Biochem. J. (1975) 152, 23-32 23 Printed in Great Britain

The Contents of Adenine Nucleotides, Phosphagens and some Glycolytic Intermediates in Resting Muscles from Vertebrates and Invertebrates By ISIDOROS BEIS* and ERIC A. NEWSHOLME* A.R.C. Unit ofMuscle Mechanisms and Insect Physiology, Department ofZoology, University ofOxford, Oxford OXI 3PS, U.K. (Received 24 April 1975)

1. The lowest contents of ATP and the lowest ATP/AMP concentration ratios are observed in the molluscan muscles that have very low rates of energy expenditure during contraction. The highest contents of ATP are observed in the extremely aerobic insect ffight muscle and the extremely anaerobic pectoral muscle of the pheasant and domestic fowl. In general, the lowest ATP/AMP concentration ratios are observed for muscle in which the variation in the rate ofenergy utilization is small (e.g. some molluscan muscles, heart muscle); the highest ratios are observed in muscles in which this variation is large (lobster abdominal muscle, pheasant pectoral muscle, some insect flight muscles). This finding is consistent with the proposed role of AMP and the adenylate reaction in the regulation ofglycolysis. However, in the flight muscle ofthe honey-bee the ATP/AMP ratio is very low, so that may be regulated by factors other than the variation in AMP concentration. 2. The variation in the contents.ofarginine phosphate in muscle from the invertebrates is much larger than the variation in phosphate in muscle from the vertebrates. 3. The contents of hexose monophosphates and pyruvate are, in general, higher in the muscles of vertebrates than in those of the invertebrates. The contents of phosphoenolpyruvate are similar in all the muscles investigated, except for the honey-bee inwhichit is about 4-10-fold higher. 4. The mass-action ratios for the reactions catalysed by phosphoglucoisomerase and are very similar to the equilibrium con- stants for these reactions. Further, the variation in the mass-action ratios between muscles is small. It is concluded that these catalyse reactions close to equilibrium. However, the mass-action ratios for the reactions catalysed by and are much smaller than the equilibrium constants. The variation in the ratios between different muscles is large. It is concluded that these enzymes catalyse non- equilibrium reactions. Since the variation in the mass-action ratios for the reactions catalysed by the phosphagen (i.e. creatine and phosphokinases) is small, it is suggested that these reactions are close to equilibrium.

In this paper the contents of adenine nucleotides, literature for contents of the glycolytic and energy creatine phosphate (or arginine phosphate), creatine intermediates in muscle in vivo indicates that such (or arginine) and some glycolytic intermediates in a data are available only for tissues from the rat, mouse, large number of muscles from different are guinea pig and, in some cases, the frog (Williamson & reported together with the calculated mass-action Brosnan, 1974). The present findings are discussed in ratios for the following reactions: adenylate kinase relation to the function and/or control of these (EC 2.7.4.3), arginine phosphokinase (EC 2.7.3.3), enzymes in the muscle. creatine phosphokinase (EC 2.7.3.2), phospho- (EC 2.7.1.11), phosphoglucoisomerase Materials and Methods (EC 5.3.1.9) and pyruvate kinase (EC 2.7.1.40). Under resting conditions (or as near resting as Chemicals and enzymes possible), muscles were rapidly frozen by the freeze- clamping technique, the frozen muscles were ex- All chemicals and enzymes were obtained from tracted and thecontents ofthe intermediates measured Boehringer Corp. (London) Ltd., London W5 2TZ, by specific enzymic techniques. A recent survey of the U.K., except for the following: EDTA (disodium salt) and all inorganic reagents were obtained from * Present address: Department of Biochemistry, South BDH Chemicals, Poole, Dorset BH12 4NN, U.K., Parks Road, Oxford OX1 3QU, U.K. and were of the highest purity available. Nembutal Vol. 152 24 I. BEIS AND E. A. NEWSHOLME was obtained from Abbott Laboratories, Arge-Vet water) and the skin was removed from the hind limb, Division, Queenborough, Kent, U.K. Sandoz-MS222 the gastrocnemius muscle was separated from the was obtained from Thompson and Joseph, Castle other muscles and freeze-clamped; the dissection House, Castle Meadow, Norwich NOR 41D, U.K. was completed within 30s. For dogfish, the animals was prepared in this laboratory from were anaesthetized by intravenous (caudal-vein) lobster muscle by Dr. A. R. Leech. injection of Nembutal (0.1 ml/kg), and a piece of white muscle was rapidly dissected and freeze- Animals clamped; the dissection was completed within 30s. For the birds, the animals were anaesthetized by an The sources of the animals were as given by intraperitoneal injection of Nembutal, feathers and Newsholme & Taylor (1969) except for the addition skin were rapidly removed from above the pectoral of the following: rats were obtained from Charles muscles and a piece of pectoral muscle was dissected River (U.K.) Ltd., Cherry Garden Lane, Ash, and immediately freeze-clamped. From the initial Canterbury, Kent, U.K., and kept in the incision to the freeze-clamping of the muscle, the house of the Department of Biochemistry; mice were procedure was completed within 5s. For rats and bred in this Department; lobsters were obtained from mice, the animals were anaesthetized with diethyl Fisher Bros., 12 Billingsgate Street, London E.C.3, ether, the skin was dissected from the hind limb and U.K.; frogs and snails were obtained from Gerrard the exposed muscle was freeze-clamped in situ. The and Haig, East Preston, Sussex, U.K. Locusts were muscle was dissected away from the bone at liquid- used 7-14 days after the final moult. Flies were used N2 temperatures. between 7 and 14 days after emerging from pupae. For each animal, the problem of whether to use an All other insects were of undetermined age, but they anaesthetic to obtain resting muscle was considered. were known to be capable of flight. Apart from rats In some animals, experiments were performed to and mice, for which only male animals were used, compare anaesthetic methods or to compare the muscle tissue was obtained from male and female results obtained with and without anaesthetization animals indiscriminately. (see the Results section and Table 1).

Freeze-clamping ofmuscle Measurement ofadenine nucleotides and intermediates All muscle tissues used in the measurements of The frozen muscle was powdered in a percussion contents of adenine nucleotides and intermediates mortar at -70°C, and the powdered muscle was were frozen rapidly with the aid of aluminium tongs extracted by the addition of 4-5 vol. of frozen HC104 cooled in liquid N2 (see the Results section). The (6 %, w/v). The extraction took place in a mortar, and method of removing and freeze-clamping the muscle continual mixing with the pestle thawed the mixture depended on the animal and the muscle under of HC104 and frozen muscle powder. The precipi- investigation. In each case, the method that was tated was removed by centrifugation and the finally adopted represented a compromise between extract was neutralized with 3 M-KHCO3. The rapidity of freezing, selection of a specific muscle and metabolic intermediates were measured by enzymic freezing the muscle under resting conditions (see also techniques, which are simple, sensitive and, in the Results section). For the snail, a piece of the foot particular, specific for the compounds under investi- was rapidly dissected and immediately freeze- gation. Glucose 6-phosphate, fructose 6-phosphate, clamped. For the other molluscs, the shell was fructose diphosphate and adenine nucleotides were opened by cutting through the muscle with a scalpel determined by using the methods described by and a piece of muscle was rapidly dissected and Gevers & Krebs (1966), and pyruvate was deter- freeze-clamped. From opening the shell to freezing mined in the same assay as phosphoenolpyruvate the muscle, the procedure was completed in 5-lOs. (Czok & Eckert, 1963). Creatine phosphate and For the lobster, the animal was 'anaesthetized' by arginine phosphate were determined in the same placing in ice for 20min, then the tail was cut off and assay as that for ATP after the addition ofcreatine or a piece of abdominal muscle was cut away and arginine phosphokinase and ADP (Lamprecht & freeze-clamped within l5s from removal of the tail. Stein, 1963). For the insects, the whole animal was freeze-clamped while at rest (i.e. non-flying). This procedure resulted in much of the flight muscle being squeezed out of Results the thorax, so that the musclecould beeasily separated from cuticle and other tissues. In some insects, the Investigations into use of anaesthetics and extraction muscle was dissected away from the cuticle at liquid- techniques N2 temperatures. For frogs, the animals were Effects of anaesthetics on the contents of energy anaesthetized with Sandoz-MS222 (0.1 g/litre of metabolites. In the present investigation the amounts 1975 ADENINE NUCLEOTIDES AND PHOSPHAGENS IN MUSCLE 25

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Vol. 152 26 I. BETS AND E. A. NEWSHOLME of metabolic intermediates in certain muscles or adenine nucleotides in this extraction process has muscle groups were measured so that, in some cases, been shown to be almost 100% (Newsholme, 1962; preparation of the animal and/or dissection of the Start, 1969)]. Further, after neutralization of the muscle before freeze-clamping (see below) was extract, the amounts of the intermediates were necessary. It was considered that such procedures measured by specific enzymic techniques, which might cause sufficient trauma to modify the resting- permit the precise measurement of individual state metabolism in the muscle and the problem arose metabolites without the necessity of separation as to the use of anaesthetics. The danger in using an procedures that could result in loss of metabolites. anaesthetic is that it might interfere either directly or In view of the precautions taken in this work, it is indirectly in metabolism and modify the concentra- considered that statistically significant variations in tions of the metabolites under investigation. The amounts of metabolic intermediates in various problem of not using an anaesthetic is that the muscles are indicative of metabolic and physiological animal or the muscle may respond to the preparative differences within the muscles. Some possible procedure in such a way that the muscle can no metabolic bases for the variations are discussed in longer be described as resting. To examine if anaes- this paper. thetics were necessary, selected animals were investi- gated. The dogfish, frog and insects were chosen. The Amounts ofmetabolites dogfish was anaesthetized by stunning, intravenous injection of Nembutal (caudal vein) or addition of The amounts of ATP, ADP, AMP, creatine (or Sandoz-MS222totheseawatercontaining thefish. The arginine) and creatine phosphate (or arginine frog was anaesthetized by the addition of Sandoz- phosphate)/g of tissue in the muscles that have been MS222 to the water. The insects were anaesthetized investigated together with the calculated mass- with CO2. The contents of creatine phosphate, action ratios for adenylate kinase and arginine or creatine, ATP, ADP and AMP were measured and creatine phosphokinase, are presented in Table 2. are reported in Table 1. In the dogfish the ATP/AMP The amounts of glucose 6-phosphate, fructose 6- concentration is lowest (8) when Sandoz-MS222 was phosphate, fructose diphosphate, phosphoenol- used as an anaesthetic and highest when Nembutal pyruvate and pyruvate/g of muscle, together with the was used (170); there was, however, little difference calculated mass-action ratios of phosphoglucoiso- in the contents of creatine or creatine phosphate. In merase, phosphofructokinase and pyruvate kinase, the frog, the ATP/AMP content ratio and the creatine are presented in Table 3. For ease of comparison, phosphate content were slightly higher in the amounts of adenine nucleotides, phosphagens and anaesthetized animals (Table 1). In the two insects glycolytic intermediates (and calculated mass-action studied, there was no effect of the anaesthetic on the ratios) from heart and tissues other than muscle, ATP/AMP content ratios. On the basis of the results which were obtained from the literature, are reported in Table 1, it was decided to use an anaesthetic to in Tables 2 and 3. obtain muscle from all vertebrate species, but Amounts of adenine nucleotides and phosphagens. anaesthetics were not used for the invertebrates For the 21 muscles investigated (from 21 different except for the lobster. It was necessary to cool the animals) the variation in amount of the adenine lobster in ice to immobilize the animal sufficiently to nucleotides/g of tissue was remarkably small. This dissect the muscle. ranged from 0.76 to 8.51, 0.43 to 1.57 and 0.02 to Effects of extraction procedure. To interpret the 0.28,4mol/g for ATP, ADP and AMP respectively. amounts of metabolic intermediates measured in any The largest differences in the extreme values are tissue in relation to the metabolic flux and/or the observed for both ATP and AMP; for each nucleotide existence of near-equilibrium or non-equilibrium it is about 11-fold. If, however, the ATP content in reactions in the steady-state, it is important that the the foot of the snail is excluded from consideration, extraction procedure causes minimum changes in the variation in content ofATP is only about fourfold the concentrations of the metabolites. The procedure (2.02-8.51). The amounts of ATP/g in the insects used in the present work is very similar to that used studied are remarkably similar, 5.35-8.51 pmol/g. in many other laboratories (for general description In the vertebrate muscles, the range is slightly of technique, see Newsholme, 1962; Hess, 1965; greater, 4.47-8.05pmol/g. The range of the ratios of Start, 1969). The muscle is clamped between plates of the amounts of ATP/AMP (and similarly ATP/ aluminium cooled in liquid N2 (freeze-clamping); this ADP) is very much larger than for the range of the produces almost instantaneous cooling of the tissue amounts/g per se; the ATP/AMP concentration to very low temperatures (see Wollenberger et al., ratio ranges from 8.8 to 402.5 (i.e. about 45-fold). 1960). The frozen muscle is pulverized in a percussion From a comparison with the contents of adenine mortar at liquid-N2 temperatures and the frozen nucleotides in other tissues, the contents of ATP and muscle powder is extracted with HCl04 at 0°C. [The especially the ATP/AMP content ratio are much recovery of added glycolytic intermediates and higher in muscle tissue. 1975 ADENINE NUCLEOTIDES AND PHOSPHAGENS IN MUSCLE 27

Phosphagen (i.e. arginine phosphate or creatine The mass-action ratios for the reactions catalysed phosphate) was detected in all the muscles studied. by phosphoglucoisomerase, phosphofructokinase The amounts of arginine phosphate in invertebrate and pyruvate kinase are reportedinTable 3. The ratios muscle ranged from 0.48 to 52.16,umol/g (i.e. more for the phosphoglucoisomerase reaction range from than 100-fold); this is much greater than the variation 0.08 to 0.77 (i.e. about a 10-fold variation). The mass- in the contents of creatine phosphate in vertebrates action ratios for the tissues other than muscle range (i.e. about fivefold). The highest phosphagen amounts from 0.25 to 0.33 (Table 3). The mass-action ratios were observed in the snap muscles of Pecten, the for the phosphofructokinase reaction range from abdominal muscles of the lobster and the pectoral 0.03 to 3.58 (i.e. more than a 100-fold variation). The muscles of the domestic fowl (52, 33 and 27,umol/g mass-action ratios for the tissues other than muscle respectively). The concentration ratio of creatine range from 0.09 to 2.3 (Table 3) and those for the phosphate/creatine (or arginine phosphate/arginine) pyruvate kinase reaction range from 1.44 to 103.6 ranges from 0.2 to 9.0 approximately (white muscle (i.e. more than a 70-fold variation). The mass-action of the dogfish and snap muscle of Pecten respec- ratios for the tissues other than muscle range from tively). 0.7 to 5.4 (Table 3). Amounts of glycolytic intermediates. A large variation in the amounts of hexose phosphates/g was Discussion found in the muscles investigated; the contents of glucose 6-phosphate, fructose 6-phosphate and In the interpretation of the results, no attempt has fructose diphosphate range from 0.02 to 1.59, 0.01 to been made to distinguish between free and bound 0.35 and 0.01 to 0.41,umol/g respectively. There is adenine nucleotides or between cytoplasmic and also a large variation in the content of pyruvate mitochondrial concentrations of metabolites. At the (0.02-0.48pmol/g), but if the value for the honey-bee present time, satisfactory methods for measurement is omitted the variation in the content of phospho- of intermediates within these different cellular com- enolpyruvate is small (0.01-0.09.umol/g). The lowest partments are not available and any interpretation contents of hexose monophosphates are observed in that involves the use of a precise concentration of a some of the invertebrate muscles. It is noteworthy metabolic intermediate must be made with caution. that, except for the honey-bee, insect flight muscles In this discussion, interpretations are based primarily which contain low amounts of hexose monophos- on concentration ratios and, since it is possible that phates also contain low activities of the glycolytic compartmentation will affect, for example, the con- enzymes, and they probably rely on compounds centrations of ATP, ADP and AMP to similar other than carbohydrates for energy provision (see extents, the danger of misinterpretation of data is Crabtree & Newsholme, 1972a,b). decreased. Further, provided that compartmentation The flight muscle of the honey-bee appears to be is similar in different muscles, comparison of a metabolically unusual in a number of respects. Thus, qualitative or semiquantitative nature between although the contents of hexose monophosphates muscles should not be meaningless. are very low, those of pyruvate and phosphoenol- pyruvate are high; indeed, the content of the latter is Amounts ofadenine nucleotides andphosphagens the highest observed in the present work. Further, of the insects studied, the flight muscle of the honey-bee Rather low concentrations of ATP were observed has the lowest ATP/AMP concentration ratio (see in two types of molluscan muscle, the foot of the Table 2). snail and the posterior adductor muscle of the sea Mass-action ratios. The mass-action ratios for mussel. This finding is consistent with the low rate of adenylate kinase and creatine phosphokinase (or energy expenditure by these muscles during contrac- arginine phosphokinase) reactions, which were tion (see Ruegg, 1971). The other molluscan muscle, calculated from the data obtained in this work, are the snap muscle of Pecten, has a higher ATP content presented in Table 2. For 21 different muscles the and it is known to utilize ATP at a high rate during mass-action ratios for the adenylate kinase reaction contraction (Ruegg, 1971). In all of the other muscles range from0.22 to 1.26 (i.e. about a fivefold variation). studied, the contents of ATP are very similar. The range of the mass-action ratios for the other Although the maximum rates of energy utilization in tissues (reported from the literature) is from 0.40 to these muscles are different, it is likely that the rates 0.78, if adipose tissue is excluded from consideration. are high in comparison with the two molluscan The mass-action ratios for the arginine phospho- muscles described above (see Crabtree & Newsholme, kinase reaction ranges from 1.0 to 7.7, and those for 1972a). The highest amounts of ATP/g are present in the creatine phosphokinase reaction range from two distinct types of muscle, the flight muscle of an 3.2 to 8.5 (excluding the value for the dogfish; see insect (the rosechafer) and the pectoral muscles of the below). For both phosphokinase reactions the ratios domestic fowl (about 8,umol/g). This finding is note- vary less than 10-fold. worthy since the latter muscle contains very few Vol. 152 28 1. BETS AND E. A. NEWSHOLME

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Vol. 152 30 I. BEIS AND E. A. NEWSHOLME mitochondria (see George & Berger, 1966), whereas much larger changes in that of AMP through the insect flight muscle contains many mitochondria (see reaction catalysed by adenylate kinase (see News- Smith, 1966). holme, 1970). In this theory, the higher the initial The highest amounts of phosphagen/g of tissue resting ATP/AMP concentration ratio is, the greater were observed in the abdominal muscle of the lobster the change in concentration of AMP for a given and the snap muscle of Pecten. This finding is con- change in that of ATP (see Newsholme & Start, sistent with the function of phosphagen in provision 1973). Muscles that require a large change in the rate of a significant proportion of the total energy of glycolysis to satisfy the energy demands of the required during the short violent bursts ofmechanical contractile apparatus should possess a high resting activity that these muscles are known to perform. ATP/AMP concentration ratio; mechanical activity The phosphagen content is generally rather low in the willcausea decrease in the concentration ofATP and a insect flight muscle, despite the fact that the rate of large increase in AMP, which will stimulate glycolysis energy utilization is very high during flight. Further, and hence energy production. Some insect flight insect flight can be sustained for long periods of time muscles provide exceptions to the relationship so that the concentration of phosphagen would (cockchafer, dung-beetle and honey-bee); they have provide a negligible proportion of the total energy lower ATP/AMP concentration ratios than the required in these muscles. The amounts of phos- other muscles. However, the flight muscles of the phagen/g of muscle are similar in the different cockchafer and dung-beetle may depend more on vertebrate muscles examined, including that of the proline or fat oxidation than on carbohydrate rat heart, despite the fact that the physiological roles oxidation for energy production for flight (see of some of these muscles are different. This suggests Crabtree & Newsholme, 1970, 1972a,b) so that that the role of the phosphagen in these muscles, and changes in the content of AMP may be much less perhaps in the insect flight muscles also, is to provide important in the control of energy formation in the a very transient buffer of the concentration of the flight muscles ofthese particular insects. Nonetheless, ATP in the muscle. the flight muscles of the honey-bee are known to A noteworthy observation arising from this work depend on carbohydrate metabolism for energy is the high content of creatine and a low creatine production (Crabtree & Newsholme, 1972a,b) and it phosphate/creatine content ratio in the dogfish is anticipated that glycolysis will be controlled in muscle. This low ratio is found in the well-anaesthe- relation to the energy demand of the contractile tized animals despite a high ATP/AMP content process in the muscle. The very low ATP/AMP con- ratio. This suggests that the high concentration of centration ratio suggests that changes in the con- creatine is not due to degradation of creatine phos- centration of AMP are unimportant in the regulation phate. Perhaps creatine is used in the dogfish muscle of the activity of phosphofructokinase and the rate of to provide intracellular osmoregulation. glycolysis in the flight muscle of the honey-bee. Another observation consistent with this suggestion Ratio ofthe contents ofATPIAMP is that the flight muscle of the honey-bee does not possess any detectable activity of fructose diphos- The variation in the ratios of the contents of ATP/ phatase (Newsholme & Crabtree, 1973), and con- AMP in the muscles studied was about 45-fold (8.8- sequently a cycle catalysed by fructose 402.5). In general, for muscle in which the change in diphosphatase and phosphofructokinase does not the rate of energy expenditure from minimum to operate in this muscle. [The role of this cycle is to maximum values is very large, the ATP/AMP con- amplify the response of fructose 6-phosphate centration ratio is large(e.g. the snap muscle ofPecten, phosphorylation, and hence glycolytic flux, to some of the insect flight muscles, pectoral muscles of changes in the concentration ofAMP (see Newsholme the birds, leg muscles of the rat and mouse; see & Crabtree, 1973)]. The metabolic mechanism for Table 1). For muscle in which the change in rate of control of glycolysis in this muscle is unknown. energy utilization is small, the ATP/AMP con- centration ratio is small (e.g. the foot muscle of the snail, the posterior adductor muscle of the sea Mass-action ratios ofsome reactions mussel, the pectoral muscle of the pigeon, in com- The mean values of mass-action ratios for the parison with those of the pheasant and domestic phosphoglucoisomerase reaction and the adenylate fowl, and the heart muscle of the rat, see Table 2). kinase reaction are 0.3 and 0.5 respectively, for the Similarly, in tissues other than muscle, in which the muscles used in this investigation. The equilibrium change in the rate of glycolysis is probably not very constants for these reactions are 0.3 (Kahana et al., large, the ATP/AMP concentration ratios are very 1960) and 0.44 (Eggleston & Hems, 1952) respec- low (a range of4-23, see Table 2). These relationships tively. Thus the values for the mass-action ratios and are consistent with the theory of control of glycolysis the equilibrium constants are very similar, which in which small changes in ATP concentration produce indicates that these reactions are close to equilibrium 1975 ADENINE NUCLEOTIDES AND PHOSPHAGENS IN MUSCLE 31 in all the muscles that have been investigated (under Table 4. Mean mass-action ratios and S.E.M. values for the conditions of rest). The near-equilibrium nature of reactions catalysed by adenylate kinase, phosphofructo- the adenylate kinase reaction is important, since this kinase, 'phosphagen' kinase andpyruvate kinase indicates that it can play an amplification role in the The mean values ± S.E.M. are calculated from the data in regulation of glycolysis (Newsholme, 1970) in all of Tables 2 and 3. The numbers of individual results used in these muscles. the calculation are given in parentheses. The results also indicate that, for all the muscles studied, the variation in the ratios of these two Reaction Mean mass-action ratio reactions is small, whereas the variation in the Adenylate kinase 0.48 ± 0.037 (21) contents of the individual metabolites is large Phosphofructokinase 0.42±0.19 (18) (e.g. the mass-action ratios for the adenylate Phosphagen kinase 4.8 ± 0.52 (17) kinase reaction vary about 11-fold whereas the Pyruvate kinase 24.8 ± 5.60 (20) ATP/AMP concentration ratio varies about 45-fold). On the other hand the mass-action ratios for the reactions catalysed by phosphofructokinase and 4). These considerations suggest that the phosphagen pyruvate kinase are very much smaller than the kinase reaction is close to equilibrium. The dis- equilibrium constants (the equilibrium constants are crepancy between the mass-action ratios and the approx. 1 x 103 and 1 x 104 respectively), which apparent equilibrium constant for this reaction may indicates that, in all the muscle studied, these enzymes be explained by a difference between the value of the catalyse non-equilibrium reactions. Moreover, for equilibrium constant as measured in vitro and that both reactions the variation in the mass-action ratios which applies to this reaction intracellularly. Factors between the different muscles is very large; it is more such as the ionic strength, pH or Mg2+ concentration than 100-fold for the phosphofructokinase reaction could be different in the cell from those that were used and more than70-foldforthepyruvatekinasereaction. in the measurement of the constant in vitro. It is The concentration ratios of the products/substrates important to know whether the 'phosphagen kinase' (i.e. mass-action ratio) is constrained in a near- enzymes catalyse a reaction close to equilibrium, equilibrium reaction by the value of the equilibrium since the accepted role of phosphagens in buffering constant; any large variation in the substrate con- small changes in the ATP/ADP ratio requires the centration must be 'balanced' by a similar variation in reaction to be near equilibrium. the . No such constraint need apply to non- This work has provided a further method for equilibrium reactions. These considerations predict a discovering whether reactions are near equilibrium or much smaller variation in the mass-action ratios far-removed from equilibrium; mass-action ratios between different tissues for a near-equilibrium for the reaction are measured in a given tissue in a reaction compared with a non-equilibrium reaction. large number of animals and a small variation in the The importance ofthis for the creatine phosphokinase ratios as indicated by the value of the S.E.M. suggests and arginine phosphokinase reactions is discussed that the reaction is near equilibrium. below. The mean mass-action ratio for the arginine We thank Professor J. W. S. Pringle, F.R.S., for his phosphokinase and creatine phosphokinase reactions interest and encouragment. I. B. was the recipient of a from this investigation is 6.0. The equilibrium Wellcome Trust Training Fellowship. constant for the creatine phosphokinase reaction is 100 (Noda et al., 1954); it is assumed that the equili- brium constant for the arginine phosphokinase References reaction is similar. The difference between the Crabtree, B. & Newsholme, E. A. (1970) Biochiem. J. 117, equilibrium constant and mass-action ratios (about 1019-1021 16-fold) might indicate that the reaction is non- Crabtree, B. & Newsholme, E. A. (1972a) Biochem. J. 126, equilibrium in the muscle (see Rolleston, 1972). 49-58 However, the variation between the ratios in 18 Crabtree, B. & Newsholme, E. A. (1972b) Biochem. J. 130, different muscles (and in brain, see Table 2) is very 697-705 small. If the dogfish muscle is excluded, the variation Czok, R. & Eckert, L. (1963) in Methods of Enzymatic is about eight fold, whereas the variation in the Analysis (Bergmeyer, H.-U., ed.), pp. 229-233, Aca- content of different demic Press, New York and London arginine phosphate between Eggleston, L. V. & Hems, R. (1952) Biochem. J. 52, muscles is greater than 100-fold. Further, variation in 156-160 the mass-action ratios for the 'phosphagen' kinase George, J. C. & Berger, A. J. (1966) Avian Myology, reaction is, as indicated by the S.E.M. values for the pp. 30-32, Academic Press, New York and London results presented in Table 2, similar to that for Gevers, W. & Krebs, H. A. (1966) Biochem. J. 98, 720-735 adenylate kinase and is much less than for those for Hems, D. A. & Brosnan, J. T. (1970) Biochem. J. 120, pyruvate kinase or phosphofructokinase (see Table 105-111 Vol. 152 32 I. BEIS AND E. A. NEWSHOLME

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