Regulation of enzyme function in freeze tolerance
The wood frog (Rana sylvatica) is one of the few vertebrate species that can survive whole-body freezing during the cold winter months. The frog endures the freezing of 65-70% of total body water as extracellular ice and, while frozen, shows no respiration, heart beat, or brain activity. Consequently, the frogs experience anoxia and ischemia throughout the freeze followed by oxidative stress when oxygen is reperfused. Enzymes, the biochemical catalysts of cells, must be appropriately controlled to ensure survival. This thesis explores the properties and regulation of key enzymes of adenylate metabolism (AMP-deaminase, AMPD; creatine kinase, CK) and glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH; hexokinase, HK). The studies showed that changes in pH, temperature, inhibitor and activator concentrations, and binding to myofibrils are involved in regulating these enzymes in the transition to the frozen state. Moreover, reversible protein phosphorylation appears to be a key regulatory mechanism, altering enzyme activity and substrate affinity to suit physiological needs during freezing. Analysis of kinetic parameters showed an increase in enzyme activity for CK and decreased activity for HK. Affinity of CK for one of its substrates, creatine, increased, whereas HK, G6PDH, and myofibril-bound AMPD showed reduced substrate affinity in the transition to the frozen state. These changes in kinetic parameters were the result reversible protein phosphorylation; bound AMPD and CK both increased in phosphorylation state in frozen frogs, whereas G6PDH and HK both decreased in phosphorylation state. Changes in enzyme activity as a result of reversible phosphorylation were analyzed by in vitro stimulation of endogenous protein kinase and protein phosphatase activities. Native phosphorylation states of these enzymes, and changes between control and frozen frogs were further confirmed by elution profiles off DEAE-Sephadex ion-exchange columns that were interconverted between the two physiological states, as well as SDS-PAGE studies that compared phosphoprotein levels to total protein levels. Though phosphorylation states of these enzymes changed, protein levels remained constant in the transition to the frozen state. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly reversible protein phosphorylation, control enzyme function and the reorganization of metabolic pathways in freeze-tolerance.