Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog
- 206 Downloads
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 inKm for fructose-6-P, a 60% decrease inKa 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.
Unable to display preview. Download preview PDF.
- Cohen P (1980) Recently discovered systems of enzyme regulation by reversible phosphorylations. Elsevier, North HollandGoogle Scholar
- Engstrom L (1978) The regulation of liver pyruvate kinase by phosphorylation-dephosphorylation. Curr Top Cell Regul 13:29–51Google Scholar
- Keppler D, Decker K (1974) Glycogen determination with amyloglucosidase. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic Press, New York, pp 1127–1131Google Scholar
- Lowry OH, Passonneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New YorkGoogle Scholar
- Sakakibara R, Uyeda K (1983) Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver. J Biol Chem 258:8656–8662Google Scholar
- Schaftingen E van (1984) D-Fructose-2,6-P2. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Chemie-Verlag, Weinheim, pp 335–341Google Scholar
- Schmid WD (1982) Survival of frogs in low temperature. Science 215:697–698Google Scholar
- Storey JM, Storey KB (1985a) Adaptations of metabolism for freeze tolerance in the gray tree frog,Hyla versicolor. Can J Zool 63:49–54Google Scholar
- Storey JM, Storey KB (1985a) Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog,Rana sylvatica. J Comp Physiol B156:191–195Google Scholar
- Storey KB (1984) Freeze tolerance in the frog,Rana sylvatica. Experientia 40:1261–1262Google Scholar
- Storey KB (1985) Freeze tolerance in terrestrial frogs. Cryo Lett 6:115–134Google Scholar
- Storey KB (1986) Freeze tolerance in vertebrates: biochemical adaptation of terrestrially hibernating frogs. In: Heller HC, Musacchia XJ, Wang LCH (eds) Living in the cold. Physiological and biochemical adaptations. Elsevier, North Holland, pp 131–138Google Scholar
- Storey KB, Storey JM (1984) Biochemical adaptation for freezing tolerance in the wood frog,Rana sylvatica. J Comp Physiol B155:29–36Google Scholar
- Storey KB, Storey JM (1986a) Freeze tolerance and intolerance as strategies of winter survival in terrestrially-hibernating amphibians. Comp Biochem Physiol 83A:613–617Google Scholar
- Storey KB, Storey JM (1986b) Freeze tolerant frogs: Cryoprotectants and tissue metabolism during freeze/thaw cycles. Can J Zool 64:49–56Google Scholar
- Williamson JR (1970) General features of metabolic control as applied to the erythrocyte. Adv Biol Med 6:117–136Google Scholar