9], who suggested that in the presence of oxygen, ATP was responsible for inhibition of gly- colysis. They reported that ni t rophenols uncouple phosphorylat ion from respira- tion and prevent the formation of ATP, which results in the increased rate of gly- colysis in these cells.

T I B S - February 1978 31

The experimental evidence

In 1943, Engelhardt and Sakov 1,10] for the first time suggested that phosphofructo- kinase which catalyzes the phosphoryla- tion of fructose 6-phosphate to fructose 1,6-bisphosphate in the presence of ATP, could be the site of the Pasteur effect. They reasoned that hexokinase could not be in- hibited in the presence of oxygen because glucose 6-phosphate which is the product of hexokinase reaction was required in the oxidative shunt pathway of glucose deg- radation and therefore the formation of glucose 6-phosphate could not be inhibited under aerobic conditions. Furthermore, since fructose 1,6-bisphosphate was readily fermented in the presence of oxygen, which inhibited glucose fermentation, none of the reactions involved in the fermentation of fructose 1,6-bisphosphate could be the site of inhibition by oxygen. Thus by eliminat- ing other stages they concluded that phos- phofructokinase must be the site o f the Pasteur effect.

Their hypothesis was supported by the experiments of Aisenberg and Potter [11] who, in 1957, observed that addition of a respiring mitochondrial preparation to the brain cytosol inhibited the conversion of glucose to lactate in brain cytosol. They postulated that an intermediate in oxi- dative phosphorylation, probably ATP, was responsible for this inhibition. It is noteworthy that about a year earlier Lardy and Parks [12] had observed the inhibi- tion of partially purified rabbit skeletal muscle phosphofructokinase by ATP. This was conclusively proved by the studies of Lynen et al. i-13] who observed a decrease in ATP and increase in P~ and fructose 1,6-bisphosphate concentrations upon transition from aerobic to anaerobic con- ditions in yeast, thus supporting the idea that a decrease in the concentration of ATP was largely responsible for the in- creased activity at phosphofructokinase step.

Abrahams and Younathan[27] suggested that that concentration of NH~, which is a powerful activator of phosphofructokin- ase, rises in certain tissues under anoxia. Cyclic AMP is shown to be an activator ofphosphofructokinase and recently it has been shown that cyclic G M P inhibits this enzyme 1,28]. In this context, it is interest- ing to note that recently Kobayashi et al. 1,29], observed 5 to 13-fold increase in the concentration of cyclic AMP, and a 5-fold decrease in the concentration of cyclic G M P in brain during ischemia. A high concentration of NH~ and a high ratio of cyclic A M P to cyclic G M P would increase the activity of phosphofructokinase, im- plicating this enzyme further in the mech- anism of the Pasteur effect.

Regulation of phosphofructokinase activity by its allosteric effectors

Soon after the report of inhibition of phosphofructokinase by ATP yet another product of respiration was shown to be involved in the inhibition of this enzyme. Passonneau and Lowry observed that ATP 1,14] and citrate [21] inhibited the activity of phosphofructokinase and this inhibition was reversed by AMP, ADP and P~. Salas et al. 1,15] identified citrate as the mitochondrial product that increases in aerobic conditions and inhibits phospho- fructokinase in yeast. Since then, the num- ber of metabolites which are known to regulate the activity of phosphofructokin- ase has increased to about 20 (Table I) and their action on this enzyme may serve as a mechanism by which glycolysis can be regulated in vivo. Thus the activity of phosphofructokinase is inhibited by one of its substrates, ATP, which also binds at an allosteric site resulting in the inhibition of enzyme.

TABLE I Effectors of phosphofructokinase °.

The control of phosphofructokinase activity by ATP is visualized by Atkinson 1,16] in terms of the 'energy charge' of the adenylate system I-(ATP + ½ADP)/(ATP + A D P + A M P ) ] and it is defined as the fundamental metabolic control parameter that inversely alters phosphofructokinase activity. The energy charge of living sys- tems is about 0.8-0.9. The inhibition of phosphofructokinase activity by ATP, cit- rate or phosphoenolpyruvate is overcome by increasing the concentrations of posi- tive effectors, such as, fructose 1,6-bisphos- phate, fructose 6-phosphate, and P~. A de- crease in the energy charge facilitates the reaction of this enzyme by a decrease in the concentration of ATP and increase in concentrations of ADP and AMP. In 1966, Stadtman 1,17] also had suggested that activation of the phosphofructokinase re- action under anaerobic conditions is ef- fected by a decrease in ATP concentration and increase in the concentrations of ADP, AMP and P~, all positive effectors of en- zyme in the cell [17]. Facilitation of phos-

Inhibitors Activators b Deinhibitors of ATP, citrate, or Mg 2+ ¢

ATP NH4 + d Y,5'-cyclic AMP

Citrate 5'-A M P Mg 2+ K +d ADP C a2 + P~ Fructose 6-P P-creatine Pi 3- P-glycerate 5'-A M P P-enolpyruvate 3',5'-cyclic AMP Fructose- 1,6-bis- P 2- P-glycerate AD P Glucose- 1,6-bis- P 2,3-bis-P-glycerate Fructose- 1,6-bis- P Mannose-l,6-bis-P Oleate or Palmitate peptide stabilizing Fructose 1,6-bis- factor

phosphatas¢ 3',5'-cyclic GMP

' Modified from [4]. b Activators increase the velocity of the phosphofructokinase reaction at noninhibitory cOncentrations of ATP. c Deinhibitors increase the velocity of phosphofructokinase reaction at inhibitory concentrations of ATE d N H.~ and K ÷ also increase the maximum velocity of enzyme.

TABLE II Substrate levels in mouse brain after 25 secOnds of ischemia'. All values are recorded as mieromoles per kg wet weight.

Substrate Concentrations Change as a result of

Initial Ischemia ischemia (~,)

Glucose 2560 1930 -25 Glucose-6- P 224 91 - 59 Fructose-6-P 50 27 -46 Fructose- 1,6-bis- P 27 153 + 467 Dihydroxyacetone-P 13 39 +200 Glyceraldehyde-P 0.9 3.3 b +267 1,3-bis-P-glycerate < l < l 0 3-P-glycerate 25 85 + 240 2,3-bis-P-glycerate 29 29 0 "2-P-glycerate 2.8 8.8 +214 P-pyruvate 3.5 8.5 + 151 Pyruvate 39 72 + 85 Lactate 770 1820 + 136

"Data taken from Lowry et al. 1964 [18]. bSixty seconds after decapitation.

32

TABLE III Effect of positive effectors separately and in combination on the activity of phosphofructokinase'.

Relative activity

AMP ADP Pi NH.~ ATP ATP (mM) (mM) (raM) (mM) (0.2 mM) ( 1.6 raM)

0 0 0 0 0 0 0.20 0 0 0 8 0 0 0.80 0 0 6 3 0 0 . 1.0 0 16 3 0 0 0 2.0 6 0 0.20 0 1.0 2.0 97 74 0.20 0.80 1.0 2.0 100 93

° Modified from [2]. 0.2 m M fructose 6-phosphate was used in assay system. For details of assay system see [2].

TABLE IV Effect of positive effectors separate y and in combina ion on the (F6P)o.5 value of phosphofructokinase ~.

AMP ADP P~ NH. + SO. z- (F6P)o.s va ue b (mM) (raM) (mM) (raM) (raM) relative to control

0 0 0 0 0 1.0 0 0 0 0.30 0 1.0 0 0.76 0 0 0 0.46 0 0 0 0 0.50 0.40 0 0 0.50 0 0 0.32 1.0 0 0 0 0 0.28 0 0 0 0.30 0.50 0.32 0 0 0.50 0.30 0 0.22 1.0 0 0.50 0 0 0.16 0 0.95 0.50 0.30 0 0.09 1.0 0 0.50 0.30 0 0.08

• Data taken from [2]. The enzyme was assayed with varying concentrations of fructose-6-P and 0.2 mM ATP. b(F6P)o.s value refers to the concentration of fructose 6-phosphate required for half-maximal activity of enzyme.

phofructokinase reaction during anoxia has been demonstrated in almost all the tissues in which the Pasteur effect is ob- served [2].

Phosphofructokinase links with hexokinase and pyruvate kinase

One of the reasons why phosphofructo- kinase activity is the limiting step in gly- colysis is that regulation of activity of this enzyme in turn affects the activities of hexokinase and pyruvate kmase. In 1964, Lowry et al. [18] reported that the gly- colytic flux was increased by about 6-fold in mouse brain during ischemia (anaerobic conditions). This effect was associated with a decrease in the intracellular concentra- tions of glucose, glucose 6-phosphate and fructose 6-phosphate, and by increase of all substrates from fructose 1,6-bisphosphate to lactate, indicating that the activity of phosphofructokinase is increased during ischemia (Table II). Activation of phos- phofructokinase under anaerobic condi- tions is also coupled to activation of hexo- kinase, because the decrease in the con- centration of fructose 6-phosphate result- ing from the activation of phosphofructo- kinase leads to a decrease in the concentra- tion of glucose 6-phosphate (Table II). A decrease in the concentration of glucose

6-phosphate which is a potent inhibitor of hexokinase results in the activation of hexokinase [22]. In addition, Pi and K + increase the activity of hexokinase as well as that of phosphofructokinase.

The activation of phosphofructokinase is also associated with activation of pyruv- ate kinase which is inhibited by alanine and ATP [23]. This inhibition of pyruvate kinase is overcome by fructose 1,6-bis- phosphate, the concentrations of which increase during activation of phosphofruc- tokinase (Table II).

Mechanisms of action of effectors

When glucose is a main source of energy, undergoing either oxidation or fermenta- tion, the concentrations of ATP, ADP, A M P and P~ are ideal signals for the con- trol of glucose degradation. In this situa- tion, the maintenance of a high concentra- tion of ATP by resynthesis from ADP and P~ is the physiological reason for glucose degradation. Phosphofructokinase from all mammalian tissues and various other sources is inhibited with higher concentra- tion of ATP and this inhibition is over- come by increasing the concentrations of AMP, ADP and Pi in the cell during anaerobic conditions. For example, as shown in Table III, the activity of mucosal

TIBS- February 1978

phosphofructokinase with 0.2 mM fruc- tose 6-phosphate is zero with ATP con- centrations of 0.2 mM or 1.6 mM. In the presence of any single positive effector the activity of the enzyme with 0.2 mM ATP is a small fraction of maximum activity and in the presence of 1.6 mM, ATP, it is only 0 -3% of maximum activity. How- ever, in the presence of AMP, ADP, P~ and NH~ together, the maximum activity of enzyme is obtained with 0.2 mM ATP or 1.6 mM ATP. Such synergism among the positive effectors in deinhibiting the phos- phofructokinase from skeletal muscle [19] and various other tissues has also been reported [26].

Inhibition of phosphofructokinase by ATP at the allosteric site results in an increase in the (F6P)o.s value, that is, the concentration of fructose 6-phosphate re- quired for half-maximal activity of enzyme. AMP, ADP, Pi, NH~, SO42- increase the activity of enzyme by decreasing its (F6P)o.5 value (Table IV). These effectors decrease the (F6P)o.s value of phospho- fructokinase in a synergistic manner by 13- fold with noninhibitory concentration of ATP (Table IV) and more than 20-fold with inhibitory concentration o fATP [20~.

Physiological significance

Anaerobic glycolysis is a primitive mode of life and probably evolved when the atmosphere was devoid of oxygen. In an- aerobic glycolysis much of the chemical energy of sugar is wasted by the cell with carbon by-products such as lactate or eth- anol. Lactate formation can decrease the cellular pH leading to toxic effects and ethanol can have narcotic effects. Combus- tion of one mole of glucose, under an- aerobic conditions, produces 2 moles of ATP, compared to 36 moles of ATP pro- duced in the presence of oxygen. The existence of the Pasteur effect in most of the systems, therefore, means that when- ever oxygen is available respiration plays a major role as a more economical and potentially less harmful mechanism for the synthesis of ATP. This is confirmed again by the fact that under anaerobic condi- tions, the rate of consumption of sugar by cells is six to eight times faster than under aerobic conditions [6].

There are certain tissues in which the rate of conversion of glucose to lactate is not affected by the presence of oxygen and therefore they normally have a high rate of aerobic glycolysis. These systems include striated muscle, intestinal mucosa or jeju- num, renal medulla, erythrocytes, fetal tissues during parturition, malignant tu- mors, retina and leukocytes, etc. However, not much is known about the mecha- nism(s), which can explain the high aerobic glycolysis in these tissues [3-5].

TIBS- February 1978 33

Phosphofructokinase from jejunal mucosa

Our efforts to explain the lack of the Pasteur effect in terms of properties of phosphofructokinase led us to partially purify this enzyme from rat-jejunal mucosa [2]. We also measured the concentrations of various effectors of this enzyme in the jejunum [20]. The jejunal phosphofructo- kinase, like the enzyme from many other sources, is inhibited by higher concentra- tions of ATP and this inhibition is reversed by the positive effectors such as AMP, ADP, Pi and NH,~ (Table III). However, the concentrations of NHI in the jejunum is very-high and it is about 4, 5 and 150 times higher than the concentration of NH,~ in the brain, liver and resting skeletal muscle respectively 120]. A high concen- tration of NH~ deinhibits the enzyme by acting synergistically with other positive effectors and maintains a high aerobic glycolytic rate. In fact, the assay of jejunal phosphofructokinase with its effectors maintained at physiological concentra- tions resulted in maximal activity of the en- zyme,whichwasnot increased further byin- creasing the concentrations of positive effectors [20].

Thus it appears that phosphofructokin- ase in jejunal mucosa is maximally active. However, the enzyme activity in vivo can be still regulated. The phosphofructokin- ase activity and the glycolytic rate in in- testinal mucosa is mainly controlled by the concentration of fructose 6-phosphate, which is a substrate and a positive effector of phosphofructokinase [20]. In jejunum as much as 50% of the absorbed fructose is immediately phosphorylated and ulti- mately appears as lactate. It is noteworthy that glycolytic enzymes are adaptive in this tissue. The activities of hexokinase, glucokinase, fructokinase, phosphofructo- kinase and pyruvate kinase in rat jejunum decrease on fasting and increase on feed- ing glucose or fructose [24].

Tissues lacking the Pasteur effect

In most ofthe tissues, the function of gly- colysis is to provide ATP and other inter- mediates required for the biosynthetic pathways, which is then stringently con- trolled to suit the immediate demands of the cell. However, there are many other tissues where the Pasteur effect is not ob- served. Although no single mechanism can satisfactorily explain the lack of the Pasteur effect in all cases, it appears that high aerobic glycolysis is needed for the immediate survival of these tissues.

For example, in the intestinal mucosa, the glycolytic pathway serves the addi- tional function of converting glucose and other sugars to lactate, an obligatory step in their transport.from the intestine to liver

[25]. During severe exercise the oxidation of glucose via the tricarboxylic acid cycle is not sufficient to provide ATP at a rate required for contraction of muscle and is therefore supplemented by aerobic glycol- ysis in striated muscle.

Retina has a sparse population of mito- chondria which is correlated with the ab- sence of blood vessels and therefore most of the ATP is produced through aerobic glycolysis. If respiration were to be the major source of ATP in retina, light ab- sorption by the red blood cells, and de- crease in transparency of retina by light scattering caused by mitochondria would interfere with visual perception [3].

Large glycogen reserves accumulate in fetal tissues towards the end of term. In the liver the glycogen reserves can rise to 10% of the wet weight. This is matched by a high glycolytic capacity of fetal tissues and the organs of the new-born. The fact that human babies can survive an- aerobiosis for 30 min is no doubt connect- ed with the high glycolytic capacity of the new-born. Lastly, the proportion of energy derived from glycolysis is very high in neo- plastic tissue with contributes to their uncontrolled growth [3,5].

In summary

The inhibition of glycolysis in the pres- ence of oxygen, that is, the Pasteur effect, remained unexplained until recently when the properties of key glycolytic enzymes were studied in detail. It is fair to con- clude that the allosteric properties of phos- phofructokinase can account for most as- pects of the Pasteur effect. The glycolysis is regulated mainly by alteration in the concentrations of numerous effectors of phosphofructokinase in the cell. Although no single mechanism can satisfactorily ex- plain the lack of Pasteur effect, that is, high aerobic glycolysis observed in many tis- sues, it appears that the high aerobic giy- colysis is needed for the very survival of these tissues. In the intestinal mucosa, high aerobic glycolysis may be the result of high activity ofphosphofructokinase maintain- ed by the favorable ratio of positive to negative effectors of this enzyme.

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