Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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TABLE I

Total PKA Activity and Effect of Hibernation on the Percentage of PKA Present as the Free Catalytic Subunit in Bat Tissues

Tissue           Total Activity              % PKAc
                                    Control     Hibernation
Skeletal Muscle  4.5 ± 0.6        35.4 ± 2.7   52.0 ± 1.9a
Liver           21.6 ± 4.0        14.8 ± 0.8   11.7 ± 0.7
Thymus          29.5 ± 2.2        36.6 ± 3.2   18.8 ± 0.6a
Spleen          28.0 ± 3.2        89.2 ± 3.0   43.1 ± 2.6a
White Adipose   17.3 ± 1.0        30.0 ± 2.5   47.0 ± 4.8a
Brown Adipose   41.2 ± 3.7        11.9 ± 0.6   12.1 ± 0.4
Brain           32.9 ± 4.0        37.4 ± 5.1   36.1 ± 4.6
Heart           12.6 ± 1.1        10.4 ± 1.1    7.3 ± 0.7
Kidney          16.7 ± 1.2        16.5 ± 1.1   14.7 ± 0.5

Data are means ± SEM, n = 3. Total PKA activity in tissues from euthermic, control bats is reported as units/gram wet weight at 22°C; total activity in hibernator tissues was not significantly different in any instance. ^a Significantly different from the corresponding control value using the Student's t-test, p<0.05. TABLE II

Purification of Bat Skeletal Muscle PKA Free Catalytic Subunit

Purification    Total     Total    % Yield  Fold   Specific
Step            Protein   Activity          Purif. Activity
                (mg)      (units)                 (units/mg)
Crude           24.72     11.51       --      --     0.466
DE-52 cellulose 10.11     280.5      100     59.5    27.7
Hydroxylapatite 2.63      328.8      100     268     125.0
Prot. agarose   1.18      170.1       52     310     144.4
Blue dextran    0.48       98.3       30     439     204.7

Enzyme assays conducted at 22°C.

Substrate Affinity Constants and I₅₀ Values for Purified Bat Skeletal Muscle PKAc and Commercial Porcine Heart PKAc at Two Assay Temperatures

                          Bat Muscle                    Porcine Heart
                      37°C           5°C             37°C          5°C       
Km (µM)
Kemptide          9.1 ± 0.2      3.4 ± 0.1a     11.9 ± 1.2      2.2 ± 0.03a
Mg-ATP           94.1 ± 4.5     49.2 ± 4.5a     25.0 ± 1.2     14.7 ± 0.9a
I50 (mM)
NaCl              461 ± 2.6      487 ± 13.7      388 ± 3.9      461 ± 13.3a
KCl               456 ± 8.8      582 ± 26.8a     378 ± 11.8     606 ± 10.7a
NH4CL             349 ± 13.5     347 ± 22.8      232 ± 0.9      410 ± 9.1a
(NH4)2SO4          108 ± 2.0      196 ± 8.6a     74.9 ± 4.0      186 ± 2.4a
NaF              37.8 ± 0.8     38.4 ± 1.1      38.9 ± 0.5     11.4 ± 0.7a

Data are means ± SEM, n = 3. ^aSignificantly different from corresponding value at 37°C using the Student's t-test, p< 0.05.

Protein Kinase Inhibitors of Bat Skeletal Muscle PKAc

Inhibitor  Concentration  Activity  % Inhibition  Protein Target



Control                      205          0



PKAi          0.1 µM         118          42        PKA
              2.0 µM         18           91



H-89         0.05 µM         150          27        PKA
              1.0 µM         55           73



Calphostin C  0.1 µM         86           58        PKC
             20.0 µM         77           62



PKCi         0.05 µM         168          17        PKC
              1.0 µM         182          11



Lav A         1.0 nM         173          15        TPK
             20.0 nM         164          21



KT-5823       0.1 µM         168          18        PKG
             20.0 µM         150          27

Data are n = 1. Activities are units/mg protein at 22°C. PKC is calcium/ phospholipid dependent protein kinase, TPK is tyrosine protein kinase, and PKG is 3'-5'-cyclic GMP dependent protein kinase.

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Fig. 1: Arrhenius plots for bat skeletal muscle and porcine heart PKAc.

FIG. 1. Arrhenius plots for (A) purified bat skeletal muscle PKAc and (B) purified porcine heart PKAc. Maximal activities were measured at 5-10° C intervals over the range 0- 46°C. Bat PKAc showed a distinct break in the plot at 10°C. For the pig enzyme, the Arrhenius relationship was linear over the full temperature range tested with a calculated activation energy of E_a of 15.9 ± 0.3 kJ/mol (n=3). By contrast, bat PKAc showed a distinct break in the relationship at 10°C. The E_a for the reaction above 10°C was 5.6 ± 0.3 kJ/mol but below 10°C was 5-fold higher at 29.5 ± 1.7 kJ/mol (n=3). Data are means ± SEM, n = 3 separate enzyme preparations.

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Fig. 2: Effect of temperature on pH for purified PKAc from Myotis lucifugus skeletal muscle

FIG. 2. The effect of assay pH on the activity of purified bat PKAc at 5°C (A) and 37°C (B). Total Mg²⁺ and ATP concentrations were varied to maintain constant concentrations of Mg-ATP and free Mg²⁺ at each temperature and pH. For pH curves, the standard 60µl assay mixture was modified by the addition of a 10µl aliquot of a buffer mixture (5 mM KH₂PO₄ + 5 mM K₂HPO₄, 10 mM imidazole, and 10 mM Tris base) that was pre-adjusted to one of the desired pH values. This changed assay pH to the desired value and this was confirmed by pH measurement after each assay was completed. The pH optimum was 8.5 at 37°C but this dropped dramatically to pH 5.5 when the assay temperature was decreased to 5°C. Data are means ± SEM for n = 3 separate enzyme preparations.

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Fig. 3: Effect of temperature on pH for purified PKAc from porcine cardiac muscle

FIG. 3. The effect of pH on purified porcine heart PKAc activity at 5°C (A) and 37°C (B). Porcine PKAc showed a different response to temperature. A broad optimum from pH 6.5-8.0 occurred at 37°C whereas at 5°C a very narrow optimum was observed at pH 6.0. PKAc from both animals showed relatively broad pH optimal ranges at euthermic body temperature and quite narrow ranges at the lower temperature. Other information as in Fig. 3.

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