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 in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with 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) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM 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(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation 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 role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.
Department of Pharmacology and Therapeutics, University of Manitoba Faculty of Medicine, 753 McDermot Avenue, Winnipeg, Manitoba, R3E 0T6, Canada.
C P
CP
Holden
K B
KB
Storey
D000064:Acclimatization / Q000502:physiology; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008112:Liver Glycogen / Q000378:metabolism; D008970:Molecular Weight; D011898:Ranidae / Q000378:metabolism*; D013379:Substrate Specificity
D008112:Liver Glycogen; D017868:Cyclic AMP-Dependent Protein Kinases