Protein Kinase A from Bat Skeletal Muscle: A Kinetic Study of the Enzyme from a Hibernating Mammal

The catalytic subunit of adenosine 3ʹ–5ʹ-cyclic monophosphate-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°C. 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 μM at 37°C, respectively. Both values decreased significantly at 5°C, falling 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 (I50 values >400 mM) but NaF had an I50 of 38 mM; except for fluoride, ions were less inhibitory at 5°C, compared with 37°C. Arrhenius plots showed evidence of a temperature-dependent conformational change; a distinct break in the plot occurred at 10°C giving calculated activation energies of 5.6 ± 0.46 kJ/mol at temperatures above 10°C and 29.5 ± 2.0 kJ/mol below 10°C. 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°C to 5.5 at 5°C, 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 I50 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.