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Protein Kinase A from Bat Skeletal Muscle: A Kinetic Study of the Enzyme from a Hibernating Mammal

Contact Person: Clark P. Holden (cholden@cc.umanitoba.ca)

Materials and Methods

Animals. Little brown bats (Myotis lucifugus) were collected in mid-January from caves near Sherbrooke, P.Q. where they had been hibernating for at least 3 months. In the laboratory, half of the bats were placed in an environmental chamber at 5C, allowed to re-enter hibernation, and then sampled after 3 d while torpid. The other animals were kept awake for 3 d at 22C and maintained at a body temperature of about 37C, as measured by a rectal thermometer. Animals were sacrificed by decapitation and tissues were rapidly excised, frozen in liquid nitrogen, and then transferred to -80C for storage. Lyophilized porcine heart PKAc from Sigma Chemical Co. was reconstituted in buffer containing 10 mM K2P04, 10 mM KH2PO4, 2 mM 2-mercaptoethanol and 1 mM EDTA, pH 6.5, and dialyzed for 2 h before use.

Assay and purification of skeletal muscle PKAc. PKA activity was measured using a radioactive assay that monitored the incorporation of radiolabeled 32P-ATP onto the phosphate-accepting artificial substrate, Kemptide (10). Frozen skeletal muscle (2 g) was homogenized 1:20 w/v in buffer containing 5 mM K2HP04, 5 mM KH2PO4, 2 mM 2-mercaptoethanol, 1 mM EDTA, pH 6.8. Cyclic AMP (1mM) and phenylmethylsulfonyl fluoride (1mM) were added prior to homogenization using a Polytron PT 10 homogenizer. PKAc was purified to homogeneity through columns of DE-52 cellulose (10 x 1 cm), hydroxylapatite (2 x 1 cm), protamine agarose and Blue Dextran (4 x 1 cm). Pooled fractions from the final column served as the enzyme source for all kinetic studies. Protein concentrations were determined using the Bradford method (11). Kinetic studies on bat skeletal muscle PKAc were performed at 37C and 5C, the former representing euthermic the body temperature of both species and the latter representing the measured core body temperature of hibernating bats. Arrhenius plots and pH curves for both enzymes were determined under Vmax assay conditions. Statistical analysis used the Student t-test.

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Holden, C.P.; Storey, J.; Storey, K.B.; (1998). Protein Kinase A from Bat Skeletal Muscle: A Kinetic Study of the Enzyme from a Hibernating Mammal. 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/holden0436/index.html
© 1998 Author(s) Hold Copyright