Characterization of γ-glutamyltranspeptidase in the liver of the frog: 1. Comparison to the rat liver enzyme

The characteristics of the enzyme γ-glutamyltranspeptidase were determined in frog liver and compared to those of the rat. In Rana pipiens, tissue distribution studies indicated the order of activity to be: kidney >>> liver >> nerve > egg > lung > heart > skeletal muscle in homogenates. In the Rana pipiens relative to the Fischer 344 rat, the activity of the liver enzyme was somewhat greater (1·8-fold) and the kidney enzyme substantially less (25-fold). Frog liver γ-glutamyltranspeptidase displayed strain-dependent differences in activity with Rana pipiens and Rana sylvatica exhibiting comparable activities and Xenopus laevis exhibiting 20-fold lower activities. No influence of sex was apparent in Rana pipiens in contrast to the sex dependent differences observed in the Fischer 344 rat: ♀ : ♂ = 7:1. In homogenates and plasma membrane fractions of Rana pipiens, Xenopus laevis and the Fischer 344 rat, high, and comparable relative specific activities, were observed, 8–11, coupled with protein yields of 2·2–2·5 per cent indicating the enzyme to be plasma membrane bound and associated with the sinusoidal surface of the liver cell. Both the frog Rana pipiens and Xenopus laevis and Fischer 344 rat liver plasma membrane enzymes displayed comparable temperature-induced activation (1·51–1·74-fold) but with a peak for the frogs at 60°C and for the rat at 50°C. Both Acivicin and maleate inhibited the liver plasma membrane γ-glutamyltranspeptidase of both Rana pipiens and the Fischer 344 rat, but the frog enzyme was less sensitive (89 per cent decrease versus 97 per cent decrease) to 150 μM Acivicin and more sensitive (65 per cent decrease versus 35 per cent decrease at 150 mM maleate) to maleate. Kinetic studies indicated that the liver plasma membrane enzyme from Rana pipiens had a K_m of 0·61 mM and V_max of 55·6 nmol mg^?1 min^?1 and that from the Fischer 344 rat had a K_m of 3·57 mM and V_max of 71·4 nmol mg^?1 min^?1.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 2. Response to season,temperature and thyroid hormone in Rana pipiens

The impact of season and temperature on frog liver γ-glutamyltranspeptidase was assessed by measuring the activity of this enzyme in plasma membranes isolated from the livers of Rana pipiens obtained as summer and winter frogs; subjected to short-term (3 weeks) temperature acclimation; and subjected to multiple-temperature shifts. Plasma levels of T₃ were determined. γ-Glutamyltranspeptidase was found to be 2·2-fold higher in the summer frog relative to the winter frog; decreased by 44 percent in the summer frog by cold acclimation and increased by 1·7-fold in the winter frog by warm acclimation; and increased by 1·9-fold in the summer frog and 2·8-fold in the winter frog subjected to multiple-temperature shifts. Plasma T₃ levels were found to be 42-fold higher in the summer frog relative to the winter frog; decreased by 42 percent by cold acclimation and increased by 2·9-fold by warm acclimation; and decreased by 39 percent and 38 percent in the summer and winter frogs subjected to multiple temperature shifts. T₃ replacement during the last phase of the multiple-temperature shift protocol, restored the plasma T₃ levels to 75 percent of the control levels and prevented the increase evoked by the multiple-temperature shifts in γ-glutamyl-transpeptidase activity. Indeed, enzyme activity in the T₃ replaced state was 19 percent lower than in the control state. The involvement of thyroid hormone as a negative regulator of enzyme activity is discussed.     

Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog

The responses of mitogen-activated protein kinase (MAPK) family members, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH₂-terminal kinase), and p38, in the metabolic responses to whole animal freezing (up to 24 h frozen at –2.5°C) and thawing (up to 4 h at 5°C after a 12 h freeze) were examined in four organs (liver, kidney, heart, brain) of the freeze-tolerant wood frog Rana sylvatica. Levels of the active phosphorylated form of p38 increased within 20 min as an early response to freezing in liver and kidney but rose later (after 12 h) in heart. Both JNK and p38 were activated during thawing in liver, kidney and heart with temporally-distinct patterns in each organ. The only MAPK response to freeze/thaw in frog brain was a transient elevation of p38 after 90 min thawing. ERK activity did not respond to freeze/thaw in any organ. The levels of c-Fos increased during freezing in kidney and brain whereas c-Jun was unaffected by freeze/thaw. Organ-specific responses by MAPKs, particularly p38, suggest that these may have roles in regulating metabolic or gene expression responses that may be adaptive in dealing with freezing stress or metabolic recovery during thawing.     

Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells. Accepted: 28 April 1999     

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.     

Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica

Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation.     

Survival and metabolism of <Emphasis Type="Italic">Rana arvali</Emphasis>s during freezing

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of −3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and −1°C). Lactate accumulation was only observed in the liver (4.1 ± 0.8 against 16.6 ± 2.4 μmol g⁻¹ fresh weight (FW) between 4 and −1°C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 ± 0.7 μl O₂ g⁻¹ FW h⁻¹ at −4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.     

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

Hepatic changes in the freeze-tolerant turtle Chrysemys picta marginata in response to freezing and thawing

Select hepatic changes in the freeze-tolerant hatchling turtle, Chrysemys picta marginata, were studied in response to freezing at -2.5 degrees C and thawing. Upon freezing, a small, selective increase in the liver weight with no increase in body weight was seen suggestive of an hepatic capacitance response. In all turtles studies, lobular differences in the hepatic content of glycogen were evident: the smaller lobe contained twice as much glycogen as the larger lobe. The response to freezing and thawing was comparable. Total hepatic glycogen levels of turtles were reduced approximately 60 per cent from control levels in the frozen state and recovered to >80 per cent of control levels in the thawed state. Compared to the control state, turtle blood glucose levels were: unchanged after 12 h in the cool state; reduced 28 per cent after 24 h and increased two-fold after 48 h in the frozen state; and increased 4.5-fold in the thawed state. Thus, changes in hepatic glycogen metabolism occur without large changes in blood glucose levels. In turtle liver plasma membranes, the hepatic alpha(1)-adrenergic receptor was barely detectable and did not change. The beta(2)-adrenergic receptor was expressed at high levels and, compared to control levels, was: unchanged after 12 h in the cool state; reduced 20 per cent after 24 h and 40 per cent after 48 h in the frozen state. On thawing, this receptor was 50 per cent of control levels. While catecholamines working through the beta(2)-adrenergic receptor may effect early hepatic glycogen breakdown in response to freezing, other factors must be involved to complete the process. The plasma membrane-bound enzyme gamma-glutamyltranspeptidase displayed a different pattern of changes indicative of selective modulation: it was increased 2.7-fold over control levels in the cool state; unchanged in the frozen state; and increased 1.8-fold in the thawed state. The activity of the kidney enzyme was decreased in the cool state and slightly increased in the frozen and thawed states emphasizing the tissue-specific nature of the changes in the activity of gamma-glutamyltranspeptidase in response to freezing and thawing. The similarities and differences of the hepatic changes in response to freezing and thawing in the freeze-tolerant hatchling turtle to those we have previously reported for the freeze-tolerant frog are discussed.     

DNA replication in eucaryotic nuclei. Evidence suggesting a specific model of replication

Preimplantation-stage mouse embryos suspended in dimethyl sulfoxide (DMSO) have been used as a model to study details of the response of a simple multicellular system to freezing and thawing. Rapid freezing to ?196 °C kills the embryos unless they have first been cooled very slowly to at least below ?50 °C. The survival of both 2-cell and 8-cell embryos has been found to depend as critically on the rate at which the frozen embryos were thawed as on the rate at which they were first frozen. The damaging consequences of thawing frozen embryos too rapidly have been shown to occur between ?70 and ?20 °C. Finally, the survival of embryos as a function of the time in DMSO prior to freezing and thawing has been compared with their volume changes as a function of time in DMSO. This comparison leads to the tentative conclusion that dimethyl sulfoxide need not permeate the embryos to protect them against freezing damage. Overall, the embryos' response to freezing and thawing is qualitatively similar to that displayed by many other cell types.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Endothelial retinoblastoma protein reduces abdominal aortic aneurysm development via promoting DHFR/NO pathway-mediated vasoprotection

Carbamoyl phosphate synthetase I (CPS1) represents an important regulatory enzyme of the urea cycle that mediates the ATP-driven reaction ligating ammonium, carbonate, and phosphate to form carbamoyl phosphate. The freeze-tolerant wood frog (Rana sylvatica or Lithobates sylvaticus) accumulates high concentrations of urea during bouts of freezing to detoxify any ammonia generated and to contribute as a cryoprotectant thereby helping to avoid freeze damage to cells. Purification of CPS1 to homogeneity from wood frog liver was performed in control and frozen wood frogs by a three-step chromatographic process. The affinity of CPS1 for its three substrates was tested in the purified control and freeze-exposed enzyme under a variety of conditions including the presence and absence of the natural cryoprotectants urea and glucose. The results demonstrated that affinity for ammonium was higher in the freeze-exposed CPS1 (1.26-fold) and that with the addition of 400 mM glucose it displayed higher affinity for ATP (1.30-fold) and the obligate activator N-acetylglutamate (1.24-fold). Denaturation studies demonstrated the freeze-exposed enzyme was less thermally stable than the control with an unfolding temperature approximately 1.5 °C lower (52.9 °C for frozen and 54.4 °C for control). The control form of CPS1 had a significantly higher degree of glutarylated lysine residues (1.42-fold increase) relative to the frozen. The results suggest that CPS1 activation and maintenance of urea cycle activity despite the hypometabolic conditions associated with freezing are important aspects in the metabolic survival strategies of the wood frog.

     

Freeze-thaw effects on metabolic enzymes in wood frog organs.

To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.     

Physical changes in specimens of five species of Cyprinidae preserved in ethanol and frozen

Preservation in 30% ethanol and freezing to a temperature of ?20 ± 2° C is an appropriate method for measurement of fish eggs, larvae and juveniles. Egg diameter of the common carp Cyprinus carpio increased insignificantly by 1·32% after preservation compared with live size. The total length (L_T) of 1 day post‐hatching (dph) larvae as well as the standard length (LS) of 16 dph larvae of C. carpio increased significantly (2·95 and 1·50%, respectively) after preservation. Egg diameter as well as the L_T of 1 dph larvae of barbel Barbus barbus increased significantly after preservation, by 1·74 and 1·96%, respectively over their original size. The standard length (L_S) of 14 dph larvae of B. barbus as well as juveniles of B. barbus, crucian carp Carassius carassius, common nase Chondrostoma nasus and tench Tinca tinca decreased significantly after preservation (?0·56 to ?5·54%), whereas their body mass increased significantly (11·46–18·57%). Preserved eggs of C. carpio and B. barbus were hard, round and transparent. The larvae and juveniles of examined fishes, preserved in frozen ethanol, were straight, flexible and easily measurable after 60 days. Integrity of body surface and fins, as well as preservation of colours were much better in larvae or juveniles frozen and thawed only once than in specimens frozen and thawed thrice. Cooling in 30% ethanol to a temperature of 6 ± 2° C and freezing in water to a temperature of ?20 ± 2° C are not appropriate preservation methods for eggs and larvae of C. carpio (1 and 16 dph).     

Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog

Summary Wood frogs,Rana sylvatica, were sampled after freezing at –4°C (a short time course from 2 to 70 min after the appearance of the freezing exotherm) and thawing (20 h at 3°C after 70 min of freezing) and the regulation of liver glycolysis with respect to cryoprotectant glucose synthesis was examined. Within 5 min of the initiation of freezing, cryoprotectant concentrations in blood and liver had begun to increase. This was correlated with a rapid rise in the levels of hexose monophosphates in liver, including a 2.5 fold increase in glucose-6-P and 10 fold rise in fructose-6-P contents within the first 5 min post-exotherm. Contents of fructose-1,6-P₂, fructose-2,6-P₂, triose phosphates, P-enolpyruvate, and pyruvate did not significantly change over the course of freezing. Thawing sharply reduced the levels of hexose monophosphates in liver but raised P-enolpyruvate content by 2.3 fold. Changes in the contents of glycolytic intermediates over the freeze/thaw course are consistent with an inhibitory block of glycolysis at phosphofructokinase during freezing in order to facilitate a rapid glycogenolysis and production of cryoprotectant; during thawing, however, glycolysis appears to be inhibited at the level of pyruvate kinase.Possible regulatory control of cryoprotectant synthesis by covalent modification of liver glycolytic enzymes was examined. Glycogenolysis during freezing was facilitated by an increase in the percentage of glycogen phosphorylase in the activea (phosphorylated) form and also by an increase in the total amount (a+b) of enzyme expressed. For phosphofructokinase, kinetic changes as a result of freezing included a 40% reduction inK _m for fructose-6-P, a 60% decrease inK _a for fructose-2,6-P₂, and a 2 fold increase in I₅₀ for ATP. These changes imply a freezing-induced covalent modification of the enzyme but are not, apparently, the factors responsible for inhibition of glycolytic flux at the phosphofructokinase locus during glucose synthesis. Kinetic parameters of pyruvate kinase were not altered over the freeze/thaw course.     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

A hydrogen peroxide safety valve: The reversible phosphorylation of catalase from the freeze-tolerant North American wood frog,Rana sylvatica

BackgroundThe North American wood frog, Rana sylvatica, endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H₂O₂ may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H₂O₂ into molecular oxygen and water, was investigated as a result.MethodsThe present study investigated the purification and kinetic profile of CAT in relation to the phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica.ResultsCatalase from skeletal muscle of frozen wood frogs showed a significantly higher V_max (1.48 fold) and significantly lower K_m for H₂O₂ (0.64 fold) in comparison to CAT from control frogs (5 °C acclimated). CAT from frozen frogs also showed higher overall phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (1.27 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the K_m for H₂O₂ of CAT, whereas protein phosphatase 2B or 2C action significantly increased the K_m.ConclusionThe physiological consequence of freeze-induced CAT phosphorylation appears to improve CAT function to alleviate H₂O₂ build-up in freezing frogs.General significanceAugmented CAT activity via reversible phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.