The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates.

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 flight 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 of energy 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 kinase reaction in the regulation of glycolysis. However, in the flight muscle of the honey-bee the ATP/AMP ratio is very low, so that glycolysis may be regulated by factors other than the variation in AMP concentration. The variation in the contents of arginine phosphate in muscle from the invertebrates is much larger than the variation in creatine phosphate in muscle from the vertebrates. 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 in which it is about 4-10-fold higher. The mass-action ratios for the reactions catalysed by phosphoglucoisomerase and adenylate kinase are very similar to the equilibrium constants for these reactions. Further, the variation in the mass-action ratios between muscles is small. It is concluded that these enzymes catalyse reactions close to equilibrium. However, the mass-action ratios for the reactions catalysed by phosphofructokinase and pyruvate kinase are much smaller than the equilibrium constants. The variation in the ratios between different muscles is large. It is concluded that these enzymes catalyse nonequilibrium reactions. Since the variation in the mass-action ratios for the reactions catalysed by the phosphagen kinases (i.e. creatine and arginine phosphokinases) is small, it is suggested that these reactions are close to equilibrium.     

The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and the glycerol 3-phosphate dehydrogenase in muscles from vertebrates and invertebrates

1. The maximum activities of hexokinase, phosphorylase and phosphofructokinase have been measured in extracts from a variety of muscles and they have been used to estimate the maximum rates of operation of glycolysis in muscle. These estimated rates of glycolysis are compared with those calculated for the intact muscle from such information as oxygen uptake, glycogen degradation and lactate formation. Reasonable agreement between these determinations is observed, and this suggests that such enzyme activity measurements may provide a useful method for comparative investigations into quantitative aspects of maximum glycolytic flux in muscle. 2. The enzyme activities from insect flight muscle confirm and extend much of the earlier work and indicate the type of fuel that can support insect flight. The maximum activity of hexokinase in some insect flight muscles is about tenfold higher than that in vertebrate muscles. The activity of phosphorylase is greater, in general, in vertebrate muscle (particularly white muscle) than in insect flight muscle. This is probably related to the role of glycogen breakdown in vertebrate muscle (particularly white muscle) for the provision of ATP from anaerobic glycolysis and not from complete oxidation of the glucose residues. The activity of hexokinase was found to be higher in red than in white vertebrate muscle, thus confirming and extending earlier reports. 3. The maximum activity of the mitochondrial glycerophosphate dehydrogenase was always much lower than that of the cytoplasmic enzyme, indicating that the former enzyme is rate-limiting for the glycerol 3-phosphate cycle. From the maximum activity of the mitochondrial enzyme it can be calculated that the operation of this cycle would account for the reoxidation of all the glycolytically produced NADH in insect flight muscle but it could account for only a small amount in vertebrate muscle. Other mechanisms for this NADH reoxidation in vertebrate muscle are discussed briefly.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

The activities of lipases and carnitine palmitoyl-transferase in muscles from vertebrates and invertebrates

1. The activities of tri-, di- and mono-glyceride lipase and carnitine palmitoyltransferase were measured in homogenates of a variety of muscles. These activities were used to estimate the rate of utilization of glycerides and fatty acids by muscle. In muscles whose estimated rates of fat utilization can be compared with rates calculated for the intact muscle from such information as O₂ uptake, there is reasonable agreement between the estimated and calculated rates. 2. In all muscles investigated the maximum rates of hydrolysis of glycerides increase in the order triglyceride, diglyceride, monoglyceride. The activity of diglyceride lipase is highest in the flight muscles of insects such as the locust, waterbug and some moths and is lowest in the flight muscles of flies, bees and the wasp. These results are consistent with the utilization of diglyceride as a fuel for some insect flight muscles. 3. In many muscles from both vertebrates and invertebrates the activity of glycerol kinase is similar to that of lipase. It is concluded that in these muscles the metabolic role of glycerol kinase is the removal of glycerol produced during lipolysis. However, in some insect flight muscles the activity of glycerol kinase is much greater than that of lipase, which suggests a different role for glycerol kinase in these muscles.     

Phosphagen kinase evolution. Expression in echinoderms

Arginine kinase and creatine kinase that catalyze the transfer of a phosphate group between ATP and arginine and creatine, respectively, play an important role in cellular energetics. In contrast to most animals which exhibit a single phosphagen kinase activity (creatine kinase in chordates and arginine kinase in protostomians), echinoderms exhibit both arginine kinase and creatine kinase activities, sometimes in the same tissue. In contrast to chordates in which creatine kinases are dimers (consisting of two subunits of 40 kDa) and protostomians in which arginine kinases are usually monomers (40 kDa), echinoids contain specific phosphagen kinases: a dimeric arginine kinase (consisting of two subunits of 42 kDa) in eggs and a monomeric creatine kinase (145 kDa) in sperm. We have examined echinoderms from the five existing classes (echinoids, asteroids, ophiuroids, holothurians and crinoids) for the expression of these specific phosphagen kinases in different tissues. Gel filtration was used to determine the molecular masses of the native enzymes. Antibodies specific for arginine kinase or for creatine kinase were used to characterize the subunit composition of arginine kinase and creatine kinase after SDS/PAGE and transfer. In all echinoderms analyzed, arginine kinase always occurred as an enzyme of about 81 kDa consisting of two subunits of 42 kDa and creatine kinase as a monomeric enzyme of 140-155 kDa. The occurrence in echinoderms of both phosphagen kinases with distinct specificities and specific molecular structures is discussed from both a developmental and evolutionary point of view.     

31P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creatine kinase activities similar to those found in mammalian heart muscle. 31P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06 and 0.1 measured directly in cell extracts.     

Inverse metabolic engineering with phosphagen kinase systems improves the cellular energy state

Inverse metabolic engineering attempts to identify or construct desired phenotypes of applied interest to endow them on appropriate host organisms. A particular desirable phenotype is the ATP homeostasis exhibited by animal cells with high and variable ATP turnover through temporal and spatial energy buffering. This buffering is achieved by phosphagen kinase systems that consist of a specific kinase and its cognate phosphagen, which functions as a large pool of 'high-energy phosphates' that are used to replenish ATP during periods of high energetic demand. This review discusses recent advances and potentials of inverse metabolic engineering of cell types that do not normally contain such systems--bacteria, yeast, plants, and liver--with creatine or arginine kinase systems. Examples are discussed that illustrate how microbial metabolism can be tailored for large-scale industrial processes with imperfect mixing and how the liver can be protected from metabolic insults or stimulated for better regeneration.     

Regulation of creatine kinase during steady-state isometric twitch contraction in rat skeletal muscle

In vivo 31P-NMR saturation transfer measurements of the creatine kinase exchange flux in the direction creatine phosphate----ATP were made in the gastrocnemius muscle of rats at rest and during steady-state isometric twitch contraction at frequencies from 0.25 to 2 Hz. There was no correlation between creatine kinase exchange flux and either free [ADP] or oxygen consumption, both of which increase with stimulation frequency. The flux was found to be nearly constant over all conditions at about 16 mM X s-1, 10-times greater than the highest estimated ATP turnover in this study. The kinetic properties of skeletal muscle creatine kinase in vivo are similar to, but not completely predictable from, the equilibrium exchange fluxes measured on the isolated enzyme. These results are not consistent with strong functional coupling between ATP synthesis and mitochondrial creatine kinase.     

Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.     

Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed on a diet containing 1% beta-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. Maximal activities of creatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions of the gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. Fast-twitch muscles were smaller in GPA-fed animals than in controls, but the size of the soleus muscle was unchanged. The activities of aerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. The proportion of Type I fibres in the soleus muscle increased from 81% in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response.     

Glycerol kinase activities in muscles from vertebrates and invertebrates

1. Glycerol kinase (EC 2.7.1.30) activity was measured in crude extracts of skeletal muscles by a radiochemical method. The properties of the enzyme from a number of different muscles are very similar to those of the enzyme from rat liver. Glycerol kinase from locust flight muscle was inhibited competitively by l-3-glycerophosphate with a K(i) of 4.0x10(-4)m. 2. The activity of glycerol kinase was measured in a variety of muscles from vertebrates and invertebrates in an attempt to explain the large variation in the activity of this enzyme in different muscles. 3. In vertebrates glycerol kinase activities were generally higher in red muscle than in white muscle; the highest activities (approx. 0.2mumole/min./g. fresh wt.) were found in the red breast muscle of some birds (e.g. pigeon, duck, blue tit) whereas the activities in the white breast muscle of the pheasant and domestic fowl were very low (approx. 0.02mumole/min./g.). 4. On the basis of glycerol kinase activities, muscles from insects can be classified into three groups: muscles that have a low enzyme activity, i.e. <0.3mumole/min./g. (leg muscles of all insects studied and the flight muscles of cockroaches and the tsetse fly); muscles that have an intermediate enzyme activity, i.e. 0.3-1.5mumoles/min./g. (e.g. locusts, cockchafers, moths, water-bugs); and muscles that have a high enzyme activity, i.e. >1.5mumoles/min./g. (e.g. bees, wasps, some blowflies). 5. The function of glycerol kinase in vertebrate and insect muscles that possess a low or intermediate activity is considered to be the removal of glycerol that is produced from lipolysis of triglyceride or diglyceride by the muscle. Therefore in these muscles the activity of glycerol kinase is related to the metabolism of fat, which is used to support sustained muscular activity. A possible regulatory role of glycerol kinase in the initiation of triglyceride or diglyceride lipolysis is discussed. 6. The function of glycerol kinase in the insect muscles that possess a high activity of the enzyme is considered to be related to the high rates of glycolysis that these muscles can perform. The oxidation of extramitochondrial NADH, and therefore the maintenance of glycolysis, is dependent on the functioning of the glycerophosphate cycle; if at any stage of flight (e.g. at the start) the rate of mitochondrial oxidation of l-3-glycerophosphate was less than the activity of the extramitochondrial glycerophosphate dehydrogenase, this compound would accumulate, inhibit the latter enzyme and inhibit glycolysis. It is suggested that such excessive accumulation of l-3-glycerophosphate is prevented by hydrolysis of this compound to glycerol; the latter would have to be removed from the muscle when the accumulation of l-3-glycerophosphate had stopped, and this would explain the presence of glycerol kinase in these muscles and its inhibition by l-3-glycerophosphate.     

Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice

To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.     

Myofibrillar creatine kinase: reversible binding to contractile proteins, stoichiometric ratio to myosin and its functional role

Rat heart myofibrils were isolated and purified in three different media: sucrose medium; EGTA medium; EGTA+ATP medium. All preparations were characterized by similar Ca2+-sensitive ATPase activities and were practically free of mitochondrial and sarcolemmal contaminations. However, they contained different amounts of creatine kinase. In preparations which showed the most intact ultrastructure, the activity of creatine kinase was 0.99 +/- 0.12 IU/mg. It was found that creatine kinase can be bound to myofibrils in a reversible manner with Kd = 0.16 mg/ml = 1.8 X 10(-6) M; the creatine kinase/myosin ratio was estimated to be approximately 1:10. The localization of creatine kinase was found to be a basis for the high turnover rate of ATP in the coupled creatine kinase and ATPase reactions occurring in cardiac myofibrils.     

Studies on phosphagen synthesis by mitochondrial preparations

Mitochondrial preparations from muscles of a crab (Cancer pagurus), two fish (Trachurus trachurus and Scyliorhinus canicula) and a bird (Columba livia) are able to synthesise, through ATP, the phosphagen related to that species. This indicates the presence of a bound phosphagen kinase. Addition of creatine kinase and creatine to crab mitochondria results in the synthesis of phosphocreatine. Similarly, the addition of arginine kinase and arginine to mitochondrial preparations from the fish and bird results in the synthesis of phosphoarginine. In the crab, the mitochondrial form of arginine kinase released by sonication had the same kinetic affinity constants and electrophoretic mobility and could not be distinguished immunologically from the cytosolic form. The close similarity of bound and cytosolic forms of arginine kinase in this crustacean suggests that the two forms have not evolved separately as has creatine kinase in the mammal.     

Analysis of compartmentation of ATP in skeletal and cardiac muscle using 31P nuclear magnetic resonance saturation transfer.

We have developed a model for the analysis of the forward creatine kinase reaction in muscle as measured by the nuclear magnetic resonance (NMR) technique of magnetization transfer. The model, accounting for the double-exponential behavior observed in some NMR magnetization transfer data, allows for the existence of two ATP pools, one that is NMR-visible (NMR-VIS) and another that is NMR-invisible (NMR-INVIS). We have applied the model to experimental data for the forward creatine kinase reaction in skeletal and cardiac muscles to study the dependence of the creatine kinase rate constants and fluxes on workload and to account for the differences between heart and skeletal muscle. The results suggest that an NMR-distinct ATP pool exists in both heart and skeletal muscles, and that phosphate exchange with this pool catalyzed by creatine kinase increases with increased workload. The results also agree with previously published estimates of the rates of mitochondrial translocase and net ATP synthesis obtained by traditional biochemical methods.     

Impaired intracellular energetic communication in muscles from creatine kinase and adenylate kinase (M-CK/AK1) double knock-out mice

Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency.     

Comparison of the two different phosphagen systems in the lugworm Arenicola marina

The different phosphagen systems in the lugworm Arenicola marina, the phosphotaurocyamine/taurocyamine kinase system of the body wall and the phosphocreatine/creatine kinase system of the spermatozoa, have been investigated to answer the question whether the change reflects different functional modes of these phosphagen systems. Enzyme analyses have shown that in contrast to the body wall taurocyamine kinase, creatine kinase of spermatozoa exists in at least two different forms which are compartmented in the mitochondria (creatine kinase I) and in the flagellum (creatine kinase II). Creatine kinase I is strongly attached to cell structures which require detergents and high phosphate concentrations for solubilization. The affinities of taurocyamine kinase and creatine kinase for all substrates are very similar except the extremely high K _m for creatine of both creatine kinase I and II. The level of creatine in spermatozoa is fivefold higher than taurocyamine in the body wall at similar phosphorylation potential (ATP/ADO_free) and ATP-buffer capacity (phosphagen/ATP), reflecting the higher equilibrium constants of the creatine kinase reaction compared to that of the taurocyamine kinase reaction (Ellington 1989). The high creatine concentration gives the phosphocreatine/creatine kinase system an advantage over the phosphotaurocyamine/taurocyamine kinase system for transport of energyrich phosphate at high phosphorylation potential by increasing the radial diffusion flux. The maximum diffusive flux of free ADP in spermatozoa is three orders of magnitude below the respiratory ATP production while the creatine flux would allow an unlimited energy transport over the long diffusion distance. In lugworm body wall, however, the low ATP turnover and the low diffusion distances between mitochondria and myosin-ATPases do not require a phosphagen shuttle.Abbreviations ADP _free cytoplasmic adenosine diphosphate - Ap ₅ A P₁, P₅-di(adenosine-5-) pentaphosphate - AK arginine kinase - CK creatine kinase (EC 2.7.3.2) - DTT dithiothreitol - GAPDH glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) - HOADH 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) - IEP isoelectric point - MIM mitochondria isolating medium - P _i-free cytoplasmic inorganic phosphate - (P)Arg (phospho)arginine - (P)Cr (phospho)creatine - (P)Tc (phospho)taurocyamine - SEM scanning electron microscopy - TK taurocyamine kinase - TEM transmission electron microscopy     

Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement

Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1-catalyzed ss-phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up-regulation of high-energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1-deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1(-/-) muscles. Thus, AK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.