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.     

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.     

The role of creatine kinase and arginine kinase in muscle.

Arginine and creatine kinase activities in different muscles are compared with calculated maximum rates of ATP turnover. The magnitude of the kinase activities decreases in the following order: anaerobic muscles and vertebrate skeletal muscles greater than heart muscle greater than insect flight muscle. The maximum activity of phosphagen kinases (i.e. creatine kinase and arginine kinase), in the direction of phosphagen formation, is lower than the calculated maximum rate of ATP turnover in insect flight muscle or rat heart.     

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.     

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     

The structure of lombricine kinase: implications for phosphagen kinase conformational changes

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.     

Induced fit in guanidino kinases--comparison of substrate-free and transition state analog structures of arginine kinase

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 A with a final R of 22.3% (R(free) = 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.     

Estimation of effective concentrations of ATP-regenerating enzymes in cilia of Paramecium caudatum

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP‐regenerating enzymes, we attempted to reconstitute certain ATP‐regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high‐energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP‐regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 μM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 μM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.     

Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.     

Creatine ethyl ester: a new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K(m) and V(max) values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with d-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.     

Creatine ethyl ester: A new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K _m and V _max values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Variable intron/exon structure in the oligochaete lombricine kinase gene

Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family.     

Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.     

Preservation of phosphagen kinase function during transient hypoxia via enzyme abundance or resistance to oxidative inactivation

Mammalian lactate dehydrogenase and phosphofructokinase are more susceptible in vitro to superoxide and hydroxyl (·OH) radicals than pyruvate kinase and glucose-6-phosphate dehydrogenase, suggesting that differential inactivation of regulatory enzymes contributes to the metabolic disintegration in stenoxic tissues during transient hypoxia. Like-wise, creatine kinase in smooth muscle from porcine ileum is significantly reduced by hypoxia-reoxygenation ex vivo from 300 (±18.2 SE,n=8) to 196 U·g wet wt^-1 (±16.7,P0.001, ANOVA). Conversely, arginine kinase, from the myocardium ofLimulus polyphemus, a species that tolerates anoxia for days was 2.9-fold less susceptible to oxidative inactivation. To examine whether preservation of kinase function is related to euryoxic capacity, a combination of non-invasive³¹P-NMR spectroscopy and enzyme-linked assays was used to follow ATP and phosphagen status during hypoxia-reoxygenation in porcine ileum smooth muscle,L. polyphemus myocardium, and the myocardium ofArgopecten irradians, a scallop species tolerant of hypoxia for only 24 h. Despite wide differences in phylogeny, euryoxic capacity and oxidative vulnerability of the phosphagen kinases, in all three tissues, the phosphagen pool recovered concomitant with ATP during reoxygenation, thereby revealing competent kinase function. In the mammalian tissue, such preservation of kinase function is facilitated by a 2400-fold excess of enzyme activity.     

Investigations on the function of creatine kinase in Ehrlich ascites tumor cells

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.     

Arginine kinase in Phytomonas, a trypanosomatid parasite of plants

Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer.     

Relative abilities of phosphagens with different thermodynamic or kinetic properties to help sustain ATP and total adenylate pools in heart during ischemia

Hearts of chicks fed the creatine analog, 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine), accumulated 15 mumol/g wet wt of the synthetic phosphagen, cyclocreatine-3-P; had total creatine levels reduced from the normal 6 mumol/g to only 1.8 mumol/g; and had their glycogen levels tripled. During total ischemia in vitro these hearts utilized the cyclocreatine-P for synthesis of ATP, had greatly prolonged glycolysis, and exhibited a two- to fivefold delay in depletion of both ATP and the total adenylate pool, relative to controls. Accumulation from the diet of comparable levels of the closely related 1-carboxyethyl-2-imino-3-phosphonoimidazolidine (homocyclocreatine-P) by heart was accompanied by only slight lowering of total creatine to 4.2 mumol/g, and a tripling of glycogen levels. During ischemia these hearts exhibited prolonged glycolysis, but they did not utilize the very stable homocyclocreatine-P (200,000-fold less reactive than creatine-P) and thus formed less Pi; most significantly, there was no delay in depletion of ATP levels relative to controls. Feeding of creatine doubled total creatine levels in heart, but had no marked effect on ATP depletion during ischemia; in all dietary groups creatine-P pools had fallen to less than or equal to 1.2 mumol/g by first tissue sampling. Although adaptive responses were also involved, maximal conservation of ATP and total adenylate pools in heart during ischemia apparently required, in addition to adequate glycogen reserves, substantial levels of a kinetically competent phosphagen that is thermodynamically poised to continue to assist glycolysis in buffering decreases and oscillations in the [ATP]/[free ADP] ratio at the lower phosphorylation potentials and more acid pH characteristic of later stages of ischemia. Decreases and oscillations in the [ATP]/[free ADP] ratio cannot be buffered effectively late in ischemia by the creatine-P system for thermodynamic reasons, or by the homocyclocreatine-P system because of kinetic limitations.     

Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification,tuning and sensing of energetic signals in the compartmentalized cellular environment

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+ (KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes.     

Freezing and cellular metabolism in the gall fly larva,Eurosta solidaginis

Summary The effects of extracellular freezing on intracellular metabolism were monitored over both a short (9 h) and long (12 weeks) time course using the freeze tolerant larvae of the gall fly,Eurosta solidaginis.The process of freezing, monitored over the short time course, had no effect upon cellular energy levels (adenylates, arginine phosphate) but initiated a rise in glucose-6-P and lactate levels. This suggests that freezing initiates a shift towards glycolysis as the predominant mode of energy production. The process of thawing at 3°C (after 24 h at –16°C) also had no effect, even transient, on cellular energy levels demonstrating that thawing and the rapid redistribution of water and solutes which must accompany it does not disrupt cellular metabolism. During thawing accumulated lactate was quickly cleared with a t 1/2 of 20–30 min.Long term freezing at –16°C had dramatic effects on energy metabolism. Freezing for up to 1 week had minimal effects with only a small drop in arginine phosphate reserves and an increase in lactate content noted. Between 1 and 2 weeks of freezing, however, larvae showed strong signs of energy stress. The arginine phosphate pool fell from 75% to 30% of control levels, ATP content dropped by 50% and energy charge dropped to 0.75. This state, with continued lactate accumulation, was maintained through 4 weeks of freezing. Between 6 and 12 weeks of freezing energy stress became even greater. Phosphagen and ATP contents dropped to 5 and 25% of control values and energy charge decreased to about 0.50. Despite this stress, however, 94% of larvae survived 12 weeks of freezing with an 86% hatch rate of adults. The data demonstrate that the larvae can survive prolonged periods of winter freezing drawing upon glycolysis and phosphagen reserves to supply the continued basal energy demands of the cell.