Cell Biology Poster Session
Storey, J. (Department of Chemistry and Biology, Carleton University, Canada)
Storey, K.B. (Department of Chemistry and Biology, Carleton University, Canada)
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 22°C, respectively. Both Km values dropped significantly at 5°C, 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 5°C, 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 10°C. Calculated activation energies were 51 ± 4 kJ/mol at temperatures above 10°C 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.
Back to the top.
| Discussion Board | Next Page | Your Poster Session |
|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|