Cell Biology Poster Session
Discussion and Conclusion
The elevated percentage of PKA present as the catalytic subunit during hibernation in skeletal muscle (a rise from 35% in control to 52% in hibernator muscle) suggested that the enzyme might be active in controlling some aspect of the hibernation process in muscle, such as a role in sustaining metabolic rate suppression. The % PKAc was also elevated in white adipose tissue during hibernation. Lipids mobilized from white adipose are the primary fuel supporting metabolism in all organs during hibernation as well as thermogenesis during arousal. Hence, elevated PKAc may be necessary to sustain continuous lipid export from white adipose during torpor.
The purification scheme developed in the present study was highly reproducible and resulted in a stable purified enzyme preparation with an overall yield of 30% (Table II). The homogeneous enzyme had a final specific activity of 205 nmol PI/min/mg protein at 22°C (Table II). This value was 5-fold higher than that for purified PKAc from snail foot muscle (12) but lower than values from most other sources, including bovine heart and liver (13,14-16). The molecular weight of bat muscle PKAc was estimated at 52.1-54.6 kDa by three methods and this is very similar to values reported for the enzyme from many other sources.
Km values of bat muscle PKAc for the phosphate-acceptor, Kemptide, were very similar to those reported for PKAc from other mammalian sources, including porcine heart PKAc (Table III). However, the bat enzyme showed a considerably higher Km Mg-ATP than has been reported for other mammals; indeed, Km Mg-ATP was nearly 4-fold higher at 37°C and 2-fold higher at 5°C than the corresponding values for porcine heart (Table III). Km values for both substrates decreased significantly at the lower assay temperature (5°C). This feature was shared by both bat and pig enzymes, as well as frog PKAc (17), and hence, is not unique to the hibernator enzyme.
The break in the Arrhenius plot for bat PKAc at 10°C indicates a conformational change in the bat enzyme at lower temperatures (Fig. 1). This differs distinctly from the porcine enzyme which showed a linear relationship over the entire temperature range. Body temperatures below 10°C frequently occur during hibernation and the sharp increase in Q10 for the reaction that is also indicated by these data would result in a differential reduction in maximal enzyme activity at lower versus higher temperatures. A differentially reduced maximal activity at low temperature may contribute to the overall metabolic rate depression of the hibernating state. Such low temperature effects could suppress the overall activity state of signal transducing enzymes while hibernating and have a general role in the maintenance of the torpid state.
The pH optimum for bat PKAc at 37°C (Fig. 2) was much higher than that found for either invertebrate or other mammalian forms of the enzyme (16), including the porcine enzyme (Fig. 3). However, when assayed at 37°C, both bat and porcine PKAc retained near-optimum activity over a broad optimum range of pH values, a characteristic common to many PKAc enzymes (17-19). At 5°C, the optimum for both enzymes became much sharper and was focused at about pH 5.8-6. Cellular pH in torpid mammals at low body temperature is influenced by two factors: a) the effect of temperature on intracellular histidine buffers (resulting in a 0.018 pH unit increase per 1°C decrease), and b) the development of respiratory acidosis due to apnoic breathing patterns (20). The net result for most tissues of hibernating mammals is a relative acidosis: pH rises slightly during hibernation but not as much as would be expected due to temperature effects on the dissociation of alpha-imidazole groups on histidine buffers (21). Intracellular pH values in muscle of hibernators are about 6.9-7.1 for most hibernators (21). Hence, the temperature effect in shifting the pH curve for both bat and porcine PKAc to an acidic optimum of around 6.0 would have the effect of reducing enzyme activity in vivo.
Bat skeletal muscle PKAc was susceptible to inhibition by inorganic salts. As Table III shows, changing the cation (with Cl- as the anion) had virtually no effect on the I50 and, hence, inhibitory effects must reside with the anions. Indeed, sulphate anion was substantially more inhibitory than chloride and fluoride anion was the strongest inhibitor of all. However, relative to the physiological concentrations of these ions in vivo, none of these ions is likely to have a major role in regulating enzyme activity in the cell. Notably, though, inhibitory effects by most ions decreased at the lower assay temperature (opposite to the increased substrate affinities) which is consistent with changing enzyme conformation with temperature.Artificial inhibitors of mammalian PKAc, PKA-I and H89, also inhibited M. lucifugus PKAc with % inhibition values similar to those reported for the enzyme from other sources (Table IV). This confirms that the enzyme is a typical PKAc (Table IV). Relatively poor inhibition of the enzyme by protein inhibitors of PKC (Calphostin C) and PKG (KT5823) further supports this identification.
It is clear from the above kinetic data that the purified free catalytic subunit of PKA from M. lucifugus skeletal muscle is very similar to other vertebrate and invertebrate forms of the enzyme. This is consistent with the general conclusion, that has been drawn before, that the structure and function of PKAc has been highly conserved through evolution (22), undoubtedly due to its extremely important role in signal transduction in all cells. Temperature effects on M. lucifugus muscle PKAc were mixed; some would enhance low temperature function (effects on Km and I50, changes in % PKAc) whereas others should suppress function (increased activation energy at low temperatures, acidic shift of the pH curve). Overall, however, the former positive effects of low temperature on enzyme function would suggest an important role for continued PKA action in signal transduction in the hibernating animal.
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|Holden, C.P.; Storey, J.; Storey, K.B.; (1998). Protein Kinase A from Bat Skeletal Muscle: A Kinetic Study of the Enzyme from a Hibernating Mammal. 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/holden0436/index.html|
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