Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

The catalytic subunit of adenosine 3'-5'-cyclic monophosphate-dependent protein kinase (PKAc) was purified to homogeneity from skeletal muscle of the little brown bat, Myotis lucifugus. Final specific activity was 205 nmol phosphate transferred/min/mg protein at 22°C. Identification of the enzyme as a protein kinase A was confirmed through the use of specific PKA inhibitors. PKAc molecular weight was 54.6 ± 3.5 kDa. Km values for Kemptide and Mg-ATP were 9.1 ± 0.2 micromolar and 94.1 ± 4.5 micromolar at 37°C, respectively; both values decreased significantly at 5°C. Neutral salts had little effect on enzyme activity (I50 values > 400 mM), but NaF did with an I50 value of 38 mM. Arrhenius plots showed evidence of a temperature-dependent conformational change in PKAc. Calculated activation energies were 5.6 ± 0.46 kJ/mol at temperatures above 10°C and 29.5 ± 2.0 kJ/mol below. The pH optimum of bat PKAc also changed dramatically with temperature falling from 8.5 at 37°C to 5.5 at 5°C, an effect that could substantially limit enzyme activity in vivo at the low body temperatures seen during hibernation. Overall, low temperature had both positive (increased %PKAc, reduced Km values, increased I50 values for salts) and negative (increased activation energy, acidic shift of pH optimum) effects on PKAc. However, the substantial positive effects of low temperature on the enzyme suggest a critical role for continued PKAc action in signal transduction processes in the hibernating bat.

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Poster Number PAholden0436
Keywords: hibernation, Signal, transduction, PKA, skeletal muscle