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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)

Materials and Methods

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 1C 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 l: 20 mM phosphate, 150 M kemptide, 237 M unlabeled ATP, 2.1 mM Mg2+, 1.0 mM EGTA, and 2.2 x 106 dpm/assay (1.0 Ci/assay) 32P-ATP [6]. The temperature and final pH of the assay mix were 22C 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 22C. Total PKA activity was measured by adding 1 M 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 5C 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 22C and 5C and kinetic parameters (Km, I50) were analyzed using computer software [8]. An Arrhenius plot of log relative activity versus 1/K x 10-3 was completed under Vmax assay conditions by monitoring reaction rates from 1-35C at intervals of 5C. Kinetic data were analyzed using the Student's t-test or one-way analysis of variance with a Student Newman Keuls (SNK) test.

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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