Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

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; V_max activities with different protein kinase substrates at 22°C and 5°C

Substrate                     22°C            5°C
                          Activity  A.R.    Activity  A.R
Kemptide (300 µM)         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 P_i 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 K_m values for both substrates decreased significantly when assay temperature was lowered. K_m for Mg-ATP decreased by 50 % from 51.8 ± 1.0 µM at 22°C to 24.8 ± 1.4 µM at 5°C (mean ± SEM, n=3, p<0.05) whereas the K_m for kemptide decreased by 33 % from 9.0 ± 0.1 µM at 22°C to 6.4 ± 0.3 µM at 5°C (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                22°C            5°C
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 22°C by a one way ANOVA with an SNK test, p<0.05; ^b- significantly different from the corresponding value at 22°C, p<0.05.

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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.

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

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

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

Fig. 3. The effect of pH on PKAc activity at 22°C (A) and 5°C (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 22°C but at 5°C 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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