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Arch Biochem Biophys. 1998 Oct 15;358(2):243-50.

Protein kinase A from bat skeletal muscle: a kinetic study of the enzyme from a hibernating mammal.

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Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6.


The catalytic subunit of adenosine 3'-5'-cyclic mono-phosphate-dependent protein kinase (PKAc) was purified to homogeneity from skeletal muscle of the little brown bat, Myotis lucifugus. The purification procedure was highly reproducible, resulting in a final activity of 205 nmol phosphate transferred/min/mg protein at 22 degreesC. Identification of the enzyme as a protein kinase A was confirmed through the use of specific PKA inhibitors. The catalytic subunit had a molecular weight of 54.6 +/- 3.5 kDa.Km values for Kemptide and Mg-ATP were 9.1 +/- 0.2 and 94.1 +/- 4.5 microM at 37 degreesC, respectively. Both values decreased significantly at 5 degreesC to 37 and 52% of their values at the higher temperature. Similar temperature effects on Km values were found with the purified commercial pig heart enzyme. Neutral salts had little effect on enzyme activity (I 50 values >400 mM) but NaF had an I 50 of 38 mM; except for fluoride, ions were less inhibitory at 5 degreesC, compared with 37 degreesC. Arrhenius plots showed evidence of a temperature-dependent conformational change; a distinct break in the plot occurred at 10 degreesC giving calculated activation energies of 5.6 +/- 0. 46 kJ/ mol at temperatures above 10 degreesC++and 29.5 +/- 2.0 kJ/mol below 10 degreesC. Porcine PKAc, by contrast, showed a linear Arrhenius plot over the entire temperature range tested and an intermediate activation energy of 15.9 +/- 0.3 kJ/mol. The pH optimum of bat PKAc also changed dramatically with temperature falling from 8.5 at 37 degreesC to 5.5 at 5 degreesC, an effect that could substantially change enzyme activity in vivo at the low body temperature of the hibernating state. Overall, low temperature had both positive (increased the percentage of PKAc, reduced Km values, increased I 50 values for salts) and negative (increased activation energy, acidic shift of pH optimum) effects on PKAc but the substantial positive effects of low temperature on the enzyme suggest an important role for continued PKA action in signal transduction in the hibernating animal.

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