Regulation of an important glycolytic enzyme, pyruvate kinase, through phosphorylation in the larvae of a species of freeze-tolerant insect, Eurosta solidaginis.

Larvae of the goldenrod gall fly, Eurosta solidaginis, rely on a freeze tolerance strategy to survive the sub-zero temperatures of Canadian winter. Critical to their survival is the accumulation of polyol cryoprotectants and global metabolic rate depression, both of which require the regulation of glycolysis and reorganization of carbohydrate metabolism. This study explored the role that pyruvate kinase (PK) regulation plays in this metabolic reorganization. PK was purified from control (5 °C-acclimated) and frozen (-15 °C-acclimated) larvae and enzyme kinetic properties, structural stability, and post-translational modifications were examined in both enzyme forms. The Km phosphoenolpyruvate (PEP) of frozen PK was 20% higher than that of control PK, whereas the Vmax of frozen PK was up to 50% lower than that of control PK at the lowest assay temperature, suggesting inhibition of the enzyme during the winter. Additionally, the activity and substrate affinity of both forms of PK decreased significantly at low assay temperatures, and both forms were regulated allosterically by a number of metabolites. Pro-Q™ Diamond phosphoprotein staining and immunoblotting experiments demonstrated significantly higher threonine phosphorylation of PK from frozen animals while acetylation and methylation levels remained constant. Together, these results indicate that PK exists in two structurally distinct forms in E. solidaginis. In response to conditions mimicking the transition to winter, PK appears to be regulated to support metabolic rate depression, the accumulation of polyol cryoprotectants, and the need for extended periods of anaerobic carbohydrate metabolism to allow the animal to survive whole-body freezing.

Insect cold hardiness: the role of mitogen-activated protein kinase and Akt signalling in freeze avoiding larvae of the goldenrod gall moth, Epiblema scudderiana.

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of cold hardiness to survive the winter. Here we report that protein kinase-dependent signal transduction featuring mitogen-activated protein kinase (MAPK) signalling cascades (extracellular signal regulated kinase, c-jun N-terminal kinase and p38 MAPK pathways) and the Akt (also known as protein kinase B, or PKB) pathway could be integral parts of the development of cold hardiness by E. scudderiana. We used Luminex technology to assess the protein levels and phosphorylation status of key components and downstream targets of those pathways in larvae in response to low temperature acclimation. The data showed that MAPK pathways (both total protein and phosphorylated MAPK targets) were inhibited after 5°C acclimation, but not -15°C exposure, as compared with the 15°C control group. However, total heat shock protein 27 (HSP27) levels increased dramatically by ∼12-fold in the -15°C acclimated insects. Elevated HSP27 may facilitate anti-apoptotic mechanisms in an Akt-dependent fashion. By contrast, both 5 and -15°C acclimation produced signs of Akt pathway activation. In particular, the inhibitor phosphorylated Glycogen Synthase Kinase 3a (p-GSK3) levels remained high in cold-exposed larvae. Additionally, activation of the Akt pathway might also facilitate inhibition of apoptosis independently of GSK3. Overall, the current study indicates that both MAPK and Akt signal transduction may play essential roles in freeze avoidance by E. scudderiana.

Amplification and quantification of cold-associated microRNAs in the Colorado potato beetle (Leptinotarsa decemlineata) agricultural pest.

The Colorado potato beetle [Leptinotarsa decemlineata (Say)] is an important insect pest that can inflict considerable damage to potato plants. This insect can survive extended periods of cold exposure, and yet the molecular switches underlying this phenomenon have not been fully elucidated. A better characterization of this process would highlight novel vulnerabilities associated with L. decemlineata that could serve as targets for the management of this devastating pest. Using high-throughput sequencing, the current work reveals a cold-associated signature group of microRNAs (miRNAs) in control (15 °C) and -5 °C-exposed L. decemlineata. The results show 42 differentially expressed miRNAs following cold exposure including miR-9a-3p, miR-210-3p, miR-276-5p and miR-277-3p. Functional analysis of predicted targets associated with these cold-responsive miRNAs notably linked these changes with vital metabolic and cellular processes. Overall, this study highlights the miRNAs probably responsible for facilitating cold adaptation in L. decemlineata and implicates miRNAs as a key molecular target to consider in the development of novel pest management strategies against these insects.

Genes and associated peptides involved with aestivation in a land snail.

Some animals can undergo a remarkable transition from active normal life to a dormant state called aestivation; entry into this hypometabolic state ensures that life continues even during long periods of environmental hardship. In this study, we aimed to identify those central nervous system (CNS) peptides that may regulate metabolic suppression leading to aestivation in land snails. Mass spectral-based neuropeptidome analysis of the CNS comparing active and aestivating states, revealed 19 differentially produced peptides; 2 were upregulated in active animals and 17 were upregulated in aestivated animals. Of those, the buccalin neuropeptide was further investigated since there is existing evidence in molluscs that buccalin modulates physiology by muscle contraction. The Theba pisana CNS contains two buccalin transcripts that encode precursor proteins that are capable of releasing numerous buccalin peptides. Of these, Tpi-buccalin-2 is most highly expressed within our CNS transcriptome derived from multiple metabolic states. No significant difference was observed at the level of gene expression levels for Tpi-buccalin-2 between active and aestivated animals, suggesting that regulation may reside at the level of post-translational control of peptide abundance. Spatial gene and peptide expression analysis of aestivated snail CNS demonstrated that buccalin-2 has widespread distribution within regions that control several physiological roles. In conclusion, we provide the first detailed molecular analysis of the peptides and associated genes that are related to hypometabolism in a gastropod snail known to undergo extended periods of aestivation.

Transcript expression of the freeze responsive gene fr10 in Rana sylvatica during freezing, anoxia, dehydration, and development.

Freeze tolerance is a critical winter survival strategy for the wood frog, Rana sylvatica. In response to freezing, a number of genes are upregulated to facilitate the survival response. This includes fr10, a novel freeze-responsive gene first identified in R. sylvatica. This study analyzes the transcriptional expression of fr10 in seven tissues in response to freezing, anoxia, and dehydration stress, and throughout the Gosner stages of tadpole development. Transcription of fr10 increased overall in response to 24 h of freezing, with significant increases in expression detected in testes, heart, brain, and lung when compared to control tissues. When exposed to anoxia; heart, lung, and kidney tissues experienced a significant increase, while the transcription of fr10 in response to 40% dehydration was found to significantly increase in both heart and brain tissues. An analysis of the transcription of fr10 throughout the development of the wood frog showed a relatively constant expression; with slightly lower transcription levels observed in two of the seven Gosner stages. Based on these results, it is predicted that fr10 has multiple roles depending on the needs and stresses experienced by the wood frog. It has conclusively been shown to act as a cryoprotectant, with possible additional roles in anoxia, dehydration, and development. In the future, it is hoped that further knowledge of the mechanism of action of FR10 will allow for increased stress tolerance in human cells and tissues.

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus.

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h=1.70 and S0.5=6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K+ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h=1.16) and GLK affinity for substrate was reduced (S0.5=23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h=1.60, S0.5=9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h=1.20, S0.5=45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.

Identification of differentially regulated micrornas in cold-hardy insects.

Freeze tolerance in insects is associated with cryoprotectant synthesis and strong metabolic suppression. Freeze avoidance, an alternative strategy in cold-hardy insects, is also characterized by hypometabolism, but possesses significant cellular and physiological differences when compared with freeze tolerance. We hypothesized that microRNAs, non-coding transcripts that bind to mRNA, could play a role in the regulation of energy-expensive mRNA translation in insects exposed to low temperatures. Expression levels of microRNA species were evaluated during cold acclimation of freeze tolerant Eurosta solidaginis and freeze-avoiding Epiblema scudderiana, comparing control (5 degree C) conditions with larvae given sequential exposures to -5 degree C and -15 degree C. MiR-1 levels were significantly elevated in frozen E. solidaginis larvae at -15 degree C, whereas miR-34 levels were unchanged. MiR-1 and miR-34 levels remained stable in E. scudderiana. These data demonstrate differential microRNA expression in frozen versus control insect larvae and highlight contrasting microRNA signatures between freeze tolerant and freeze avoiding species.

Regulation of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) in turtle muscle and liver during acute exposure to anoxia.

The freshwater turtle Trachemys scripta elegans naturally tolerates extended periods of anoxia during winter hibernation at the bottom of ice-locked ponds. Survival in this anoxic state is facilitated by a profound depression of metabolic rate. As calcium levels are known to be elevated in anoxic turtles, and ion pumping is an ATP-expensive process, we proposed that activity of the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) would be reduced in muscle and liver of T. s. elegans during acute (up to 20 h) exposure to anoxia. SERCA activity decreased approximately 30% in liver and approximately 40% in muscle after 1 h anoxia exposure and was approximately 50% lower after 20 h of anoxia exposure in both tissues, even though SERCA protein levels did not change. SERCA kinetic parameters (increased substrate K(m) values, increased Arrhenius activation energy) were indicative of a less active enzyme form under anoxic conditions. Interestingly, the less active SERCA in anoxic turtles featured greater stability than the enzyme from normoxic animals as determined by both kinetic analysis (effect of low pH and low temperatures on K(m) MgATP) and conformational resistance to urea denaturation. The quick time course of deactivation and the stable changes in kinetic parameters that resulted suggested that SERCA was regulated by a post-translational mechanism. In vitro experiments indicated that SERCA activity could be blunted by protein phosphorylation and enhanced by dephosphorylation in a tissue-specific manner.

Identification of novel and conserved microRNA and their expression in the gray mouse lemur, Microcebus murinus, a primate capable of daily torpor.

MicroRNA (miRNA) are endogenous small noncoding RNA gene products, on average 22 nt long, that play important regulatory roles in mediating gene expression by binding to and targeting mRNAs for degradation or translational repression. In this paper we identify both novel and conserved miRNA sequences present in the genome of the gray mouse lemur, Microcebus marinus. In total, 122 conserved and 44 novel miRNA were identified with high confidence from the lemur genome (Mmur_2.0) and were used for expression analysis. All conserved and novel miRNA were subjected to relative quantification by RT-qPCR in liver samples from control and torpid lemurs. A total of 26 miRNA (16 conserved and 10 novel) showed increased levels during primate torpor, whereas 31 (30 conserved and 1 novel) decreased. Additional in silico mapping of the predicted mRNA targets of torpor-responsive mature miRNA suggested that miRNA that increased during torpor were collectively involved in cell development and survival pathways, while miRNA that decreased were enriched in targeting immune function. Overall, the study suggests new regulatory mechanisms of primate torpor via miRNA action.

Differential peptide expression in the central nervous system of the land snail Theba pisana, between active and aestivated.

Hypometabolism is a physiological state of dormancy entered by many animals in times of environmental stress. There are gaps in our understanding of the molecular components used by animals to achieve this metabolic state. The availability of genomic and transcriptome data can be useful to study the process of hypometabolism at the molecular level. In this study, we use the land snail Theba pisana to identify peptides that may be involved in the hypometabolic state known as aestivation. We found a total of 22 neuropeptides in the central nervous system (CNS) that were differentially produced during activity and aestivation based on mass spectral-based neuropeptidome analysis. Of these, 4 were upregulated in active animals and 18 were upregulated in aestivation. A neuropeptide known to regulate muscle contractions in a variety of molluscs, the small cardioactive peptide A (sCAPA), and a peptide of yet unknown function (termed Aestivation Associated Peptide 12) were chosen for further investigation using temporal and spatial expression analysis of the precursor gene and peptide. Both peptides share expression within regions of the CNS cerebral ganglia and suboesophageal ganglia. Relative transcript abundance suggests that regulation of peptide synthesis and secretion is post-transcriptional. In summary, we provide new insights into the molecular basis of the regulation of aestivation in land snails through CNS peptide control.

Characterisation of two conopressin precursor isoforms in the land snail, Theba pisana.

Increased understanding of the molecular components involved in mollusc reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring. The neuropeptide conopressin, a member of the vasopressin/oxytocin-like peptide family, can modulate various reproductive activities in invertebrates. In this study, we used the hermaphroditic land snail, Theba pisana, to investigate the presence and tissue-specific distribution of a conopressin gene. Our transcriptomic analysis of T. pisana CNS sheath tissue has revealed two conopressin gene transcripts (Tpi-conopressin-1 and Tpi-conopressin-2), each encoding for precursors containing an identical conopressin nonapeptide and a variable neurophysin. T. pisana conopressins share high identity with other land snails and slugs, as well as other mollusc and vertebrate vasopressin/oxytocin, supported by phylogenetic analysis. Conserved residues in the T. pisana neurophysin are important for peptide binding, and we present molecular dynamic models demonstrating the most likely stable structure of the Tpi-conopressin-1 peptide when associated with neurophysin. RT-PCR shows that Tpi-conopressin-1 is additionally expressed in reproductive tissues, including the dart sac, where abundant spatial expression throughout the sac region is found; this implies a role in 'love' dart synthesis or dart injection during mating. The presence of a conopressin receptor in the CNS sheath indicates CNS neural excitation. In summary, this study represents a detailed molecular analysis of conopressin in a land snail.

Multi-tissue transcriptomics for construction of a comprehensive gene resource for the terrestrial snail Theba pisana.

The land snail Theba pisana is native to the Mediterranean region but has become one of the most abundant invasive species worldwide. Here, we present three transcriptomes of this agriculture pest derived from three tissues: the central nervous system, hepatopancreas (digestive gland), and foot muscle. Sequencing of the three tissues produced 339,479,092 high quality reads and a global de novo assembly generated a total of 250,848 unique transcripts (unigenes). BLAST analysis mapped 52,590 unigenes to NCBI non-redundant protein databases and further functional analysis annotated 21,849 unigenes with gene ontology. We report that T. pisana transcripts have representatives in all functional classes and a comparison of differentially expressed transcripts amongst all three tissues demonstrates enormous differences in their potential metabolic activities. The genes differentially expressed include those with sequence similarity to those genes associated with multiple bacterial diseases and neurological diseases. To provide a valuable resource that will assist functional genomics study, we have implemented a user-friendly web interface, ThebaDB (http://thebadb.bioinfo-minzhao.org/). This online database allows for complex text queries, sequence searches, and data browsing by enriched functional terms and KEGG mapping.

Global metabolite analysis of the land snail Theba pisana hemolymph during active and aestivated states.

The state of metabolic dormancy has fascinated people for hundreds of years, leading to research exploring the identity of natural molecular components that may induce and maintain this state. Many animals lower their metabolism in response to high temperatures and/or arid conditions, a phenomenon called aestivation. The biological significance for this is clear; by strongly suppressing metabolic rate to low levels, animals minimize their exposure to stressful conditions. Understanding blood or hemolymph metabolite changes that occur between active and aestivated animals can provide valuable insights relating to those molecular components that regulate hypometabolism in animals, and how they afford adaptation to their different environmental conditions. In this study, we have investigated the hemolymph metabolite composition from the land snail Theba pisana, a remarkably resilient mollusc that displays an annual aestivation period. Using LC-MS-based metabolomics analysis, we have identified those hemolymph metabolites that show significant changes in relative abundance between active and aestivated states. We show that certain metabolites, including some phospholipids [e.g. LysoPC(14:0)], and amino acids such as l-arginine and l-tyrosine, are present at high levels within aestivated snails. Further investigation of our T. pisana RNA-sequencing data elucidated the entire repertoire of phospholipid-synthesis genes in the snail digestive gland, as a precursor towards future comparative investigation between the genetic components of aestivating and non-aestivating species. In summary, we have identified a large number of metabolites that are elevated in the hemolymph of aestivating snails, supporting their role in protecting against heat or desiccation.

Phosphofructokinase from a vertebrate facultative anaerobe: effects of temperature and anoxia on the kinetic parameters of the purified enzyme from turtle white muscle.

The effects of low temperature and anoxia were determined on phosphofructokinase (PFK) purified from white skeletal muscle of the freshwater turtle, Pseudemys scripta. These effects were assayed by comparing PFK kinetic constants measured at a high (20 degrees C) and low (6 degrees C) temperature using enzyme obtained from animals held under normoxic and anoxic conditions. When assayed at 20 degrees C, PFK from anoxic animals had a lower Ka for phosphate, a lower Ka for AMP and showed no inhibition with increasing concentrations of ATP (up to 10 mM) when compared to enzyme from normoxic animals. At 6 degrees C, anoxic enzyme had a higher Km for fructose 6-phosphate and a higher I50 value for citrate with respect to normoxic enzyme. Decreasing temperature also had a differential effect on PFK kinetic parameters depending on the source of the enzyme. When normoxic enzymes were compared at 20 and 6 degrees C, the enzyme measured at 6 degrees C showed a lower Km for ATP and a lower Ka for AMP. Comparison of anoxic enzymes at these two temperatures showed that anoxic PFK at 6 degrees C had a higher Ka for phosphate, a higher Ka for AMP, and a larger Hill coefficient. A comparison of maximal velocities at varying temperature showed that normoxic enzyme (Q10 = 2.22) was more temperature sensitive than the anoxic enzyme (Q10 = 1.80). It is possible to interconvert the normoxic and anoxic forms of PFK by incubating normoxic enzyme with the active subunit of protein kinase, suggesting that the kinetic changes observed during anoxia resulted from enzyme phosphorylation. These data are discussed with respect to the mechanisms underlying white muscle function during diving and hibernation in red-eared turtles.

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase.

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.

Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties.

Phosphofructokinase was purified and characterized from the white skeletal muscle of rainbow trout Oncorhynchus mykiss. Purification involved three steps: ion-exchange chromatography on hydroxyapatite and affinity chromatography on phosphocellulose and ATP-agarose. A final specific activity of 75 units per mg of protein at 22 degrees C and pH 7.2 with 40% recovery was obtained. The purified enzyme gave a single band on SDS-PAGE with a subunit molecular mass of 76.5 +/- 0.6 kDa. Based on gel filtration analysis, the active form of the enzyme was found to be composed of six identical subunits. A high isoelectric point (7.1) was found for this enzyme. Arrhenius plots of the enzyme activity showed a sharp transition at 15-16 degrees C. The pH optimum of the enzyme was 8.0-8.5 at physiological level of ATP and positive modulators shifted the optimum to lower pH values. Amino-acid analysis revealed a lower content of the aromatic residues Phe, Tyr and Trp and higher level of Ser residue than in the rabbit muscle enzyme.

Differential expression of adipose- and heart-type fatty acid binding proteins in hibernating ground squirrels.

The up-regulation of heart- and adipose-type fatty acid binding proteins (H-FABPs and A-FABPs) was detected during hibernation in brown adipose tissue (BAT) of 13-lined ground squirrels, Spermophilus tridecemlineatus, using a commercial rat cDNA array. Full length cDNAs encoding H-FABPs and A-FABPs were subsequently retrieved from a BAT cDNA library. These cDNAs were used to probe Northern blots of total RNA from tissues of euthermic versus hibernating ground squirrels. H-FABP mRNA transcripts increased in BAT, skeletal muscle and heart of hibernating animals whereas A-FABP transcripts, which are normally expressed exclusively in adipose tissue, increased in both BAT and heart during torpor. It is proposed that the increased expression of H-FABPs and A-FABPs during hibernation accelerates the rate at which fatty acids can be transported to the mitochondria for oxidization, particularly in support of the huge increase in thermogenesis by BAT and rapid increase in heart rate that are required during arousal from torpor. Comparison of the deduced polypeptide sequence of ground squirrel H-FABP with that from other mammals also revealed three unique amino acid differences which may be important for protein function at low body temperatures during hibernation.

6-Phosphofructo-2-kinase and control of cryoprotectant synthesis in freeze tolerant frogs.

A critical part of natural freeze tolerance is the production of low molecular weight cryoprotectants; in freeze tolerant frogs this involves a freezing-induced activation of liver glycogenolysis that leads to the accumulation of glucose as the cryoprotectant, in amounts up to 300 mM, in all organs. The present study shows that the synthesis and maintenance of high organ glucose pools is facilitated by changes in the levels of fructose-2,6-bisphosphate (F2,6P2) and an inhibition of liver 6-phosphofructo-2-kinase (PFK-2) activity that blocks the catabolism of glucose by glycolysis. Freezing exposure (24 h at -2.5 degrees C) resulted in a sharp drop in F2,6P2 levels in four organs, to 23-75% of control values, but F2,6P2 rebounded when frogs were thawed. Freezing also stimulated changes in the properties of liver PFK-2 including a decrease in maximal velocity, a basic shift in pH optimum, a 10-fold increase in Km for fructose-6-phosphate, and increased I50 values for enzyme inhibitors. I50 values for glycerol-3-phosphate and phosphoenolpyruvate were 60- and 2.4-fold higher, respectively, for liver PFK-2 from frozen frogs compared with controls. Changes in liver PFK-2 properties are consistent with a freezing-induced phosphorylation of the enzyme to produce a less active enzyme form, resulting in reduced organ F2,6P2 levels and a decrease in 6-phosphofructo-1-kinase activity.

Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress.

The gene Aat coding for ADP/ATP translocase (AAT) was cloned from liver of the freeze-tolerant wood frog, Rana sylvatica, via differential screening of a cDNA library from liver of frozen frogs and using probes from control versus frozen frogs. Sequence analysis showed that clone pBfFR07 bearing the AAT cDNA contained a 1318-bp insert with one full-length open reading frame. The deduced amino acid sequence included 317 residues, with 81-86% identities to mammalian AAT. A 1750-nt transcript from the Aat gene was detected using pBfFR07 probe and a putative frog AAT of over 30 kDa was visualized by immunoblotting using a polyclonal antibody raised against chicken AAT. Analysis of liver samples from a time course of freezing showed a maximal 4.5-fold increase in mRNA after 8 h with AAT protein peaking in 24-h frozen frogs. Freezing also induced Aat expression in bladder and lung. In liver, mRNA expression also responded positively to anoxia stress but not to experimental dehydration of the animals. These results suggest that AAT induction during freezing may be stimulated by the ischemia that develops when plasma freezes; changes in AAT may contribute to stabilizing energetics in mitochondrial versus cytosolic pools over freeze/thaw cycles.

Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog.

As an adaptation for overwinter survival, the wood frog, Rana sylvatica is able to tolerate the freezing of extracellular body fluids. Tolerance is made possible by the production of very high amounts of glucose in liver which is then sent to other organs where it acts as a cryoprotectant. Cryoprotectant synthesis is under the control of glycogen phosphorylase which in turn is activated in response to ice formation. To determine the mechanism of phosphorylase activation, a quantitative analysis of phosphorylase protein concentration and enzymatic activity in liver was carried out following separation of the phosphorylated a and nonphosphorylated b forms of the enzyme on native polyacrylamide gels. The results suggest that in gels, the b form is completely inactive, even in the presence of AMP and sodium sulfate, whereas the a form is active and stimulated 3-fold by these substances. Further, phosphorylase activation appears to arise solely from conversion of the b to a form of the enzyme without an increase in phosphorylase concentration or activation of a second isozyme. The quantitative analysis presented here should prove generally useful as a simple and rapid method for examining the physiological and genetic regulation of phosphorylase in animal cells.