The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

474

Biochimica etBiophysica Acta, 719 (1982) 474-479 Elsevier Biomedical Press

BBA 21290

THE REGULATORY ROLE OF SPERMINE AND FATTY ACID IN THE INTERACTION OF AMP DEAMINASE WITH PHOSPHOFRUCTOKINASE M A S A T A K A Y O S H I N O a and K E I K O M U R A K A M I b

"Department of Biochemistry, Yokohama City University School of Medicine, Urafune-cho 2-33, Minami-ku, Yokohama 232 and b Department of Laboratory Medicine, St. Marianna University School of Medicine, Kawasaki 213 (Japan) (Received June 21 st, 1982)

Key words: AMP deaminase; Fatty acid," Spermine," Phosphofructokinase," Energy charge," (Yeast)

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.

Introduction AMP deaminase (EC 3.5.4.6) is responsible for the stimulation of glycolysis [1,2] and for the control of the adenylate energy charge [3]. AMP deaminase activity is modulated by a variety of effectors [4]; fatty acids, in particular unsaturated ones, can inhibit the enzyme under the in vitro and in situ conditions [5-7], and polyamines, whose accumulation is accompanied with increased cellular proliferation [8-10], act as effective activators of AMP deaminase [11,12], resulting in the stimulation of glycolysis [1,2]. Furthermore, spermine specifically prevented AMP deaminase in situ from the inhibition by fatty acid [13]. In this study we analyzed the effect of spermine on the inhibition of glycolysis by fatty acid and of the control of the energy charge using permeabilized yeast [14]: linolenate lowered the phosphofructokinase activity 0304-4165/82/0000-0000/$02.75 © t982 Elsevier Biomedical Press

through the inhibition of AMP deaminase, and the addition of spermine specifically cancelled the inhibition of AMP deaminase, resulting in the enhancement of phosphofructokinase and glycolysis. The physiological role of the action of spermine on the inhibition by fatty acid is discussed in relation to the metabolic interconversion between glycolysis and respiration. Materials and Methods

Materials. All chemicals were obtained from commercial sources as described previously [1-3,13]. Incubation conditions and determination of metabolites. Baker's yeast cells (Saccharomyces cerevisiae) were permeabilized as described previously [ 14]. Reaction mixture for the experiments of glycolytic control and the determination of

475

metabolites were essentially identical to those described previously [2,13]. NAD and Pi were excluded in the reaction mixture for the determination of phosphofructokinase activity in situ. Fatty acid solution was prepared by sonication for 5 min with a Branson sonifier [7].

A

~3 rl

ca2

O-

Results

Effect of spermine on the inhibition by linolenate of the energy charge-control system. The effect of linolenate and spermine on the control of the adenylate energy charge was investigated in permeabilized yeast cells. Fig. 1 shows the variation of the concentrations of adenylates and ammonium ion, and the energy charge values. After the addition of ATP and glucose, the decrease in ATP and the increase in ADP and AMP (Fig. 1A) with the phosphorylation of glucose dropped the adenylate energy charge (Fig. 1B). The decrease in total adenylates was accompanied by the stoichiometric production of ammonium ion under these conditions (Fig. 1C), suggesting that the depletion of adenylates was dependent on the AMP deaminase reaction because there is little or no adenosine deaminase activity in yeast cells [3,15]. The addition of linolenate retarded the regeneration of ATP, degradation of adenylates, and recovery of the energy charge; however, the concomitant addition of spermine cancelled these changes but rather stimulated the recovery of the energy charge in the presence of linolenate. Changes in the energy charge and adenylate degradation were not at all affected by the addition of KCI in the presence of linolenate (data not shown). Effect of spermine and linolenate on glycolysis. Concentrations of glycolytic intermediates were also determined under the same conditions. Formation of fructose 1,6-bisphosphate and pyruvate was markedly inhibited by the addition of linolenate, and the concomitant addition of spermine cancelled the inhibition of glycolytic flux by linolenate within 20 min of the reaction (Fig. 2). After 20 min, the level of fructose 1,6-bisphosphate is considerably higher in the presence of linolenate than in its absence or in the presence of linolenate plus polyamine. Time-dependent accumulation of sugar phosphates may be due to the inactivation of phosphoglycerate kinase (EC 2.7.2.3) by linolenate [16].

IlL

0

C 4

0-

It l

Time (rain)

60

90

Fig. 1. Effect of linolenate with or without spermine on the changes in the adenine nucleotides, adenylate energy charge and ammonium ion in permeabilized yeast cells. Toluenized yeast cells (10 mg/ml) were incubated at 37°C in the reaction mixture consisting of 5 mM glucose, 5 mM ATP, 10 mM MgC12, 2 mM Pi, 0.1 mM NAD, 10 mM cacodylate buffer (pH 7.1) and 2 mM linolenate with or without 1 mM spermine in a total volume of 3 ml. After aliquots of 0.2 ml were deproteinized at appropriate intervals and neutralized, the supernatant was utilized for the determination of adenine nucleotides and ammonium ion. A. ATP (©, ~ , e), ADP (zx, A, A) and AMP (D, I!, II). B. Adenylate energy charge (O, no addition; O, + linolenate + spermine; ~ , + linolenate). The values were calculated as follows: energy charge = ([ATP]+ 1/2. [ADP])/([ATP]+[ADP]+[AMP]). C. Total adenylates (O, ~ , o) and ammonium ion (zx, &, A). Open symbols, no addition; semiclosed symbols, 2 mM linolenate included; closed symbols, 2 mM linolenate plus 1 mM spermine included.

A quantitative representation of the changes in the concentration of glycolytic intermediates and adenylates is given in Fig. 3. As demonstrated previously, glucose added was almost completely recovered as glycolytic intermediates including glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, triose phosphates and pyruvate within 10 min [2]. Metabolite levels were determined in the presence of 2 mM linolenate with or without 1 mM spermine after 10

476 rain. U s i n g the c r o s s o v e r t h e o r e m [17], these results suggest t h a t (a) the i n h i b i t i o n o f p h o s phofructokinase activity by linolenate paralleled i n h i b i t i o n o f A M P d e a m i n a s e , a n d t h a t (b) sperm i n e c a n c e l l e d t h e l i n o l e n a t e - i n h i b i t i o n o f these e n z y m e s , r e s u l t i n g in a s t i m u l a t i o n o f glycolysis.

3~

Z

h

&2 _u

Effect of linolenate on the in situ activities of AMP deaminase and phosphofructokinase and the reversal by spermine of the inhibition. T h e effect o f

dJ u

o

B 6

~E2 to

a~

't

2

IL

30

60

Time(rain)

9"00

Fig. 2. Effect of linolenate with or without spermine on the changes in glycolytic intermediates in permeabilized yeast cells. Toluenized yeast cells were incubated at 37°C as described in the legend to Fig. 1. Glycolytic intermediates were determined enzymatically [2]. A. glucose (D, ill, II) and glucose 6-phosphate plus fructose 6-phosphate (C), ~, e). B. Fructose 1,6-bisphosphate (C), ~, e) and pyruvate (zx, A, *). Open symbols, no addition; semiclosed symbols, 2 mM linolenate included; closed symbols, 2 mM iinolenate with 1 mM spermine included.

s p e r m i n e a n d l i n o l e n a t e o n the a c t i v i t y o f A M P d e a m i n a s e a n d p h o s p h o f r u c t o k i n a s e was investig a t e d . Fig. 4 s h o w s the i n h i b i t i o n of the A M P d e a m i n a s e a c t i v i t y b y l i n o l e n a t e in the p r e s e n c e of various concentrations of KCI and spermine. Only a s m a l l i n c r e a s e in t h e c o n c e n t r a t i o n s n e c e s s a r y f o r 50% i n h i b i t i o n o f t h e e n z y m e , I0. 5 v a l u e s for l i n o l e n a t e was o b s e r v e d e v e n w h e n K + , an activat o r o f A M P d e a m i n a s e , was e l e v a t e d f r o m 0 to 100 m M (Fig. 4A). H o w e v e r , the a d d i t i o n of s p e r m i n e e f f e c t i v e l y p r e v e n t e d the e n z y m e f r o m i n h i b i t i o n

0.2e

~

29(

o10( c

Linolenate(raM) Qt~ ~p ~r:~-P~ 6r AIP AbP A~P N~,,:

F(~PTrios¢-P

Fig. 3. Percentage changes in glycolytic intermediates and adenine nucleotides in the presence of linolenate with (e) or without ((3) spermine. The data were taken from Figs. l and 2. Metabolite levels, determined in the presence of 2 mM linolenate with or without 1 mM spermine 10 rain after initiation of the reaction, were expressed as a percentage of the control (no addition) values. G6P, glucose 6-phosphate; F6P. fructose 6-phosphate; Fru-P2, fructose 1,6-bisphosphate; Pyr, pyruvate.

Fig. 4. Effect of concentration of linolenate on the in situ activity of AMP deaminase in the presence of various concentration of KCl and spermine. The reaction mixture of 0.5 ml consisted of l0 mM cacodylate buffer (pH 7.1), 10 mM AMP, various concentrations of KCI or spermine, and the permeabilized yeast cells (4 mg/ml) as the enzyme. A. Inhibition of AMP deaminase by linolenate in the presence of KC1. ©, no addition; [2, 20 mM KCI added; zx, 100 mM KC1 added; e, 100 mM KC1 plus 1 mM spermine added. B. Inhibition of AMP deaminase by linolenate in the presence of spermine. C), no addition; D, 0.1 mM spermine added; v, 0.2 mM spermine added; A, l mM spermine added; e, 1 mM spermine plus 100 mM KC1 added.

477

by linolenate (Fig. 4B). The inhibition curves with respect to linolenate became more sigmoid in shape and the inhibition became partial with increase in the concentration of spermine from 0 to 1 mM. The activity fell to a definite limit with increase in the inhibitor concentration. We examined the effect of spermine and linolenate on the interaction of AMP deaminase with phosphofructokinase activity: the reaction was started with MgATP and glucose but Pi and NAD were excluded in the reaction mixture so that fructose 1,6-bisphosphate, the reaction product of the phosphofructokinase reaction and triose phosphates formed from it, cannot be metabolized further. The production of ammonium ion and fructose 1,6-bisphosphate decreased with increase in the concentration of linolenate. The addition of

].0 E

=

~

0.5

E o -i-

z

0

to

o

Linolenat¢

(mM)

Fig. 5. Effect of concentration of linolenate with or without spermine or NH4C1 on the formation of ammonium ion and of fructose 1,6-bisphosphate in permeabilized yeast cells. A. AMP deaminase activity in situ. B. Phosphofructokinase in situ. The reaction mixture of 0.2 ml contained 10 mM cacodylate buffer (pH 7.1), 10 mM glucose, 5 mM ATP, 10 mM MgCI2, the indicated concentration of linolenate and the toluenized yeast cells (10 m g / m l ) in the absence and presence of 1 mM spermine or 1.8 raM NH4C1. Fructose 1,6-bisphosphate and ammonium ion were determined after incubation for 15 rain. ©, no addition; zx, 1 mM spermine added; D, !.8 mM NH4CI added.

spermine completely removed the inhibition of these enzymes; on the other hand, the presence of increasing concentration of ammonium ion effectively prevented the fructose 1,6-bisphosphate formation from the inhibition by linolenate without changes in the AMP deaminase activity (Fig. 5). We can conclude that the inhibition of phosphofructokinase by linolenate is indirect, that is, via the inhibition of AMP deaminase and that spermine can enhance the phosphofructokinase activity by elevating NH~- level. Discussion

Inhibition of glycolysis by fatty acids may be due to the inhibition of phosphofructokinase by citrate, a metabolic product increased during fatty acid oxidation [18,19] or due to direct inhibition of hexokinase (EC 2.7.1.1), phosphofructokinase and pyruvate kinase (EC 2.7.1.40) by fatty acids and their CoA esters [16,20]. However, as is well known, fatty acids are strong surfactants, and fatty acid inhibition of glycolytic enzymes was shown to be due to enzyme inactivation as a result of nonspecific detergent effect [21]. Thus, doubt is thrown on the physiological significance of the irreversible inactivation of enzymes by fatty acids, since reversibility is one of the requirements for an effector. The lack of inactivation of phosphofructokinase by fatty acid in the presence of physiological level of the substrate, ATP or fructose 6-phosphate suggests that fatty acids cannot participate in direct control of glycolytic flux [21]. Our previous papers [5-7] showed that free fatty acids, in particular, unsaturated ones, act as powerful and reversible inhibitors of AMP deaminase as a control system of glycolysis [1,2]. Linolenate could regulate the activity of phosphofructokinase and glycolytic flux by changing the level of NH~- as a result of AMP deaminase inhibition without direct inhibition of phosphofructokinase. It should be noted that glycolytic flux is inhibited by fatty acid below the level of 2 mM. Since concentrations of total free fatty acids were reported to be 3-4/xmol/g fresh yeast [22], the inhibitory effect of fatty acid on glycolysis demonstrated in this paper is of physiological relevance. Polyamines show a variety of biological effects;

478

a stimulation of nucleic acid synthesis, protein synthesis [8-10], and conversion of glucose to carbon dioxide [23-25]. Recently, we presented evidence suggesting that polyamine can be responsible for the stimulation of phosphofructokinase and pyruvate kinase [1,2] and threonine dehydratase [26] by increasing the level of ammonium ion produced through the activation of AMP deaminase. The results presented here showed that spermine prevented the in situ AMP deaminase from the linolenate-induced inhibition, but the increase in K ÷ and other monovalent cations had little effect on the linolenate inhibition of the enzyme. Polyamine and alkali metals seemed to affect AMP deaminase with the same mechanism: kinetic studies suggested that these ligands could interact with the enzyme at the identical sites [12]. However, as shown in this study, some differences were observed in the action of spermine and K ÷ on the linolenate-induced inhibition of the enzyme: (a) the inhibition by linolenate was complete type without cooperativity in the presence of K ÷ , and (b) the addition of spermine resulted in the partial inhibition of the enzyme with cooperativity, and the inhibition was nearly completely cancelled in its presence at higher concentration. These results suggest that spermine-induced deinhibition of the enzyme is not simply due to the activation of the enzyme by polyamine but due to the specific interaction of polyamine with the enzyme at the inhibitory sites for fatty acid, although a decrease in the effective fatty acid concentration resulting from the complex formation with polyamines may be considered. The lack of reversal of fatty acid inhibition of AMP deaminase by K ÷ suggest that the enzyme can be regulated by fatty acid in the presence of the high intracellular K ÷ concentration, and polyamine can effectively release the fatty acid inhibition of the enzyme in cells. The addition of spermine completely removed the inhibition of AMP deaminase by linolenate: the depletion of total adenylates, the production of ammonium ion and the recovery of the energy charge were almost identical to those without linolenate. The inhibition of phosphofructokinase by linolenate was shown to be indirect, that is, via the inhibition of AMP deaminase, and spermine can prevent the phos-

phofructokinase from the linolenate inhibition by elevating NH~- level (Fig. 5). However, glycolytic rate remained relatively inhibited under these conditions; this retardation may be accounted for by the inhibition or inactivation by fatty acid of 3-phosphoglycerate kinase [16]. Accumulation of fructose 1,6-bisphosphate in the presence of linolenate after 20 min (Fig. 2) suggest the time-dependent inactivation of the enzyme. The effects of polyamine and fatty acid were analyzed from the experiments in which only a single polyamine, spermine and only a single fatty acid, linolenate were used. Unsaturated fatty acids act as powerful inhibitors of AMP deaminase in vitro and in situ, whereas saturated ones have little inhibitory effects [7]. All the polyamines are activators of the enzyme: spermine is the most effective, followed by spermidine, putrescine, cadaverine, and 1,6-diaminohexane in that order [11,12]. The inhibitory and the reversal effects were general for unsaturated fatty acids and for polyamines, respectively, although the effectiveness varies depending on the ligands. AMP deaminase activity strongly correlates with the activity of glycolytic flux in several tissues and cells; by contrast, the activity of AMP deaminase is lower in aerobic tissues with a higher activity of phosphorylation coupled to respiration [27,28]. Fatt3~ acid inhibition of glycolysis through the control of the AMP deaminase has a physiological meaning in metabolic regulation under the conditions where energy metabolism depends on the fatty acid oxidation and respiration [29]. When glucose can be utilized as the energy source [30], polyamine can act as effective activators of AMP deaminase [11,12]. These ligands may contribute to the metabolic interconversion between glycolysis and respiration.

Acknowledgements This work was supported in part by Grants-inAid for Scientific Research (No. 56570119 and No. 56770119) from the Ministry of Education, Science and Culture of Japan. We are grateful to Dr. K. Tsushima from the Department of Biochemistry, Yokohama City University School of Medicine for his interest and encouragement.

479

References 1 Yoshino, M. and Murakami, K. (1980) Biochem. Int. 1, 84-90 2 Yoshino, M. and Murakmai, K. (1982) J. Biol. Chem. 257, 2822-2828 3 Yoshino, M. and Murakami, K. (1981) Biochim. Biophys. Acta 672, 16-20 4 Zielke, C.L. and Suelter, C.H. (1971) in The Enzymes (Boyer, P.D., ed), 3rd Ed., Vol. 4, pp. 47-78, Academic Press, New York 5 Yoshino, M., Miyajima, E. and Tsushima, K. (1976) FEBS Lett. 72, 143-146 6 Yoshino, M., Miyajima, E. and Tsushima, K. (1979) J. Biol. Chem. 254, 1521-1525 7 Yoshino, M. and Murakami, K. (1981) Biochim. Biophys. Acta 660, 199-203 8 Tabor, H. and Tabor, C.W. (1972) Advl Enzymol. 36, 203-268 9 Tabor, H. and Tabor, C.W. (1964) Pharmacol. Rev. 16, 245-300 10 Tabor, C.W. and Tabor, H. (1974) Annu. Rev. Biochem. 45, 285-306 11 Yoshino, M., Murakami, K. and Tsushima, K. (1978) Biochim. Biophys. Acta 542, 177-179 12 Yoshino, M. and Murakami, K. (1980) Biochim. Biophys. Acta 616, 82-88 13 Yoshino, M. and Murakami, K. (1981) Biochem. Biophys. Res. Commun. 102, 905-910 14 Murakami, K., Nagura, H. and Yoshino, M. (1980) Anal. Biochem. 105, 407-413 15 Yoshino, M., Murakarni, K. and Tsushima, K. (1979) Biochim. Biophys. Acta 570, 157-166

16 Tortora, P., Hanozet, G.M., Guerritore, A., Vincenzini, M.T. and Vanni, P. (1978) Biochim. Biophys. Acta 522, 297-306 17 Williamson, J. (1971) in Red Cell Metabolism and Function (Brewer, G.J., ed.), pp. 117-137, Plenum Press, New York 18 Newsholme, E.A. and Start, C. (1973) Regulation in Metabolism, pp. 279-281, John Wiley & Sons, London 19 Williamson, J.R., Browning, E.T., Scholz, R., Kreisberg, R.A. and Fritz, I.B. (1966) Diabetes 17, 194-208 20 Weber, G., Lea, M.A. and Stamm, N.B. (1968) Adv. Enzyme Regul. 6, 101-123 21 Ramadoss, C.S., Uyeda, K. and Johnston, J.M. (1976) J. Biol. Chem. 251, 98-107 22 Hunter, K. and Rose, A.H. (1972) Biochim. Biophys. Acta 260, 639-653 23 Lockwood, D.H., Lipsky, J.J., Meronk, F., Jr. and East, L.E. (1971) Biochem. Biophys. Res. Commun. 44, 600-607 24 Lockwood, D.H. and East, L.E. (1974) J. Biol. Chem. 249, 7717- 7722 25 Amatruda, J.M. and Lockwood, D.H. (1974) Biochim. Biophys. Acta 372, 266-273 26 Yoshino, M. and Murakami, K. (1981) Biochim. Biophys. Acta 672, 313-316 27 Sugden, P.H. and Newsholme, E.A. (1975) Biochem. J. 150, 113-122 28 Winder, W.W., Terjung, R.L., Baldwin, K.M. and Holloszy, J.O. (1974) Am. J. Physiol. 227, 1411-1414 29 Yoshino, M. and Murakami, K. (1981) Biochem. Int. 3, 173-179 30 Hirai, M., Shimizu, S., Teranishi, Y., Tanaka, A. and Fukui, S. (1972) Agric. Biol. Chem. 36, 2335-2343