Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica

Cryobiology xxx (2017) 1e9

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Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica Michael B. Smolinski, Jessica J.L. Mattice, Kenneth B. Storey* Institute of Biochemistry, Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 January 2017 Received in revised form 25 May 2017 Accepted 5 June 2017 Available online xxx

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (Km ¼ 0.098 ± 0.003 and Km ¼ 0.092 ± 0.008) than PK from control and anoxic conditions (Km ¼ 0.065 ± 0.003 and Km ¼ 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress. © 2017 Elsevier Inc. All rights reserved.

Keywords: Pyruvate kinase Rana sylvatica Freeze tolerance Metabolic rate depression

1. Introduction The freeze tolerant wood frog (Rana sylvatica) displays some of the most remarkable survival mechanisms of all cold-hardy animals. Sheltering in the subnivean space beneath an insulating layer of leaf litter and snow, it is one of the few vertebrates known to survive long-term whole-body freezing at temperatures as low as 18  C [21]. Besides low temperatures, wood frogs are also faced with a cessation of breathing and blood circulation, resulting in ischemic and anoxic conditions that ultimately contribute to oxidative damage and impaired access to nutrients [11,18,34]. Unsurprisingly, then, a variety of metabolic and physiological changes are necessary for the animals' survival. These include a global metabolic rate depression, the coordinated accumulation of up to 70% of the animal's total body water as ice in extracellular spaces, and the accumulation of intracellular glucose to both depress cellular freezing temperatures and limit cellular dehydration [7,34,37]. As a colligative cryoprotectant, glucose limits the amount

* Corresponding author. E-mail address: [email protected] (K.B. Storey).

of cellular volume reduction due to extracellular ice formation and therefore prevents the shrinking of cells. It can accumulate to levels up to 400 mM during freezing, up from 1 to 5 mM when unfrozen [36,37]. The metabolism of wood frogs therefore may be highly regulated to support the accumulation and maintenance of these concentrations of glucose during freezing. As well as acting as a cryoprotectant in the wood frog, glucose, and carbohydrates in general, are a primary energy source during periods of freezing via glucose fermentation resulting in lactate production [22,36]. To ensure that the limited stores of carbohydrate energy are sufficient to meet the needs of the animals during dormancy, metabolic rate is depressed significantly and a new equilibrium of ATP-production and ATP-consumption must be established. This is in part mediated by the regulation of metabolic enzymes. Hexokinase (HK), for example, exhibits a significant decrease in both activity and substrate affinity in response to freezing which limits the rate of muscle glucose catabolism during freezing [16]. There is also evidence suggesting that glycolysis is further limited via phosphofructokinase inhibition in response to subzero temperatures [33]. By inhibiting these enzymes, glycolytic flux is suppressed along with the associated energy production.

http://dx.doi.org/10.1016/j.cryobiol.2017.06.002 0011-2240/© 2017 Elsevier Inc. All rights reserved.

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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M.B. Smolinski et al. / Cryobiology xxx (2017) 1e9

Indeed, enzymatic regulation plays a role in reducing the metabolic rate. However, many enzymes contributing to this phenomenon have yet to be investigated. The final catalytic enzyme of the glycolytic pathway, pyruvate kinase (PK, EC 2.7.1.40), is one of the primary regulatory points of glycolysis and carbohydrate metabolism. While PK regulation is important in controlling levels of glycolytic intermediates and ATP, studies of PK inhibition also correlated with increased rates of gluconeogenesis suggesting a mechanism of promoting intracellular glucose accumulation [20, 24, 28]. Although PK can be regulated transcriptionally and post-transcriptionally in response to dietary changes and hypoxic stress [30,43], its regulation has been primarily studied at the allosteric and post-translational levels [23, 32, 42,44]. Indeed, both of these modes of regulation have been shown to modulate PK activity in response to environmental stress, including anoxia and dehydration exposure, to conserve energy while animals are in a hypometabolic state [9,27,29]. In these cases, PK is found to be regulated commonly via reversible phosphorylation or allosteric inhibition to limit energy consumption when resources are scarce. The aim of this study is to determine what role PK may play in regulating glucose metabolism and the energetic needs of muscle tissue of R. sylvatica in response to freezing and the associated dehydration and anoxia exposure. 2. Materials and methods

2.3. Pyruvate kinase assay Pyruvate kinase activity was measured by coupling its reaction to the consumption of NADH by lactate dehydrogenase, which was measured using a microplate reader (Multiskan Spectrum, Thermo Labsystems, Finland) measuring absorbance at 340 nm. Optimal assay conditions were found to be 50 mM Tris (pH 7.2), 20 mM KCl, 2 mM ADP, 5 mM MgCl2, 1 mM PEP, 1 U of LDH and 0.15 mM NADH. Reactions were carried out in a total volume of 200 mL. Assays were initiated through the addition of PK. Vmax and Km values were determined at optimal assay conditions, as stated previously. The effects of various potential inhibitory and activating molecules (urea, ATP, AMP, FBP, alanine, aspartate, and glucose) were assayed at optimal conditions in the presence of increasing concentrations of the metabolites of interest. Low-temperature assays were carried out by placing the spectrophotometer in a VWR International BOD 2020 Incubator (Sheldon Manufacturing Inc., OR, USA) set to the desired temperature and left to equilibrate to either 13  C or 5  C. All assay reagents and buffers were also equilibrated at the corresponding temperature prior to starting the assay. Temperatures of reagents and incubator were monitored using a telethermometer (Traceable, TX, USA). To determine the activation energy (Ea) of the enzyme, the activity of PK was measured over a range of temperatures from 5  C to 25  C. The activation energies of PK purified from control and frozen muscle tissue were then determined using the Arrhenius equation, as previously described [1].

2.1. Animal treatment 2.4. Purification of pyruvate kinase Male wood frogs (R. sylvatica) were captured in early April from breeding ponds in the Ottawa area. Animals were washed and kept in plastic containers with a damp moss bed at 5  C for two weeks (control condition) before being subjected to experimental conditions. For freezing conditions, frogs were placed in a plastic box lined with damp paper towel and placed in an incubator set to -4  C for 24 h before sampling. Dehydration experiments were performed as previously described [6]. Briefly, frogs were placed in desiccators held at 5  C lined with a calcium sulfate desiccant that simulated dehydration conditions. A 1 cm-thick layer of sponge separated the frogs from the desiccant. Frogs were weighed at regular intervals until they had lost 40% of total body water. Frogs were subjected to anoxic conditions by being placed in sealed containers flushed with nitrogen gas. A small amount of water was left in the bottom of the containers to maintain hydration throughout the procedure. Animals were held in these containers for 24 h at 5  C. Control and experimental animals were sacrificed by pithing followed by rapid dissection. Tissues were flash-frozen in liquid nitrogen and stored at 80  C until use. All experiments performed were in accordance with the Carleton University Animal Care Committee (B09-22) and abided by the guidelines set down by the Canadian Council on Animal Care. 2.2. Preparations of tissue PK was isolated from both control and frozen samples of hindleg skeletal muscle of R. sylvatica. Tissue samples were homogenized 1:5 w:v with ice-cold buffer A (10 mM MES, 2 mM EDTA, 2 mM EGTA, 15 mM b-glycerophosphate, 15 mM b-mercaptoethanol (b-MeSH), 10% v:v glycerol, pH 6.0), with a few crystals of phenylmethylsulphonyl fluoride (PMSF). Samples were homogenized completely on ice using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY, USA) and centrifuged in a pre-chilled Eppendorf 5810 R tabletop centrifuge (22331 Hamburg, GER) for 30 min at 13 500  g and 5  C. The supernatant (crude homogenate) was decanted and held on ice until use.

A 1 mL aliquot of crude homogenate was added to a CM sephadex column (0.75 cm  10 cm) that had been previously equilibrated with 20 mL of buffer A. The column was then washed with 15 mL of buffer B (10 mM MOPS, 2 mM EDTA, 2 mM EGTA, 15 mM b-glycerophosphate, 15 mM b-MeSH, and 10% v:v glycerol, pH 6.75) and the enzyme was eluted with 30 mL of a 0.5 mM PEP solution made in buffer B. Using an automated fraction collector (Gilson Medical Electronics, Inc., Middleton, WI, USA) 1.2 mL fractions of column eluant were collected. To determine where PK eluted from the column, 10 mL of each fraction was taken and used to measure PK activity spectrophotometrically as stated previously. Peak activity fractions were pooled, reduced to pH 6.0 and diluted 3:1 v:v with buffer C (10 mM MES, 2 mM EDTA, 2 mM EGTA, 15 mM b-glycerophosphate, 15 mM b-MeSH, and 10% v:v glycerol, pH 5.9). The pooled fractions were then added to a second CM sephadex column, identical to the first and equilibrated in buffer A. PK was eluted with a 0e1 M KCl gradient made in buffer A and collected in 1.2 mL fractions which were then assayed for PK activity using 10 mL from each fraction, as before. Peak fractions were pooled and used for all subsequent experiments. Protein concentrations were determined using Coomassie blue G-250 dye-binding reagent (BioRad, Hercules, CA; Cat #500e0006) as instructed by the manufacturer using bovine serum albumin as a standard [2]. 2.5. Dot blots Dot blots were used to identify and quantify the posttranslational modifications on purified PK from control and frozen tissue using a Bio-Dot Microfiltration apparatus (Bio-Rad, Hercules, CA, USA). Nitrocellulose membranes were hydrated in TBST (20 mM Tris-base, pH 7.6, 140 mM NaCl, 0.05% v:v Tween-20) and placed in the Bio-Dot apparatus according to manufacturer's instructions. Purified PK samples (5 mg of protein) were applied to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and allowed to filter through the membrane by gravity flow. Membranes were then

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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washed twice with 100 mL of ddH2O and removed from the apparatus. Membranes were blocked with a 1.5% (w:v) skim milk protein solution made in TBST solution for 20 min with gentle rocking at room temperature. The blocking solution was removed and the membranes were washed three times for 5 min (3  5 min) in TBST. Membranes were then incubated overnight in one of the following primary antibodies from Invitrogen, Carlsbad, USA: Rabbit antiphosphoserine (Cat. #618100; 0.25 mg/mL), rabbit antiphosphothreonine (Cat. #718200; 0.25 mg/mL), mouse antiphosphotyrosine (Cat. #615800; 0.25 mg/mL), or rabbit anti-panacetyl (CR)-R (Cat. # sc-8663-R; 0.2 mg/mL; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All primary antibodies were diluted 1:1000 in TBST. Primary antibody was removed the following day and the blots were washed (3  5 min) in TBST prior to incubation in goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bioshop, Cat. #APA007P.2) for 45 min at room temperature with gentle rocking. Secondary antibody was diluted 1:5000 in TBST. Membranes were washed in TBST (3  5 min) before being visualized by enhanced chemiluminescence with the ChemiGenius BioImaging System. Membranes were then stained with Coomassie blue to be used to standardize the chemiluminescence signals. Blot quantification and analysis was performed using Genetools software (Syngene, v 4.02). 2.6. SDS polyacrylamide gel electrophoresis Purity and identity of PK were confirmed using SDS-PAGE and western blotting experiments. Following purification of PK, purified samples were mixed in a 1:1 (v:v) ratio of SDS-PAGE loading buffer (100 mM Tris-base, pH 6.8, 4% w:v SDS, 20% v:v glycerol, 0.2% w:v bromophenol blue and 10% v:v b-MeSH) and were boiled for 5 min before being stored at 20  C until use. Purified PK samples (2 mg of protein) were loaded onto a 10% SDS polyacrylamide gel and run at a constant voltage of 180 V for 55 min at room temperature. A protein molecular weight ladder (Froggabio, Cat. #PM005-0500) and a commercially purified sample of rabbit muscle pyruvate kinase (Sigma Life Sciences, Cat. #P1506) were also run on every gel. Following the completion of gel electrophoresis, gels were either stained directly in Coomassie blue stain to determine protein purity (0.25% w:v Coomassie brilliant blue, 7.5% v:v acetic acid, 50% methanol) or transferred to PVDF membranes for western blotting. For direct Coomassie blue staining, gels were initially fixed for 1 h in fixing solution (10% v:v acetic acid, 25% (v:v) methanol) before being rinsed gently with ddH2O and then stained completely in Coomassie blue solution overnight with gentle rocking. The stain solution was then removed, and the gels were rinsed with ddH2O before being submerged in destain solution (10% v:v acetic acid, 25% v:v methanol) with gentle rocking until protein bands could be seen clearly. The gels were then rehydrated in ddH2O and imaged using Chemi-Genius BioImaging System (Syngene, Frederick, MD). 2.7. Western blotting Western-blotting was used to compare the levels of serine phosphorylation of control, dehydrated and anoxic forms of PK. For western-blotting, after the SDS-PAGE was run the proteins were transferred to PVDF membranes using a wet transfer procedure as follows: PVDF membranes were first equilibrated in methanol prior to the protein transfer. Electroblotting was carried out in transfer buffer (25 mM Tris, pH 8.5, 192 mM glycine, and 20% v:v methanol) at a constant amperage of 160 mA for 90 min and were maintained at 5  C throughout. Membranes were then removed from the apparatus, washed in TBST (3  5 min) and blocked in 1.5% w:v milk protein solution for 20 min followed by further washing with TBST (3  5 min). All incubations in primary and secondary antibody,

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visualization and quantification procedures were performed as described previously for Dot Blots. 2.8. Incubation experiments Incubation experiments were performed to determine the stability of PK under extreme conditions of high temperature, or high urea concentrations. Aliquots of 750 mL of purified enzyme samples were incubated at 52  C using a dry block heater (Fisher Scientific, Dubuque, IA, USA) for up to 90 min, with or without 400 mM glucose present. At the specified time intervals, 25 mL aliquots of sample were removed from the stock sample and immediately placed on ice. Following the removal of the final aliquot after 90 min, samples were left to equilibrate to room temperature before being assayed at optimal conditions. The reported value (tm) is the incubation time that resulted in a Vmax reduced to 50% of the non-incubated value. Urea incubations were performed by incubating samples of purified PK in increasing concentrations of urea (up to 3 M) for a 90 min period at room temperature, with or without 400 mM glucose present. Following completion of the 90 min incubation period, samples were immediately assayed at optimal conditions. The reported value (Cm) is the concentration of urea that resulted in a Vmax that had been reduced to 50% of the original Vmax, following the 90 min incubation. 2.9. Data and statistical analyses All enzyme assays were analyzed using a microplate analysis program [4] while kinetic parameters (I50, Km, Cm and tm values) were determined using the Kinetics v. 3.51 program [3]. Data for all kinetic parameters and dot blots comparing the control and frozen samples were analyzed using the Student's t-test (two-tailed). Data for all kinetic parameters and western blots comparing the control, frozen, dehydrated and anoxic forms of PK were compared using a one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. Results were deemed significant when the resulting pvalue from t-tests or ANOVAs was less than 0.05. The statistical analysis software used for t-tests and ANOVAs was Sigmaplot version 12. 3. Results 3.1. Purification of pyruvate kinase PK was successfully purified from control and frozen samples of R. sylvatica skeletal muscle by column chromatography using two consecutive CM sephadex columns. PK was eluted from the first column using 0.5 mM PEP in buffer B (pH 6.75) followed by elution from the second CM column using a 0e1 M KCl gradient. The first CM column gave an 8.35-fold purification (47% yield) with a specific activity of 3.91 U/mg, while the second and final step resulted in a final 10.24-fold purification with a specific activity of 4.79 U/mg and a final 31% yield (Table 1). This two-step purification resulted in PK being purified to >95% purity as determined by SDSPAGE and staining with coomassie blue. The purified PK had a molecular weight of 63 kDa (Fig. 1). 3.2. Kinetic activity of PK The Vmax and total activity of PK did not differ significantly between the control and frozen forms of the enzyme at any temperature tested (5  C, 13  C, or 22  C) in either purified or crude samples (Table 2). Relative to the control condition (0.065 ± 0.003 mM), the KmPEP of PK increased significantly in the

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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M.B. Smolinski et al. / Cryobiology xxx (2017) 1e9

Table 1 Typical purification scheme of PK from control R. sylvatica muscle tissue. Purification step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Fold purification

% Yield

Crude CM PEP CM KCl

9.61 0.54 0.29

4.50 2.11 1.39

0.468 3.907 4.793

e 8.348 10.24

e 46.89 30.89

consistently found to be significantly higher in the frozen tissue than in the control tissue with one exception; the addition of 5 mM AMP. When 5 mM AMP was added the control and frozen KmPEP values did not differ significantly. Addition of both urea and ATP had an inhibitory effect on the Vmax of PK, though the resulting I50 values were the same for both control and frozen conditions (Average I50 urea ¼ 0.43 M; Average I50 ATP ¼ 11.1 mM; Table 4). Additionally, the inclusion of 400 mM glucose in the assay well resulted in a nearly 4-fold increase of the I50 urea for both control and frozen PK (Table 4). AMP, FBP, alanine and aspartate (up to 25 mM) and glucose (up to 750 mM) had no effect, activating or inhibitory, on the Vmax of PK (Table 4).

3.3. Post-translational modifications of PK

Fig. 1. 10% SDS-PAGE gel showing entire purification of PK from control muscle of R. sylvatica. Lanes (1): Commercially available purified rabbit PK (positive control; Sigma life sciences); (2) molecular weight ladder (Froggabio); (3): crude homogenate; (4) pooled peak fraction after elution from CM Sephadex column with PEP; (5) pooled peak fraction after elution from CM Sephadex column with KCl.

frozen (0.098 ± 0.003 mM; p ¼ 0.005) and dehydrated (0.092 ± 0.008 mM; p ¼ 0.0196) conditions while no significant change was observed for the anoxic (0.073 ± 0.002) condition (Table 2). No significant difference was observed in the KmADP of PK (Average ¼ 0.22 mM) or the activation energy (Average ¼ 35.12 kJ/mol) of the reaction between the control and frozen conditions (Table 2). The addition of various potential allosteric effectors (400 mM glucose, 5 mM ATP, 5 mM AMP, 2 mM FBP) did not significantly alter the KmPEP of PK of control or frozen animals. However, the presence of 100 mM urea resulted in a significant increase in the KmPEP under both control (0.5-fold increase; p ¼ 0.0012) and frozen conditions (0.7-fold increase; p ¼ 0.012). The KmPEP was

Immunoblotting using a dot blot apparatus was used to assess the differences in the post-translational modifications of purified muscle PK from control and frozen conditions. Phosphorylation via serine residues of the frozen form of PK was 0.5-fold (p ¼ 0.02) higher than the control form of PK (Fig. 2). The phosphorylation levels of threonine and tyrosine residues as well as total acetylation levels between control and frozen PK did not differ significantly. Western blotting was used to investigate serine phosphorylation under anoxic and dehydration conditions, as well. Serine phosphorylation of PK from dehydrated animals was found to be 25% higher than that from control animals (p ¼ 0.004) while no difference was observed for anoxic animals (Fig. 3).

Table 3 Effects of various substrates and allosteric effectors on the Km PEP of purified PK from control and frozen muscle samples of R. sylvatica. Data are mean ± SEM, n ¼ 4. Kinetic Parameter Km Km Km Km Km

PEP PEP PEP PEP PEP

Control

400 mM Glucose (mM) 5 mM ATP (mM) 5 mM AMP (mM) 2 mM FBP (mM) 100 mM Urea (mM)

0.07 0.06 0.07 0.07 0.10

± ± ± ± ±

0.006 0.004 0.006 0.004 0.004

Frozen

b

0.10 0.08 0.08 0.10 0.17

± ± ± ± ±

0.007 a 0.003 a 0.004 0.003 a 0.01 5 ab

a indicates significantly different from the corresponding control value, Student's ttest, p < 0.05. b indicates significantly different from the corresponding KmPEP value from Table 2 of the same condition with no additional metabolites added, Student's t-test, p < 0.05.

Table 2 Kinetic properties of purified PK from control, frozen, dehydrated, and anoxic skeletal muscle of R. sylvatica. Data are mean ± SEM, n ¼ 4. Kinetic parameter Km PEP (mM) Km ADP (mM) Vmax (kU/g wet weight) Vmax 5  C (U/mg) Vmax 13  C (U/mg) Vmax 22.5  C (U/mg) Ea (kJ/mol)

Control 0.065 ± 0.003 0.22 ± 0.02 5.63 ± 0.05 0.59 ± 0.12 0.88 ± 0.15 1.48 ± 0.20 35.70 ± 1.98

Frozen 0.098 ± 0.003 0.21 ± 0.008 4.75 ± 0.03 0.58 ± 0.07 0.75 ± 0.07 1.35 ± 0.14 34.53 ± 7.12

Dehydrated a

Anoxic a

0.092 ± 0.008

0.073 ± 0.002

a indicates significant difference from the corresponding control value, one-way ANOVA, Dunnett's post-hoc, p < 0.05. All experiments were performed on purified samples of PK except for the Vmax(kU/gww), which was determined by assaying the crude homogenate.

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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Table 4 Effects of various substrates and allosteric effectors on the Vmax of PK from control and frozen muscle samples of R. sylvatica. Data are means ± SEM, n ¼ 4.

b

Substrate

Observed effect (parameter reported)

Control

Frozen

Urea Urea þ400 mM Glucose ATP AMP FBP Alanine Glucose Aspartate

Inhibition (I50, M) Inhibition (I50, M) Inhibition (I50, mM) No effect up to 25 mM No effect up to 25 mM No effect up to 25 mM No effect up to 750 mM No effect up to 25 mM

0.44 ± 0.02 1.65 ± 0.1b 10.1 ± 0.34 N/A N/A N/A N/A N/A

0.42 ± 0.04 1.63 ± 0.07b 12.1 ± 1.13 N/A N/A N/A N/A N/A

indicates significantly different from the corresponding value of the same condition without glucose, Student's t-test, p < 0.05.

Fig. 2. Relative levels of post-translational modifications determined through dot blots of purified PK from control and frozen muscle tissue of R. sylvatica. Chemiluminescence signal intensities were standardized to protein amount, and the value for frozen PK was expressed relative to the control value that was set to 1. Data are mean ± SEM, n ¼ 5e6 determinations on purified enzyme samples. Asterisk indicates significant difference from the corresponding control value, Student's t-test, p < 0.05.

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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Fig. 3. Relative levels of serine phosphorylation of purified pyruvate kinase from control dehydrated and anoxic muscle tissue of Rana sylvatica determined through western blotting. Chemiluminescence signal intensities were standardized to corresponding coomassie protein amount, and the values for the dehydrated and anoxic forms were expressed relative to the control value that was set to 1. Data are mean ± SEM, n ¼ 4 determinations on purified enzyme samples. Asterisk indicates significant difference from the control value, one-way ANOVA, Dunnett's post-hoc, p < 0.05.

3.4. Structural stability of PK Structural stability of pyruvate kinase was investigated by examining stability under various durations of high temperature exposure and under exposure to high concentrations of urea for 90 min (Fig. 4). During incubations at 52  C, the PK from control animals was significantly less stable than that from frozen animals (p ¼ 0.001) with the Vmax being reduced by 50% after 3.00 ± 0.02 min and 13.7 ± 0.02 min, for control and frozen samples, respectively (Fig. 4A). This trend of PK from frozen muscle remaining more stable than PK from control muscle was also found when the proteins were incubated at 52  C with 400 mM glucose (p ¼ 0.003; Fig. 4B). Similarly, when PK was incubated in increasing concentrations of urea the Vmax of PK was reduced to 50% at a significantly lower concentration of urea in the control animals (0.832 ± 0.01 M) compared to the frozen animals (1.24 ± 0.03 M) (p < 0.001; Fig. 4C). When incubated with 400 mM glucose the PK purified from control tissue was still less stable than that from frozen tissue with Vmax values being reduced to 50% at 1.01 ± 0.02 M urea and 1.31 ± 0.06 M urea respectively (p ¼ 0.03; Fig. 4D). Control PK was found to be significantly more stable when incubated with glucose present at 52  C (p ¼ 0.001) or in high concentrations of urea (p ¼ 0.002), while frozen PK was not significantly affected by the presence of glucose (Fig. 4).

4. Discussion Survival of wood frogs during winter hibernation at subzero conditions, resulting in whole-body freezing, requires welldeveloped adaptations that inhibit ice crystallization and cellular dehydration, thereby limiting cellular damage. Among these adaptions is an accumulation of the colligative cryoprotectant glucose that functions by limiting intracellular dehydration and cryo-injury [31,35,41]. Secondly, there is a metabolic rate

depression to conserve limited carbohydrate energy stores in the dormant amphibian. PK is therefore an interesting enzyme to study in this animal, given its role in glucose metabolism and for its many known regulatory mechanisms. PK is a highly regulated glycolytic enzyme, serving as a control point of glycolysis and promoting gluconeogenesis when inhibited [45]. Investigation of the kinetic properties of PK from experimental and control R. sylvatica muscle demonstrated significant changes in substrate affinity (Table 2). The KmPEP was significantly higher (~0.5-fold) in both frozen and dehydrated leg muscle tissue compared to control tissue which confirms a lower affinity for PEP. The anoxic and control forms of PK did not differ significantly in their substrate affinity. Furthermore, the frozen KmPEP was consistently higher than that of the control regardless of the additions of various metabolites and potential allosteric regulators (Table 3). This strongly suggests that PK is being negatively regulated during environmental freezing and dehydration. A decrease in PK activity would be beneficial to the winter survival of R. sylvatica for multiple reasons: (1) the negative regulation of this glycolytic enzyme would contribute to an overall metabolic rate depression to conserve energy, and (2) it would limit the consumption of the vital cryoprotectant glucose by slowing the rate of glycolysis. To address the former point, significant literature has been published in support of metabolic rate depression as a primary mechanism of surviving freezing and other environmental stress, and is reviewed in Ref. [41]. This phenomenon is important for winter survival of wood frogs as it ensures that the limited fuel stores can last the entire freezing period and that sufficient energy remains postfreezing to allow for energetically expensive activities such as mating. The inhibition of PK would contribute to reducing energy expenditure directly and indirectly by suppressing substrate-level phosphorylation of ADP and limiting glycolytic flux, ultimately slowing the production of pyruvate for consumption in downstream pathways. Similar regulation has been observed in other

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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Fig. 4. Relative stability of purified PK from R. sylvatica muscle tissue determined through enzyme incubations at high temperature and in high concentrations of urea. (A) 52  C incubation; (B) 52  C incubation with 400 mM glucose; (C) 90 min urea incubation; (D) 90 min urea incubation with 400 mM glucose. The reported value is either the incubation time at 52  C (A, B) or the concentration of urea during a 90 min incubation (C, D) that resulted in a Vmax reduced to 50% of the non-incubated value. Data are mean ± SEM, n ¼ 4. a indicates significantly different from the corresponding control value, Student's t-test, p < 0.05. b indicates significantly different from the corresponding value of the same condition without glucose, Student's t-test, p < 0.05.

glycolytic enzymes in wood frog muscle, including HK, which demonstrated a decrease in total activity and affinity for its substrate following freezing exposure and directly limited glycolytic rate [16]. With respect to the conservation of the frog's cryoprotectant, glucose, the results from this study lend support to the hypothesis that glucose consumption must be limited during cold exposure to preserve cryoprotectant levels. The importance of glucose in winter survival of the wood frog has previously been well-established [7,19, 39, 40]. By accumulating to levels of up to 400 mM, wood frogs can depress their intracellular freezing point to significantly below 0  C and limit cellular dehydration caused by extracellular ice formation. By decreasing glycolytic activity, glucose levels can remain high enough that freeze tolerance can be maintained. The significant decrease in affinity for PEP suggests that PK is being regulated to restrict its activity, and the rate of glycolysis in general. Enzyme-dependent down-regulation of glucose catabolism in the wood frog has been clearly demonstrated through a series of

enzymatic studies. As previously mentioned, inhibition of HK during freezing would limit glycolytic rate [16]. Studies of enzymes also suggest that the pentose-phosphate pathway is suppressed in wood frog muscle during freezing, as the activities of both glucose6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two early enzymes of this pathway, decrease upon freeze exposure [10,15]. This has the effect of limiting the rate at which glucose-6-phoshpate is removed from the cryoprotectant pool and shunted into another metabolic pathway. There is also evidence that muscle glycogen stores are converted into glucose early in freezing. Levels of glycogen in muscle tissue have been shown to decrease by as much as 50% through the first 72 h of freezing while glucose levels increase accordingly and apparently are not consumed for energy [38]. This demonstrates the importance of accumulating this cryoprotectant for freeze survival and limiting glycolytic rate. Together with the results presented in the current study, there is significant evidence showing that wood frog metabolism is regulated during freezing and dehydration to hinder

Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002

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glycolysis and glucose catabolism in muscle tissue and support accumulation of glucose as a cryoprotectant. Furthermore, it appears that PK is one of many enzymes that are regulated to achieve this. The observation that the KmPEP increases 0.5-fold in the presence of 100 mM urea in both control and frozen tissues (Table 3) suggests that urea could act as a regulator of PK during freezing, contributing to both metabolic rate depression and conservation of glucose. Urea has been previously shown to accumulate to levels up to 100 mM in plasma and 100 mmol/g in muscle tissue of coldacclimated frogs [8], suggesting that its effect on PK could be a physiological mechanism to regulate the activity of the enzyme during stress. These results appear to support findings from a study by Muir et al. [25] in which urea-treated organs of R. sylvatica had significantly-depressed metabolic rates (up to a 50% decrease) compared to control organs. This study led to the proposal of the urea-hypometabolism hypothesis, which suggests that accumulated urea in the wood frog acts as a mediator of metabolic rate depression. The evidence presented in the current study suggests that the decrease in metabolic rate observed by Muir et al. [25,26] is caused in part by urea-mediated inhibition of metabolic enzymes. As relative concentrations of urea increase during freezing, the activity of PK decreases accordingly. Other enzymes may be inhibited similarly by urea and act together to reduce the metabolic rate of the animal during times of environmental duress. The stability of PK is significantly increased during freezing as demonstrated when PK is exposed to both high concentrations of urea and high temperatures (Fig. 4). This would be advantageous to the wood frog as cellular conditions are significantly less favorable during freezing, warranting modifications to ensure protein stability. Concentrations of urea accumulate to 100 mM in the plasma during freezing and increases in oxidative stress defenses suggest that oxidative damage is occurring [8,18]. Stress-responsive changes in protein stability have been observed previously in the wood frog. In response to freezing, MnSOD purified from muscle tissue showed an increased stability together with increased serine phosphorylation [11]. A similar effect was observed in this study, and it is consistent with other observed examples of stress-induced changes in protein stability [5,16,46]. A second reason for which increased enzyme stability is desirable during freezing stress is directly attributed to the energetic cost of protein turnover. Indeed, protein synthesis and degradation are highly energy expensive processes. In vivo measurements estimate the total energetic cost of each peptide bond to be as much as 7.5 units of ATP and mathematical modelling estimates protein turnover accounting for 19% of cellular ATP use [13,17]. By stabilizing the enzyme during environmental stress exposure, energy can be conserved by limiting the rate at which it unfolds and therefore must be resynthesized. The stabilization of PK observed in this study may represent a common mechanism of energy conservation given the abundance of other examples of increased enzyme stability during stress. An increase in serine phosphorylation of PK was observed after exposure to freezing (0.5-fold increase, Fig. 3) and dehydration (0.25-fold increase, Fig. 4) relative to the control. These results, in combination with the stress-responsive changes in PK substrate affinity and stability, may be indicative of a stress-responsive regulatory mechanism via phosphorylation of serine residues on PK. This is further supported by the observation that in the anoxic state, PK shows neither different phosphorylation patterns nor different kinetic properties compared to the control. The phosphorylation of serine residues could be causing a conformational change in the structure of PK resulting in hindered access to the active site and the observed increase in the KmPEP. There is significant evidence supporting phosphorylation as the regulatory mechanism of enzymes during periods of environmental stress, including many

instances in the wood frog. These include glucose-6-phosphate dehydrogenase, creatine kinase and catalase [12,14,15]. These results suggest that PK, too, may be a target of regulation via reversible protein phosphorylation in the wood frog during freezing. Studying the regulation of PK in the freeze tolerant frog R. sylvatica provides an excellent opportunity to better understand carbohydrate metabolism during environmental stress. PK from frozen and dehydrated frogs exhibit a lesser affinity for PEP and greater levels of serine phosphorylation. Furthermore, PK from frozen and dehydrated frogs is more stable than PK from control frogs. These differences suggest that reversible phosphorylation is used as a regulatory mechanism of PK during times of environmental stress and functions by decreasing substrate affinity and increasing protein stability. Decreased PK activity would contribute to both an overall metabolic rate depression and conserve energy as well as suppress consumption of glucose, a key cryoprotectant in the wood frog. Funding This study was supported by a discovery grant (No. 6793) from the Natural Sciences and Engineering Research Council of Canada. KBS also holds the Canada Research Chair in Molecular Physiology. MS is a Master's Student funded by an Ontario Graduate Scholarship. Conflict of interest None. Acknowledgements We thank J.M. Storey for providing us with valuable guidance and knowledge throughout this project. References [1] J. Abboud, K.B. Storey, Novel control of lactate dehydrogenase from the freeze tolerant wood frog: role of posttranslational modifications, PeerJ 1 (2013) e12. [2] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248e254. [3] S.P. Brooks, A simple computer program with statistical tests for the analysis of enzyme kinetics, Biotechniques 13 (1992) 906e911. [4] S.P. Brooks, A program for analyzing enzyme rate data obtained from a microplate reader, BioTechniques 17 (1994) 1154e1161. [5] C.L. Childers, K.B. Storey, Post-translational regulation of hexokinase function and protein stability in the aestivating frog Xenopus laevis, Protein J. 35 (1) (2016) 61e71. [6] T.A. Churchill, K.B. Storey, Dehydration tolerance in wood frogs: a new perspective on development of amphibian freeze tolerance, Am. J. Physiol. Regul. Integr. Comp. Physiol. 265 (1993) 1324e1332. [7] J.P. Costanzo, R.E. Lee, P.H. Lortz, Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica, J. Exp. Biol. 181 (1993) 245e255. [8] J.P. Costanzo, M.C.F. do Amaral, A.J. Rosendale, R.E. Lee, Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog, J. Exp. Biol. 216 (2013) 3461e3473. [9] K.J. Cowan, K.B. Storey, Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii, Mol. Cell. Biochem. 195 (1999) 173e181. [10] K.J. Cowan, K.B. Storey, Freeze-thaw effects on metabolic enzymes in wood frog organs, Cryobiology 43 (2001) 32e45. [11] N.J. Dawson, B.A. Katzenback, K.B. Storey, Free-radical first responders: the characterization of CuZnSOD and MnSOD regulation during freezing of the freeze-tolerant North American wood frog, Rana sylvatica, Biochim. Biophys. Acta, Gen. Subj. 1850 (2015) 97e106. [12] N.J. Dawson, K.B. Storey, A hydrogen peroxide safety valve: the reversible phosphorylation of catalase from the freeze-tolerant North American wood frog, Rana sylvatica, Biochim. Biophys. Acta, Gen. Subj. 1860 (2016) 476e485. [13] F.P. De Vries, The cost of maintenance processes in plant cells, Ann. Bot. 39 (1975) 77e92. [14] C.A. Dieni, K.B. Storey, Creatine kinase regulation by reversible

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Please cite this article in press as: M.B. Smolinski, et al., Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.06.002