Cell Biology Poster Session
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 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 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 µM at 22°C to 24.8 ± 1.4 µM at 5°C (mean ± SEM, n=3, p<0.05) whereas the Km 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 Vmax 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, Ea, 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 . 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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|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|
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