Purification and characterization of protein kinase a from liver of the freeze-tolerant wood frog: Role in glycogenolysis during freezing

Freeze tolerance by various amphibians includes cryoprotectant production i n the form of glucose. Activation of the catalytic subunit of liver cAMP-de pendent protein kinase (PKAc) facilitates activation of glycogenolysis, a c ritical biochemical process necessary for production of glucose. Here, we p urified PKAc from Rana sylvatica liver to determine the extent to which col d temperature, which stimulates cryoprotectant production, affected PKAc ac tivity and function. PKAc was purified to greater than 95% homogeneity, wit h a final specific activity of 71 nmol phosphate transferred/min/mg protein . The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K-m valu es for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 mu M at 22 degrees C, respectively. K-m values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA,, H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at l ow temperature. The effect of temperature on enzyme activity indicated a co nformational change in PKAc at 10 +/- 2 degrees C, with calculated activati on energies of 51 +/- 4 KJ/mol at temperatures above 10 degrees C and 110 /- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial rol e in mediating the freeze-induced glycogenolysis that is responsible for th e production of 200-300 mM levels of glucose as a cryoprotectant. Different ial effects of low temperature on enzyme function, increased substrate affi nity and reduced ion inhibition, appear to be central to this role. (C) 200 0 Academic Press.