Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Chemicals and animals

Biochemicals were purchased from Boehringer-Mannheim Corp., Sigma Chemical Co., Bio-Rad Labs, and New England Nuclear (Dupont). Wood frogs, Rana sylvatica were collected from ponds in the Ottawa area in mid-April. Frogs were placed in plastic boxes containing damp sphagnum moss and were held in incubators at 5 ± 1°C for two weeks prior to use. Animal sacrifice was by double pithing and tissues were immediately excised and frozen in liquid nitrogen.

Cyclic AMP-dependent protein kinase assay

Reactions were carried out in disposable glass tubes with standard assay conditions which gave the following reactant concentrations in a final volume of 60 µl: 20 mM phosphate, 150 µM kemptide, 237 µM unlabeled ATP, 2.1 mM Mg²⁺, 1.0 mM EGTA, and 2.2 x 10⁶ dpm/assay (1.0 µCi/assay) ³²P-ATP [6]. The temperature and final pH of the assay mix were 22°C and 6.8, respectively. One unit of PKAc activity is defined as the amount of enzyme which catalyzes the incorporation of 1 nmol of ³²P from ATP onto kemptide in 1 min at 22°C. Total PKA activity was measured by adding 1 µM cAMP to the assay mixture.

Purification of the free catalytic subunit of R. sylvatica liver PKA

A novel purification scheme was developed. Frozen liver (2 g) was homogenized 1:3 w/v in Buffer A (10 mM K₂P0₄, 2 mM b-mercaptoethanol, 1 mM EDTA, 10 % v/v glycerol, 1 mM cAMP, pH 6.8) with 1 mM phenylmethylsulfonyl fluoride added immediately prior to homogenization with a Polytron PT 10 homogenizer. The homogenate was incubated on ice for 30 min to stimulate full dissociation of the PKA catalytic subunit and then centrifuged at 27,000 g for 30 min at 5°C in a Sorvall RC-5B refrigerated centrifuge. PKAc was purified through columns of hydroxylapatite column (1.5 X 10 cm), protamine agarose column (1 X 4 cm), and a Bio-Gel P-200 gel filtration column (0.8 X 15 cm). The final peak fractions eluted from the gel filtration column were pooled and concentrated using a Centricon (Amicon) with a 30,000 M.W. cut-off. Protein was measured using the Coomassie blue G-250 dye-binding method of Bradford [7].

Molecular weight determinations and kinetic characterization of frog liver PKAc

The molecular weight of purified PKAc was determined by SDS polyacrylamide gel electrophoresis (PAGE) and gel filtration using a Bio-Gel P-200 column (0.8 x 15 cm). Studies were performed at 22°C and 5°C and kinetic parameters (K_m, I₅₀) were analyzed using computer software [8]. An Arrhenius plot of log relative activity versus 1/°K x 10^-3 was completed under V_max assay conditions by monitoring reaction rates from 1-35°C at intervals of 5°C. Kinetic data were analyzed using the Student's t-test or one-way analysis of variance with a Student Newman Keuls (SNK) test.

Back to the top.