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.