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 m olecular weight cryoprotectants; in freeze tolerant frogs this involve s a freezing-induced activation of liver glycogenolysis that leads to the accumulation of glucose as the cryoprotectant, in amounts up to 30 0 mM, in all organs. The present study shows that the synthesis and ma intenance of high organ glucose pools is facilitated by changes in the levels of fructose-2,6-bisphosphate (F2,6P2) and an inhibition of liv er 6-phosphofructo-2-kinase (PFK-2) activity that blocks the catabolis m 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. Freezi ng 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-6-phosphate, and increased I50 values f or enzyme inhibitors. I50 values for glycerol-3-phosphate and phosphoe nolpyruvate were 60- and 2.4-fold higher, respectively, for liver PFK- 2 from frozen frogs compared with controls. Changes in liver PFK-2 pro perties are consistent with a freezing-induced phosphorylation of the enzyme to produce a less active enzyme form, resulting in reduced orga n F2,6P2 levels and a decrease in 6-phosphofructo-1-kinase activity.