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Cell Biology Poster Session






Abstract

Introduction

Materials & Methods

Results

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


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


Results

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 22C; 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 22C.

TABLE III

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
                      37C           5C             37C          5C       
Km (M)
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 37C using the Student's t-test, p< 0.05.

TABLE IV

Protein Kinase Inhibitors of Bat Skeletal Muscle PKAc

Inhibitor  Concentration  Activity  % Inhibition  Protein Target


Control 205 0

PKAi 0.1 M 118 42 PKA 2.0 M 18 91

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

Calphostin C 0.1 M 86 58 PKC 20.0 M 77 62

PKCi 0.05 M 168 17 PKC 1.0 M 182 11

Lav A 1.0 nM 173 15 TPK 20.0 nM 164 21

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

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

click to enlarge

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- 46C. Bat PKAc showed a distinct break in the plot at 10C. For the pig enzyme, the Arrhenius relationship was linear over the full temperature range tested with a calculated activation energy of Ea of 15.9 0.3 kJ/mol (n=3). By contrast, bat PKAc showed a distinct break in the relationship at 10C. The Ea for the reaction above 10C was 5.6 0.3 kJ/mol but below 10C was 5-fold higher at 29.5 1.7 kJ/mol (n=3). Data are means SEM, n = 3 separate enzyme preparations.

click to enlarge

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 5C (A) and 37C (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 60l assay mixture was modified by the addition of a 10l 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 37C but this dropped dramatically to pH 5.5 when the assay temperature was decreased to 5C. Data are means SEM for n = 3 separate enzyme preparations.

click to enlarge

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 5C (A) and 37C (B). Porcine PKAc showed a different response to temperature. A broad optimum from pH 6.5-8.0 occurred at 37C whereas at 5C 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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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
© 1998 Author(s) Hold Copyright