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Cell Biology Poster Session






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Materials & Methods

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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)


Results

Table 1. Purification of Rana sylvatica liver protein kinase A catalytic subunit.

                     Total     Total        %Yield Fold     Specific
                     Protein   Activity           Purif.   Activity
                     (mg)     (nmol Pi/min)              (nmolPi/min/mg)
Crude Homogenate     137.0       113.3       --    --        0.83
Hydroxylapatite       23.1        67.9       60    3.6       2.9
Protamine Agarose      7.5        25.1       22    4.0       3.3
P-200 Gel Filtration   0.05        3.3        3   85.7      70.9

Table 2. Substrate specificity of the free catalytic subunit of Rana sylvatica liver PKA; Vmax activities with different protein kinase substrates at 22C and 5C

Substrate                     22C            5C
                          Activity  A.R.    Activity  A.R
Kemptide (300 M)         568  29  100     353  14  100
Histone IIA (1 mg/ml)     216  12a  38      31  3a   9
Histone IIIS (1 mg/ml)    121  6a  21       13  1a   4
Histone VIS (0.2 mg/ml)   125  8a  22       13  1a   4
Protamine Cl- (0.2 mg/ml)  64  3a  11        7  0.7a 2

Activity is nmol Pi transferred/min/mg protein, means SEM, n = 4 determinations on one preparation of purified enzyme. A.R.: activity ratio relative to kemptide at either temperature. a - Significantly different from the corresponding value with kemptide at the same temperature using one-way ANOVA with an SNK test, p<0.05.

Substrate affinity constants.

Substrate affinity constants for kemptide and Mg-ATP were also temperature-sensitive and Km values for both substrates decreased significantly when assay temperature was lowered. Km for Mg-ATP decreased by 50 % from 51.8 1.0 M at 22C to 24.8 1.4 M at 5C (mean SEM, n=3, p<0.05) whereas the Km for kemptide decreased by 33 % from 9.0 0.1 M at 22C to 6.4 0.3 M at 5C (mean SEM, n=3, p<0.05).

Table 3. Inhibitor coefficients (I50 values) for the purified free catalytic subunit of PKA from Rana sylvatica liver.

Inhibitor                        I50 Value
Temperature                22C            5C
KCl (mM)                 495  10       720  10b
KNO3 (mM)                426  12          --
NaCl (mM)                562  16       700  12b
NaF (mM)                19.3  1a          --
NH4Cl (mM)               396  8a          --
(NH4)2SO4 (mM)            150  12a         --
PKA-I (nM)               3.3  0.15        --
H89 (nM)                25.1  0.6         --

Data are means SEM, n = 3 separate preparations of purified frog liver PKAc enzyme. a- Significantly different from the corresponding value for KCl at 22C by a one way ANOVA with an SNK test, p<0.05; b- significantly different from the corresponding value at 22C, p<0.05.

click to enlarge

Fig. 1. Elution profiles of frog liver PKAc from 3 chromatography columns.

Fig. 1. Elution profiles showing the chromatography steps in the purification of the free catalytic subunit of cyclic AMP-dependent protein kinase A from Rana sylvatica liver: A) hydroxylapatite, B) protamine agarose, C) Bio-Gel P-200 gel filtration column. Data are means of n = 3 preparations.

click to enlarge

Fig. 2. Arrhenius plot for purified frog liver PKAc.

Fig. 2. Arrhenius plot for Rana sylvatica liver PKAc. Activities (dpm) were measured at 5C intervals between 1C to 35C under Vmax assay conditions. The effect of temperature on the maximal activity of PKAc showed a distinct break at 10C and the slopes of the lines gave calculated activation energies, Ea, of 51 4 kJ/mol for temperatures >10C and significantly higher, 110 9 kJ/mol, for temperatures <10C (n=4; p<0.05). Data are means SEM for n = 4 separate enzyme preparations.

click to enlarge

Fig. 3. pH profiles of frog liver PKAc at high and low temperature.

Fig. 3. The effect of pH on PKAc activity at 22C (A) and 5C (B). Total Mg2+ and ATP concentrations were varied to maintain constant concentrations of free Mg2+ and Mg-ATP at each temperature and pH [8]. The pH optimum was strongly influenced by assay temperature. A sharp optimum at pH 7.4 was seen for the enzyme assayed at 22C but at 5C the optimum dropped to pH 6.0. Data are means SEM for n = 3 separate enzyme preparations. pH was monitored at each temperature before and after each assay.

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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