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Cell Biology Poster Session






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Purification and Characterization of Protein Kinase A Catalytic Subunit from Liver of the Freeze-Tolerant Wood Frog: Role in Glycogenolysis During Freezing

Holden, C.P. (Department of Pharmacology and Therapeutics, University of Manitoba, Canada)
Storey, J. (Department of Chemistry and Biology, Carleton University, Canada)
Storey, K.B. (Department of Chemistry and Biology, Carleton University, Canada)

Contact Person: Clark P. Holden (cholden@cc.umanitoba.ca)


Abstract

The free catalytic subunit of cAMP-dependent protein kinase (PKAc) from liver of the freeze tolerant wood frog, Rana sylvatica, was purified to greater than 95 % homogeneity. Final specific activity of PKAc was 71 nmol phosphate transferred/min/mg protein. Frog liver PKAc molecular weight was 47.6 1.1 kDa and K-m values for the phosphate acceptor, Kemptide, and Mg-ATP were 9.0 0.1 micromolar and 51.8 1.0 micromolar at 22C, respectively. Both Km values dropped significantly at 5C, facilitating low temperature function of PKAc. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA-I, H89) and NaF, but not by several potassium, sodium and ammonium salts. Salt inhibition was reduced at 5C, which would minimize PKAc inhibition when cytoplasmic ion levels rise during the conversion of up to 65% of the frog's body water into extracellular ice. The effect of temperature on enzyme activity, analyzed via Arrhenius plots, indicated a conformational change in PKAc at 10C. Calculated activation energies were 51 4 kJ/mol at temperatures above 10C and 110 9 kJ/mol below. Wood frog liver PKAc plays a crucial role in mediating freeze-induced glycogenolysis, the metabolic pathway responsible for production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on PKAc function (increased substrate affinity, reduced ion inhibition) appear to be central to this role.

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Poster Number PAholden,0435
Keywords: glycogen phosphor, adenosine 3'-5' c, cryoprotectant sy, liver glycogenoly, low


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Holden, C.P.; Storey, J.; Storey, K.B.; (1998). Purification and Characterization of Protein Kinase A Catalytic Subunit from Liver of the Freeze-Tolerant Wood Frog: Role in Glycogenolysis During Freezing. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cellbio/holden0435/index.html
© 1998 Author(s) Hold Copyright