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Purification and Characterization of Protein Kinase A Catalytic Subunit from Liver of the Freeze-Tolerant Wood Frog: Role in Glycogenolysis During Freezing

Contact Person: Clark P. Holden (cholden@cc.umanitoba.ca)


A number of amphibian and reptile species that hibernate on land have evolved an amazing capacity, the ability to endure the freezing of body fluids. The best studied of the freeze tolerant vertebrates is the wood frog Rana sylvatica. Among the biochemical adaptations that support freeze tolerance in this species is the synthesis of massive amounts of glucose as a cryoprotectant which act in a colligative manner to minimize cell volume reduction during the growth of extracellular ice crystals [1]. As soon as ice formation begins in body extremities, a signal is transmitted to the liver which stimulates glycogenolysis. Within 2 min, blood glucose levels begin to climb and in just a few hours, glucose is distributed to all organs of the body with concentrations reaching 150-300 mM, compared with normal values of 1-5 mM [1]. This extreme hyperglycemia is supported by massive reserves of glycogen in wood frog liver and by high hepatic activities of glycogen phosphorylase.

In a series of recent studies, most parts of the signal transduction system responsible for freeze-induced cryoprotectant synthesis have been traced. The extracellular signal (which has not yet been identified) acts via a -adrenergic mechanism. Evidence supporting this includes the differential responses of liver a- and -adrenergic receptors to freezing [2], the ability of the -adrenergic blocker (propranolol), but not a-adrenergic antagonists, to suppress freeze-induced hyperglycemia [3], and the activation of glycogen phosphorylase and glucose output in vitro in frog hepatocytes by hormones and analogues that have a -adrenergic pathway [4]. Furthermore, freezing induces a rapid increase in liver cyclic AMP levels and in the percentage of cyclic AMP-dependent protein kinase (PKA) present as the free catalytic subunit (PKAc) indicating that intracellular signal transduction is supplied by PKA-mediated protein phosphorylation to coordinate the requisite responses that promote glucose synthesis and export [5].

We wondered whether any specific adaptations of liver PKA helped to facilitate the enzyme's role in activating cryoprotectant output at subzero temperatures in the wood frog. Our present analysis of wood frog liver PKAc includes a novel purification method for the enzyme and an analysis of enzyme properties that indicate adaptive modifications that would suit enzyme function in mediating the cryoprotective response by wood frog liver.

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Holden, C.P.; Storey, J.; Storey, K.B.; (1998). Purification and Characterization of Protein Kinase A Catalytic Subunit from Liver of the Freeze-Tolerant Wood Frog: Role in Glycogenolysis During Freezing. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cellbio/holden0435/index.html
© 1998 Author(s) Hold Copyright