Clark P. Holden, Ph.D. Biology, 1995

Signal transduction and the function of second messengers and protein kinases in the control of glycogenolysis and cryoprotectant production in freeze tolerant vertebrates



A unique group of lower vertebrates, termed freeze tolerant, have developed complex biochemical mechanisms that allow them to survive whole body freezing. Perhaps the most important of these mechanisms is the production of cryoprotectant, usually in the form of glucose, via an activation of glycogenolysis as a response to freezing. This thesis investigates: (1) whether protein kinase enzymes are responsible for the early activation of glycolysis that stimulates cryoprotectant production in freeze tolerant vertebrates and/or the subsequent inhibition of the pathway in the later stages of a freezing episode, (2) the comparative aspects of the responses of protein kinases elicited by the environmental stresses of dehydration and anoxia, to those of freezing, and (3) the role of the free catalytic subunit of cyclic AMP-dependent protein kinase (PKAc) and protein kinase C (PKC) in cryoprotectant production in Rana sylvatica liver. Short freezing episodes caused a dramatic increase in the percentage of free catalytic PKA (PKAc). This response diminished with increased time frozen in tissues of freeze tolerant vertebrates. The measurement of second messengers over a freezing time course revealed significant increases in adenosine 3′,5′- cyclic monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) in liver and muscle of 4 freeze tolerant animals. An activation of PKA and PKC by these molecules, in response to freezing, is likely. Dehydration stress. not anoxia stress, elicited a remarkably similar protein kinase response to that seen during freezing. Levels of PKAc and its second messenger cAMP, as well as the second messenger for PKC, IP3, rose in dehydrated liver tissue whereas anoxia did not activate PKAc and only increased liver IP3. Purified R. sylvatica liver PKAc was not inhibited by high concentrations of salt or glucose and showed an increased affinity for substrates at low temperatures. Additional evidence showed that a freezing stress did not initiate a translocation of PKC to the plasma membrane in R. sylvatica liver and brain tissues. Freeze tolerant vertebrates possess an adaptive strategy to combat freezing which links second messengers and protein kinases to the post-translational modification of glycolytic enzymes conducting the biosynthesis of cryoprotectant.