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Cell Biology Poster Session






Abstract

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Materials & Methods

Results

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Purification and Characterization of Protein Kinase A Catalytic Subunit from Liver of the Freeze-Tolerant Wood Frog: Role in Glycogenolysis During Freezing


Contact Person: Clark P. Holden (cholden@cc.umanitoba.ca)


Discussion and Conclusion

The novel purification scheme developed for the catalytic subunit of cAMP dependent protein kinase from R. sylvatica liver resulted in a purified preparation with a final specific activity of 71 nmol Pi/min/mg protein at 22C. This value is lower than many of the specific activities reported for the enzyme other sources [9-13] but when the difference in assay temperature is factored in (mammalian activities are generally measured at 37C) the resulting specific activity of the wood frog enzyme is similar to values for the homogeneous mammalian enzyme. The purification procedure developed for the enzyme was highly reproducible. Although the overall yield was low, a stable purified enzyme preparation was easily obtained.

The molecular weights of R. sylvatica liver PKAc determined by SDS-PAGE and P-200 gel filtration, 47-47.6 kDa, were very similar to those of mammalian, reptilian and invertebrate forms of the enzyme [12, 14-17]. The kinetic constants of R. sylvatica liver PKAc were also similar to those reported for mammalian PKAc and, like the enzyme from other sources, frog liver PKAc showed a much higher affinity for the synthetic substrate kemptide than for several other phosphate-accepting histone substrates [14]. The artificial protein inhibitors of mammalian PKAc (PKA-I and H89) also inhibited R. sylvatica PKAc with I50 values in the same range as those found for mammalian forms of the enzyme [18,19]. All these data support the proposal that both the overall protein structure and the catalytic site of PKAc have been highly conserved throughout evolution.

The pH optimum of wood frog PKAc was broad at room temperature with over 90% of optimal activity maintained over the range from pH 6.0-8.0, a characteristic also seen for turtle liver PKAc [14]. However, the pH optimum at 5C fell to 6.0 which was not the case for other vertebrate forms of PKAc. Since the intracellular pH of ectothermic animals rises as temperature decreases (due to temperature effects on the dissociation of alpha-imidazolium groups on histidine moities) this opposite change in the pH optimum of PKAc could, predictably, have a substantial negative effect on the relative activity of the free catalytic subunits at low temperature. Furthermore, the break in the Arrhenius plot at 10C indicates that there is a conformational change in the enzyme at lower temperatures and the increase in Ea at low temperatures indicates a stronger suppression of activity by decreasing temperatures over the lower versus higher temperature range. Both the temperature effects and the pH effects on enzyme activity would, therefore, have negative effects on enzyme function at low body temperatures. These effects on this important enzyme of signal transduction, as well as the low percentage of the total enzyme present as the catalytic subunit (7% in resting frogs at 5C) [5], could be factors that contribute to the general quiescent state of wood frogs during the winter.

Other factors, however, leave the enzyme available to be activated rapidly and strongly in response to freezing. It is, in fact, at subzero body temperatures during the freezing of body water that the enzyme has perhaps its most important function. This is the activation of glycogenolysis to support the synthesis of the cryoprotectant, glucose [1]. Indeed, within 2 min after nucleation on the skin surface, cAMP levels double and liver PKA is strongly activated [5]. The % PKAc rises from 7 % to 43 % within 2 min and reaches a maximum of 62% within 5 min [5]. This results in an equally rapid increase in glycogen phosphorylase a activity which doubles within 2 min and rises by 7-fold in 70 min and stimulates an equally rapid rise in liver and blood glucose concentrations from < 5 mM in control frogs to 35-50 mM within about an hour post-nucleation [21,22]. The ability of PKAc to rapidly activate phosphorylase at low temperature would also be aided by the effect of low temperature on the affinity of PKAc for both its substrates. Km values for both Mg-ATP and the phosphate acceptor, Kemptide, decreased substantially at 5C, as compared with 22C, and this would facilitate PKAc phosphorylation of its protein substrates whenever PKAc content rose at low body temperature.

Frog liver PKAc was susceptible to inhibition by a number of inorganic salts. These effects were similar to those reported for salt inhibition of mammalian, reptilian, and invertebrate forms of PKAc [13,14,17]. Notably, inhibition by KCl and NaCl decreased (I50 increased) at low assay temperature. This would also be advantageous for maintaining PKAc function during freeze-induced glycogenolysis for intracellular ion concentrations rise rapidly as water is withdrawn into extracellular ice crystals. The conversion of as much as 65 % of total body water into ice effectively triples ion and metabolite concentrations in remaining body fluids. Liver PKAc was not affected by high glucose, however, indicating that the product of freeze-induced glycogenolysis would not affect the signal transduction cascade that controls its synthesis, at least at the level of PKAc.

In conclusion, R. sylvatica liver PKAc clearly maintains many properties that are very similar to those of the enzyme from other vertebrate, and even invertebrate, sources. This adds further evidence to the proposal that the structure and function of PKAc has been highly conserved throughout evolution [22]. Nonetheless, selected properties of the enzyme, particularly the effect of low temperature in increasing substrate affinity and reducing ion inhibition at low temperature, and the lack of high glucose effects on the enzyme, are clearly advantageous for supporting the crucial function of the enzyme in rapidly activating cryoprotectant biosynthesis by liver during the freezing of body fluids.

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<= Results DISCUSSION & CONCLUSSIONS References =>

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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