- Email: [email protected]
Regulation of enzyme activity in the hibernator: Effect of cations on liver pyruvate kinase in the arctic ground squirrel
J. therm. Biol. Vol. 6. pp. 31 to 34 Pergamon Press Ltd 1981. Printed in Great Britain
0306-4565/81/0101-0031502.00/0
REGULATION OF ENZYME ACTIVITY IN THE HIBERNATOR: EFFECT OF CATIONS ON LIVER PYRUVATE KINASE IN THE ARCTIC G R O U N D SQUIRREL HANS W. BEHRISCHand W. A. GALSTEg* Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska 99701, U.S.A.
(Received 19 March 1980; revised 8 September 1980; accepted 7 October 1980) Abstract--l. The kinetic behavior of pyruvate kinase (PyK) from liver of the Arctic ground squirrel (Citellus undulatus) was examined over a wide temperature (5-37:C) range in the presence of its substrates and modulators, and cations that are known to influence activity of this enzyme. 2. The substrates adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) as well as the modulators fructose 1,6-bisphosphate (FBP) and adenosine triphosphate (ATP) appear to be bound in such a manner that a decrease in the S0.s value is accompanied by an increase in the Hill constant (h), indicating an enhanced degree of site-site interaction in the binding of these metabolites. 3. In marked contrast, the cations K +, Mg2+ and Mn 2÷, are bound in such a manner that falling S0.s values are accompanied by decreases in the attendant h values. 4. The significance of these results is discussed and it is suggested that only the intermediates play a role in regulating activity of liver pyruvate kinase while the ions appear to enhance the Q~0 effect on the Pyk reaction, and function as an integral part of the intracellular milieu that maintains the efficiency of enzyme regulation over a wide temperature range.
INTRODUCTION
In addition to influencing the intensity of intermediate metabolism in heterothermic animals, temperature also affects certain components of the intracellular milieu. Numerous studies demonstrate that in ectotherms a drop in temperature effects large changes in concentration and compartmentation of various ions (Catlett & Millich, 1976; Hickman et al., 1964; Houston & Madden, 1969). In the hibernating ground squirrel alterations in ion profiles are observed as the animal enters hibernation, during the short hibernation cycle, as it emerges for the summer and once again enters hibernation (Galster, unpublished data). Some of these changes may be attributable to differences in permeability characteristics of the cell membrane, and in the case of K + - N a + balance have been proposed as causal factors in the initiation of arousal (Willis et al., 1971). While many enzymes require particular cations for activity, their mode of action is largely unclear. Thus in a number of enzymes the substrate is accepted by the enzyme molecule only when it exists as an intermediate-cation complex (Melchior, 1965; DeAsua et al., 1970), while in others an ionic cofactor appears to be required for maximal activity (Behrisch, 1971). However it is not clear if these ions act as allosteric modulators or as part of a fairly stable and regulated (within limits) intracellular milieu that permits macromolecular function (see Behrisch, 1972). The enzyme pyruvate kinase (ATP:pyruvate phosphotransferase EC 2.7.1.40) catalyzes the reaction PEP+ADP-+PYR+ATP. The enzyme has a * Present address: Department of Physiology, University of Utah, Salt Lake City, Utah 84112, U.S.A. T.II. 6 | ~ ( "
31
regulatory role in glycolysis and a number of mechan~ isms exist that exert tight control over its operation (Newsholme & Start, 1973). Although metabolism of the hibernating mammal is much reduced, available evidence indicates that glycolysis is active in the liver of the hibernating ground squirrel (Burlington & Klain, 1967; Whitten & Klain, 1968; Klain & Whitten, 1968). In kinetic experiments on the regulatory behavior of liver PyK from the Arctic ground squirrel we noted that the enzyme behaves in allosteric fashion in the binding of the intermediates ADP, PEP, ATP and FBP while the binding of various cations seems to proceed in a markedly different fashion. We here propose the physiological significance of such-differences in kinetic behavior in a sometimes heterothermous mammal, the hibernator. METHODS AND MATERIALS
Experimental animals Arctic ground squirrels (C. undulatus) were captured on Augustine Island in Kamishak Bay, Alaska. Time of capture was late June. The island is volcanic in origin and has been isolated from the Alaskan mainland for about 50,000 years or so, and it may be assumed that gene flow into this population has been negligible from about that time. In this connection, we find that animals captured from this population show a very marked lack of variability in all enzyme characteristics that we have examined to date (Behrisch, unpublished data). The animals were fed natural foods and Purina lab chow ad libitum. For the experiments, ground squirrels were taken at two times in the year: during deep hibernation in the winter and during the summer when the animals were homeo-
32
HANS W. BEHRISCH a n d W. A. GALSTER
thermic and active. Immediately after death from a blow on the head liver samples were excised to be used either at once in the preparation of enzyme or stored at - 7 0 ° C until use.
Experimental methods Ground squirrel liver homogenate was prepared in three parts (w/v) of 100ram triethanolamine-HCl buffer, pH 7.50, in a VirTis model 23 homogenizer at top speed at 1-2°C. The homogenate was spun at 30,0000 for 15min at 2°C and the pellet discarded. The supernatant was brought to 20% saturation with solid (NH4)2SO4 and stirred for at least 2 h at 4-5°C. The suspension was then spun, the pellet discarded, and the supernatant brought to 70% saturation with solid (NH4)2SO4 and stirred again for at least 2 h at 4-5°C. The precipitate was collected by centrifugation at 2°C and dissolved in a minimal volume of 10 mM triethanolamine-HCI buffer, pH 7.50. The preparation was then dialysed against the same buffer, with one change in buffer after 2 h. Dialysis longer than 4 h resulted in rapid loss of activity. The enzyme preparation was then either used at once in assays or stored at -70°C. No loss of catalytic activity was observed during storage. The reaction medium contained, in 2.0ml of 10raM triethanolamine-HCI buffer of pH 7.0, various concentrations of Na +, K ÷, Mg 2+ and Mn 2 +, excess amounts of lactate dehydrogenase (Sigma Chemical Co., St Louis, MO) and the metabolites ADP, PEP and NADH. The reaction was initiated by the addition of the PyK preparation to the medium and was assayed by measuring the rate of formation of NAD ÷ with the use of a Hitachi PerkinElmer recording spectrophotometer. All experiments were carried out at pH 7.0. Enzyme activity was directly proportional to concentration of enzyme. All enzyme activities reported in this paper are the average of at least two separate determinations, with less than 5°0 variability between individual animals. RESULTS AS noted in the allosteric binding of the substrates ADP, PEP and the modulators ATP and FBP (Behrisch & Johnson, 1974), the acceptance of the cations
6,0
15
4.0
125
K +, Mg 2+ and Mn 2+ by the ground squirrel PyK seems also allosteric in nature. Values of the Hill constants (h) that accompany the saturation curves for the cations are higher than unity, suggesting homotropic interaction in the effect of these ions on the enzyme. (Fig. 1). However, carrying out these experiments over a wide temperature range (5-37°C) demonstrates differences in the binding of these ions and the intermediates. Thus with the monovalent cation K +, an increase in temperature from 5 to 26°C brings about a sharp increase in So.s values. A further rise in temperature from 26 to 37°C on the other hand, effects a fall in So.5 values for K +. The two bivalent cations Mg "+ and Mn "+ behave in an opposite manner. An increase in temperature from 5 to 26°C brings about a decrease in So.5 values for both these cations, and a further temperature increase causes a rise in So.s values. As noted in previous papers on the liver PyK from the Arctic ground squirrel (Behrisch & Johnson, 1974; Behrisch, 1974) affinity (as reflected by apparent Km or So.5) of the summer enzyme for the four intermediates ADP, PEP, ATP and FBP is markedly sensitive to temperature. In that study the So.5 values for these metabolites varied in a disparate manner with changing temperature, suggesting that perhaps the summer variant of PyK may be unsuited to heterothermic function during the hibernation season. Thus in a summer PyK the So.s for PEP rises with decreasing temperature, while that for ADP, the other substrate, falls with a drop in temperatures. Similarly, the So.5 for ATP, the allosteric inhibitor of the enzyme, rises with decreasing temperature, while for the activator FBP it remains constant o~er the same temperature range at the intervals 5, 15, 26 and 37°C. However, dissimilar though these results might appear, there is a consistent difference between the ionic effects and those of the metabolites. Thus when a decrease in temperature causes a fall in So.5 for K +, there is also a fall in the attendant Hill values. Similar results are observed in the kinetic experiments on the other two cations Mg 2+ and Mn '+. In contrast, the ground squirrel PyK accepts its four metabolites, ADP, PEP, ATP and FBP in such a fashion that a
3.0
3.0
~ l5
0 .I
.0.0 7
•
Mn2+ /~
2.0
2.C
E 1.0
2.0
0.0
5
115 Z 5
35
I 5
It5
I 25
35
20
0.05
I. 0
0.025
1.5
5
IJ5
!1.o
Temp ,*C
Fig. 1. Effects of temperature on affinity as reflected by So 5 of ground squirrel liver pyruvate kinase for the cations K ÷, Mg2÷ and Mn 2÷. Included are the values of the Hill coefficients for the saturation curves of these ions. Closed symbols denote So.5 values, open symbols h values.
Ionic control of enzyme .activity in the hibernator
33
Table 1. Effect of temperature on at~nity (as reflected by So.s) of liver PyK from the summer active ground squirrel for the substrates ADP and PEP as well as the modulators ATP and FBP, in the presence of tissue concentrations of the cations Na +, K +, Mg 2+ and Ca 2+ that were observed in the totally aroused state (between bouts of hibernation) and the deep hibernating state
Temperature (C°)
So.s for PEP (mM} Ions of Ions of Control aroused deep (10 mM MgCh) • state hibernation
37 26 15 5
0.91 1.01 I.I9
0.86 0.83 1.05 1.65
0.83 0.96 1.01 1.90
So.s for FBP (/aM) Ions of
Ions of
1.75
So.s for ADP (raM) Ions of Ions of Control aroused deep (10 mM MgCI~) state hibernation 1.33 0.78 0.03 0.02
1.53 0.82 0.03 0.04
1.45 0.65 0.03 0.02
So.s for ATP (raM) Ions of
Ions of
Temperature (C~)
Control (10 mM MgCI~)
aroused state
deep hibernation
Control (10 mM MgCI2)
aroused state
deep hibernation
37 26 15 5
0.4 0.3 0.3 0.4
0.4 0.3 0.2 0.2
0.6 0.4 0.4 0.3
1.35 1.6 2.0 3.5
1.5 1.8 1.8 4.1
1.65 1.9 2.3 4.3
Ionic concentrations: aroused state--Na +, 0.02; K +, 0.06; Mg2+, 0.02; Ca z+, 0.001. (mEq./g tissue) deep hibernation--Na +, 0.03; K +, 0.05, Mg' +, 0.01 ; Ca 2*, 0.022. (From Galster, unpublished data).
decrease in So.s values is accompanied by an increase in the attendant value of the Hill constant (Behrisch & Johnson, 1974). In kinetic experiments on liver PyK from the hibernating Arctic ground squirrel in the presence of Na +, K +, Mg 2+ and Ca 2+ together at total tissue concentrations (Galster, unpublished data) that characterize the period of early hibernation (immediately upon reentry into deep hibernation after a period of arousal), the So.s values for the substrates ADP and PEP or the modulators ATP and FBP remain unchanged (Table 1). Indeed, calculated So.s values for these experiments are very much in agreement with those previously reported for the hibernating or non-hibernating ground squirrel PyK (Behrisch, 1974; Behrisch & Johnson, 1974). The major influence of these ions is an enhancement of the effect of temperature on enzyme activity in such a way that the Q~o factor of the PyK reaction
is raised (Table 2). In the non-hibernating PyK they raise the Qlo of the reaction from below 1.5 to around 2.5, while in the hibernating PyK the ions increase the Qlo from around 2.5 to about 3.4. Moreover, because of the constant So.s values this high Q~o value is observed over the entire concentration range of substrates tested. The result of such a high Q~o effect, over a temperature range of 37°C to about 0°C, is a change in the rate of this reaction by a factor of about 100, a value in good agreement with the observed reduction of metabolic rate in the hibernating Arctic ground squirrel (Hock, 1960). DISCUSSION In their discussion of the role of allosterism in enzyme function, Monod et al. (1965) generate a model--later borne out by various studies {see Changeux & Rubin, 1968)--which states that an increase in
Table 2. Effects of the cations Na +, K ÷, Ca "+ together on the temperature coefficient (Qto) of liver pyruvate kinase from the Arctic ground squirrel. Data are given for the enzyme variants from both the hibernating and non-hibernating animals, and at Vm,, and So.s concentrations of the substrates. Ionic conditions were those determined from liver of animals in deep hibernation, and are noted in Table 1 State of animal (enzyme variant) Temperature range ("C) 37-25 25-15 15-5 Control Q1o values* (I0 mM MgCI2 only)
Hibernating Vm,. conc. of So.s conc. of ADP and PEP ADP and PEP 3.65 3.31 3.78
3.57 3.66 3.21
Non-hibernating V,,,, conc. of So.s conc. of ADP and PEP ADP and PEP 2.34 2.56 2.48
2.55
* Control values of Qlo are for the temperature range 5-37°C (from Behrisch, 1974).
2.21 2.11 2.23 1.21
34
HANS W. BEHRISCHand W. A. GAI.,VrER
an enzyme's affinity for its (physiological) substrate or modulator is accompanied by, indeed based on, an enhanced cooperation between the binding sites for that ligand. A decrease in the So.s--an apparent increase in affinity--is rooted in an enhanced degree of site-site interaction in the binding of that molecule (see for example Fig. 1). The corollary of this is that in order to be effective as a modulator of enzyme activity, a ligand must be able to affect the interaction between the enzyme's subunits in such a way that affinity for it (or another metabolite) is directly controlled. Judging from the present data, it appears that only the intermediates ADP, PEP, FBP and ATP, that are the indicators of the intensity of glycolysis, play a role in its integration. The effectiveness of these intermediates in regulating the PyK reaction over a wide, but physiological, temperature range appears to be due to the observed homeostasis of their So.s values in the face of changing temperatures. The importance of the cations, on the other hand, appears to be not in allosteric control of PyK, but rather in the enhancement of the Qto effect. Such an arrangement would allow the liver cell of the hibernator to commit the same proportion of its regulatory metabolites to the regulation of PyK, regardless of metabolic rate and temperature. Further, during hibernation, the raised temperature coefficient would appear to be important in reducing the rate of catalysis at this level of intermediary metabolism.
Acknowledgement--We gratefully acknowledge the support of the National Institutes of Health (NIGMS 10402).
REFERENCES
BEHRISCHH. W. (1971) Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king crab. Biochem. J. 121, 399-409. BEtmxsc~ H. W. 0972) Molecular mechanisms of adaptation to low temperature in marine poikilotherms, some regulatory properties of dehydrogenases from two Arctic species. Mar. Biol. 13, 267-275.
BE~ISCH H. W. (1974) Temperature and the regulation of enzyme activity in the hibernator. Isoenzymes of fiver pyruvate kinase from the hibernating and non-hibernating ground squirrel. Can. J. Biochem. 52, 894-902. BEHRISCH H. W. & JOHNSON C. E. (1974) Regulatory properties of pyruvate kinase from liver of the summer active Arctic ground squirrel. Can. J. Biochem. 52, 547-559. Bt~UNGTON R. F. & KLAn~ G. J. (1967) Gluconeogenesis during hibernation and arousal from hibernation. Comp. Biochem. Physiol. 22, 701-708. CAT~Yr R. H. & MILUCH D. R. (1976) Intracellular and extracell~lar osmoregulation of temperature-acclimated goldfish i Carassius auratus L. Comp. Biochem. Physiol. $5A, 261-269. CHANG~X J. P. & RUmN M. M. (1968) Allosteric interac. tions in aspartate trans-carbamylase. Ill. Interpretation of experimental data in terms of the model of Monod, Wyman and Changeux. Bioehem. 7, 553-561. D~AsuA J., ROZENGURTE. & CARMINATrl H. (1970) Some kinetic properties of liver pyruvate kinase (Type L). IlL Effect of monovalent cations on its allosteric behavior. 2. biol. Chem. 245, 3901-3905. HICKMANC. P., MCNABBR. A., NELSONR. A., VAN BREEMEN E. D. & COMFORTD. (1964) Effect of cold acclimation on electrolyte distribution in rainbow trout (Salmo gairdneri). Can. J. Zool. 42, 577-597. HOCK R. S. (1960) Seasonal variations in physiologic functions of Arctic ground squirrels and black bears. In Mammalian Hibernators (Edited by LYM^N C. P. & DAWE A. R.) Bull. Mus. comp. Zool. Harv. 124, 155--171. HOUSTON A. H. & M^DO~N J. A. (1969) Osmoregulatory adaptation of steelhead trout (Salmo gairdneri) to seawater. Can. J. Zool. 37, 729-748. KLAING. J. & WHI'ITENB. K. (1968) Carbon dioxide fixation during hibernation and arousal from hibernation. Comp. Biochem. Physiol. 25, 363-366. MELCHIORJ. B. (1965) The role of metal ions in the pyrurate kinase reaction. Biochem. 4, 1518-1525. M O N O D J., W Y M A N J. & CHANGEUX J. P. (1965) O n the nature of allosteric transitions: a plausible model. J. molec. Biol. 12, 88-118. NEWSHOLSm E. A. & START C. (1973) Regulation in Metabolisr~ 349 p. Wiley, London. WHrr~N B. K. & KLAIN G. J. (1968) Protein metabolism in hepatic tissue of hibernating and arousing ground squirrels. Am. J. Physiol. 214, 1360-1362. WILLIS J. S., GOLDMAN S. S. & FOSTER R. F. (1971) Tissue K concentration in relation to the role of the kidney in hibernation and the cause of periodic arousal. Comp. Biochem. Physiol. 39A, 437---445.
0306-4565/81/0101-0031502.00/0
REGULATION OF ENZYME ACTIVITY IN THE HIBERNATOR: EFFECT OF CATIONS ON LIVER PYRUVATE KINASE IN THE ARCTIC G R O U N D SQUIRREL HANS W. BEHRISCHand W. A. GALSTEg* Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska 99701, U.S.A.
(Received 19 March 1980; revised 8 September 1980; accepted 7 October 1980) Abstract--l. The kinetic behavior of pyruvate kinase (PyK) from liver of the Arctic ground squirrel (Citellus undulatus) was examined over a wide temperature (5-37:C) range in the presence of its substrates and modulators, and cations that are known to influence activity of this enzyme. 2. The substrates adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) as well as the modulators fructose 1,6-bisphosphate (FBP) and adenosine triphosphate (ATP) appear to be bound in such a manner that a decrease in the S0.s value is accompanied by an increase in the Hill constant (h), indicating an enhanced degree of site-site interaction in the binding of these metabolites. 3. In marked contrast, the cations K +, Mg2+ and Mn 2÷, are bound in such a manner that falling S0.s values are accompanied by decreases in the attendant h values. 4. The significance of these results is discussed and it is suggested that only the intermediates play a role in regulating activity of liver pyruvate kinase while the ions appear to enhance the Q~0 effect on the Pyk reaction, and function as an integral part of the intracellular milieu that maintains the efficiency of enzyme regulation over a wide temperature range.
INTRODUCTION
In addition to influencing the intensity of intermediate metabolism in heterothermic animals, temperature also affects certain components of the intracellular milieu. Numerous studies demonstrate that in ectotherms a drop in temperature effects large changes in concentration and compartmentation of various ions (Catlett & Millich, 1976; Hickman et al., 1964; Houston & Madden, 1969). In the hibernating ground squirrel alterations in ion profiles are observed as the animal enters hibernation, during the short hibernation cycle, as it emerges for the summer and once again enters hibernation (Galster, unpublished data). Some of these changes may be attributable to differences in permeability characteristics of the cell membrane, and in the case of K + - N a + balance have been proposed as causal factors in the initiation of arousal (Willis et al., 1971). While many enzymes require particular cations for activity, their mode of action is largely unclear. Thus in a number of enzymes the substrate is accepted by the enzyme molecule only when it exists as an intermediate-cation complex (Melchior, 1965; DeAsua et al., 1970), while in others an ionic cofactor appears to be required for maximal activity (Behrisch, 1971). However it is not clear if these ions act as allosteric modulators or as part of a fairly stable and regulated (within limits) intracellular milieu that permits macromolecular function (see Behrisch, 1972). The enzyme pyruvate kinase (ATP:pyruvate phosphotransferase EC 2.7.1.40) catalyzes the reaction PEP+ADP-+PYR+ATP. The enzyme has a * Present address: Department of Physiology, University of Utah, Salt Lake City, Utah 84112, U.S.A. T.II. 6 | ~ ( "
31
regulatory role in glycolysis and a number of mechan~ isms exist that exert tight control over its operation (Newsholme & Start, 1973). Although metabolism of the hibernating mammal is much reduced, available evidence indicates that glycolysis is active in the liver of the hibernating ground squirrel (Burlington & Klain, 1967; Whitten & Klain, 1968; Klain & Whitten, 1968). In kinetic experiments on the regulatory behavior of liver PyK from the Arctic ground squirrel we noted that the enzyme behaves in allosteric fashion in the binding of the intermediates ADP, PEP, ATP and FBP while the binding of various cations seems to proceed in a markedly different fashion. We here propose the physiological significance of such-differences in kinetic behavior in a sometimes heterothermous mammal, the hibernator. METHODS AND MATERIALS
Experimental animals Arctic ground squirrels (C. undulatus) were captured on Augustine Island in Kamishak Bay, Alaska. Time of capture was late June. The island is volcanic in origin and has been isolated from the Alaskan mainland for about 50,000 years or so, and it may be assumed that gene flow into this population has been negligible from about that time. In this connection, we find that animals captured from this population show a very marked lack of variability in all enzyme characteristics that we have examined to date (Behrisch, unpublished data). The animals were fed natural foods and Purina lab chow ad libitum. For the experiments, ground squirrels were taken at two times in the year: during deep hibernation in the winter and during the summer when the animals were homeo-
32
HANS W. BEHRISCH a n d W. A. GALSTER
thermic and active. Immediately after death from a blow on the head liver samples were excised to be used either at once in the preparation of enzyme or stored at - 7 0 ° C until use.
Experimental methods Ground squirrel liver homogenate was prepared in three parts (w/v) of 100ram triethanolamine-HCl buffer, pH 7.50, in a VirTis model 23 homogenizer at top speed at 1-2°C. The homogenate was spun at 30,0000 for 15min at 2°C and the pellet discarded. The supernatant was brought to 20% saturation with solid (NH4)2SO4 and stirred for at least 2 h at 4-5°C. The suspension was then spun, the pellet discarded, and the supernatant brought to 70% saturation with solid (NH4)2SO4 and stirred again for at least 2 h at 4-5°C. The precipitate was collected by centrifugation at 2°C and dissolved in a minimal volume of 10 mM triethanolamine-HCI buffer, pH 7.50. The preparation was then dialysed against the same buffer, with one change in buffer after 2 h. Dialysis longer than 4 h resulted in rapid loss of activity. The enzyme preparation was then either used at once in assays or stored at -70°C. No loss of catalytic activity was observed during storage. The reaction medium contained, in 2.0ml of 10raM triethanolamine-HCI buffer of pH 7.0, various concentrations of Na +, K ÷, Mg 2+ and Mn 2 +, excess amounts of lactate dehydrogenase (Sigma Chemical Co., St Louis, MO) and the metabolites ADP, PEP and NADH. The reaction was initiated by the addition of the PyK preparation to the medium and was assayed by measuring the rate of formation of NAD ÷ with the use of a Hitachi PerkinElmer recording spectrophotometer. All experiments were carried out at pH 7.0. Enzyme activity was directly proportional to concentration of enzyme. All enzyme activities reported in this paper are the average of at least two separate determinations, with less than 5°0 variability between individual animals. RESULTS AS noted in the allosteric binding of the substrates ADP, PEP and the modulators ATP and FBP (Behrisch & Johnson, 1974), the acceptance of the cations
6,0
15
4.0
125
K +, Mg 2+ and Mn 2+ by the ground squirrel PyK seems also allosteric in nature. Values of the Hill constants (h) that accompany the saturation curves for the cations are higher than unity, suggesting homotropic interaction in the effect of these ions on the enzyme. (Fig. 1). However, carrying out these experiments over a wide temperature range (5-37°C) demonstrates differences in the binding of these ions and the intermediates. Thus with the monovalent cation K +, an increase in temperature from 5 to 26°C brings about a sharp increase in So.s values. A further rise in temperature from 26 to 37°C on the other hand, effects a fall in So.5 values for K +. The two bivalent cations Mg "+ and Mn "+ behave in an opposite manner. An increase in temperature from 5 to 26°C brings about a decrease in So.5 values for both these cations, and a further temperature increase causes a rise in So.s values. As noted in previous papers on the liver PyK from the Arctic ground squirrel (Behrisch & Johnson, 1974; Behrisch, 1974) affinity (as reflected by apparent Km or So.5) of the summer enzyme for the four intermediates ADP, PEP, ATP and FBP is markedly sensitive to temperature. In that study the So.5 values for these metabolites varied in a disparate manner with changing temperature, suggesting that perhaps the summer variant of PyK may be unsuited to heterothermic function during the hibernation season. Thus in a summer PyK the So.s for PEP rises with decreasing temperature, while that for ADP, the other substrate, falls with a drop in temperatures. Similarly, the So.5 for ATP, the allosteric inhibitor of the enzyme, rises with decreasing temperature, while for the activator FBP it remains constant o~er the same temperature range at the intervals 5, 15, 26 and 37°C. However, dissimilar though these results might appear, there is a consistent difference between the ionic effects and those of the metabolites. Thus when a decrease in temperature causes a fall in So.5 for K +, there is also a fall in the attendant Hill values. Similar results are observed in the kinetic experiments on the other two cations Mg 2+ and Mn '+. In contrast, the ground squirrel PyK accepts its four metabolites, ADP, PEP, ATP and FBP in such a fashion that a
3.0
3.0
~ l5
0 .I
.0.0 7
•
Mn2+ /~
2.0
2.C
E 1.0
2.0
0.0
5
115 Z 5
35
I 5
It5
I 25
35
20
0.05
I. 0
0.025
1.5
5
IJ5
!1.o
Temp ,*C
Fig. 1. Effects of temperature on affinity as reflected by So 5 of ground squirrel liver pyruvate kinase for the cations K ÷, Mg2÷ and Mn 2÷. Included are the values of the Hill coefficients for the saturation curves of these ions. Closed symbols denote So.5 values, open symbols h values.
Ionic control of enzyme .activity in the hibernator
33
Table 1. Effect of temperature on at~nity (as reflected by So.s) of liver PyK from the summer active ground squirrel for the substrates ADP and PEP as well as the modulators ATP and FBP, in the presence of tissue concentrations of the cations Na +, K +, Mg 2+ and Ca 2+ that were observed in the totally aroused state (between bouts of hibernation) and the deep hibernating state
Temperature (C°)
So.s for PEP (mM} Ions of Ions of Control aroused deep (10 mM MgCh) • state hibernation
37 26 15 5
0.91 1.01 I.I9
0.86 0.83 1.05 1.65
0.83 0.96 1.01 1.90
So.s for FBP (/aM) Ions of
Ions of
1.75
So.s for ADP (raM) Ions of Ions of Control aroused deep (10 mM MgCI~) state hibernation 1.33 0.78 0.03 0.02
1.53 0.82 0.03 0.04
1.45 0.65 0.03 0.02
So.s for ATP (raM) Ions of
Ions of
Temperature (C~)
Control (10 mM MgCI~)
aroused state
deep hibernation
Control (10 mM MgCI2)
aroused state
deep hibernation
37 26 15 5
0.4 0.3 0.3 0.4
0.4 0.3 0.2 0.2
0.6 0.4 0.4 0.3
1.35 1.6 2.0 3.5
1.5 1.8 1.8 4.1
1.65 1.9 2.3 4.3
Ionic concentrations: aroused state--Na +, 0.02; K +, 0.06; Mg2+, 0.02; Ca z+, 0.001. (mEq./g tissue) deep hibernation--Na +, 0.03; K +, 0.05, Mg' +, 0.01 ; Ca 2*, 0.022. (From Galster, unpublished data).
decrease in So.s values is accompanied by an increase in the attendant value of the Hill constant (Behrisch & Johnson, 1974). In kinetic experiments on liver PyK from the hibernating Arctic ground squirrel in the presence of Na +, K +, Mg 2+ and Ca 2+ together at total tissue concentrations (Galster, unpublished data) that characterize the period of early hibernation (immediately upon reentry into deep hibernation after a period of arousal), the So.s values for the substrates ADP and PEP or the modulators ATP and FBP remain unchanged (Table 1). Indeed, calculated So.s values for these experiments are very much in agreement with those previously reported for the hibernating or non-hibernating ground squirrel PyK (Behrisch, 1974; Behrisch & Johnson, 1974). The major influence of these ions is an enhancement of the effect of temperature on enzyme activity in such a way that the Q~o factor of the PyK reaction
is raised (Table 2). In the non-hibernating PyK they raise the Qlo of the reaction from below 1.5 to around 2.5, while in the hibernating PyK the ions increase the Qlo from around 2.5 to about 3.4. Moreover, because of the constant So.s values this high Q~o value is observed over the entire concentration range of substrates tested. The result of such a high Q~o effect, over a temperature range of 37°C to about 0°C, is a change in the rate of this reaction by a factor of about 100, a value in good agreement with the observed reduction of metabolic rate in the hibernating Arctic ground squirrel (Hock, 1960). DISCUSSION In their discussion of the role of allosterism in enzyme function, Monod et al. (1965) generate a model--later borne out by various studies {see Changeux & Rubin, 1968)--which states that an increase in
Table 2. Effects of the cations Na +, K ÷, Ca "+ together on the temperature coefficient (Qto) of liver pyruvate kinase from the Arctic ground squirrel. Data are given for the enzyme variants from both the hibernating and non-hibernating animals, and at Vm,, and So.s concentrations of the substrates. Ionic conditions were those determined from liver of animals in deep hibernation, and are noted in Table 1 State of animal (enzyme variant) Temperature range ("C) 37-25 25-15 15-5 Control Q1o values* (I0 mM MgCI2 only)
Hibernating Vm,. conc. of So.s conc. of ADP and PEP ADP and PEP 3.65 3.31 3.78
3.57 3.66 3.21
Non-hibernating V,,,, conc. of So.s conc. of ADP and PEP ADP and PEP 2.34 2.56 2.48
2.55
* Control values of Qlo are for the temperature range 5-37°C (from Behrisch, 1974).
2.21 2.11 2.23 1.21
34
HANS W. BEHRISCHand W. A. GAI.,VrER
an enzyme's affinity for its (physiological) substrate or modulator is accompanied by, indeed based on, an enhanced cooperation between the binding sites for that ligand. A decrease in the So.s--an apparent increase in affinity--is rooted in an enhanced degree of site-site interaction in the binding of that molecule (see for example Fig. 1). The corollary of this is that in order to be effective as a modulator of enzyme activity, a ligand must be able to affect the interaction between the enzyme's subunits in such a way that affinity for it (or another metabolite) is directly controlled. Judging from the present data, it appears that only the intermediates ADP, PEP, FBP and ATP, that are the indicators of the intensity of glycolysis, play a role in its integration. The effectiveness of these intermediates in regulating the PyK reaction over a wide, but physiological, temperature range appears to be due to the observed homeostasis of their So.s values in the face of changing temperatures. The importance of the cations, on the other hand, appears to be not in allosteric control of PyK, but rather in the enhancement of the Qto effect. Such an arrangement would allow the liver cell of the hibernator to commit the same proportion of its regulatory metabolites to the regulation of PyK, regardless of metabolic rate and temperature. Further, during hibernation, the raised temperature coefficient would appear to be important in reducing the rate of catalysis at this level of intermediary metabolism.
Acknowledgement--We gratefully acknowledge the support of the National Institutes of Health (NIGMS 10402).
REFERENCES
BEHRISCHH. W. (1971) Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king crab. Biochem. J. 121, 399-409. BEtmxsc~ H. W. 0972) Molecular mechanisms of adaptation to low temperature in marine poikilotherms, some regulatory properties of dehydrogenases from two Arctic species. Mar. Biol. 13, 267-275.
BE~ISCH H. W. (1974) Temperature and the regulation of enzyme activity in the hibernator. Isoenzymes of fiver pyruvate kinase from the hibernating and non-hibernating ground squirrel. Can. J. Biochem. 52, 894-902. BEHRISCH H. W. & JOHNSON C. E. (1974) Regulatory properties of pyruvate kinase from liver of the summer active Arctic ground squirrel. Can. J. Biochem. 52, 547-559. Bt~UNGTON R. F. & KLAn~ G. J. (1967) Gluconeogenesis during hibernation and arousal from hibernation. Comp. Biochem. Physiol. 22, 701-708. CAT~Yr R. H. & MILUCH D. R. (1976) Intracellular and extracell~lar osmoregulation of temperature-acclimated goldfish i Carassius auratus L. Comp. Biochem. Physiol. $5A, 261-269. CHANG~X J. P. & RUmN M. M. (1968) Allosteric interac. tions in aspartate trans-carbamylase. Ill. Interpretation of experimental data in terms of the model of Monod, Wyman and Changeux. Bioehem. 7, 553-561. D~AsuA J., ROZENGURTE. & CARMINATrl H. (1970) Some kinetic properties of liver pyruvate kinase (Type L). IlL Effect of monovalent cations on its allosteric behavior. 2. biol. Chem. 245, 3901-3905. HICKMANC. P., MCNABBR. A., NELSONR. A., VAN BREEMEN E. D. & COMFORTD. (1964) Effect of cold acclimation on electrolyte distribution in rainbow trout (Salmo gairdneri). Can. J. Zool. 42, 577-597. HOCK R. S. (1960) Seasonal variations in physiologic functions of Arctic ground squirrels and black bears. In Mammalian Hibernators (Edited by LYM^N C. P. & DAWE A. R.) Bull. Mus. comp. Zool. Harv. 124, 155--171. HOUSTON A. H. & M^DO~N J. A. (1969) Osmoregulatory adaptation of steelhead trout (Salmo gairdneri) to seawater. Can. J. Zool. 37, 729-748. KLAING. J. & WHI'ITENB. K. (1968) Carbon dioxide fixation during hibernation and arousal from hibernation. Comp. Biochem. Physiol. 25, 363-366. MELCHIORJ. B. (1965) The role of metal ions in the pyrurate kinase reaction. Biochem. 4, 1518-1525. M O N O D J., W Y M A N J. & CHANGEUX J. P. (1965) O n the nature of allosteric transitions: a plausible model. J. molec. Biol. 12, 88-118. NEWSHOLSm E. A. & START C. (1973) Regulation in Metabolisr~ 349 p. Wiley, London. WHrr~N B. K. & KLAIN G. J. (1968) Protein metabolism in hepatic tissue of hibernating and arousing ground squirrels. Am. J. Physiol. 214, 1360-1362. WILLIS J. S., GOLDMAN S. S. & FOSTER R. F. (1971) Tissue K concentration in relation to the role of the kidney in hibernation and the cause of periodic arousal. Comp. Biochem. Physiol. 39A, 437---445.