6-Phosphofructo-2-kinase and control of cryoprotectant synthesis in freeze tolerant frogs

Abstract

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 cryprotectant, in amounts up to 300 nM, 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 (F2,6P 2) 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°C) resulted in a sharp drop in F2,6P 2 levels in four organs, to 23–75% of control values, but F2,6P 2 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 K m for fructose-60-phosphate, and increased I 50 values for enzyme inhibitors. I 50 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 consitent with a freezing-induced phosphorylation of the enzyme to produce a less active enzyme form, resulting in reduced organ F26P 2 levels and a decrease in 6-phosphorylation of the enzyme to produce a less active enzyme form, resulting in reduced organ F2,6P 2 levels and a decrease in 6-phosphofructo-1-kinase activity.