Oxidative Stress Poster Session



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Purification And Characterization Of A Camp-Dependent Protein Kinase >From The Tail Muscle Of The Crayfish, Orconectes Virilis.

Cowan, K.J. (COR Therapeutics, Inc., So. San Francisco, USA.)
Storey, K.B. (Institute of Biochemistry and Department of Chemistry, Carleton University, Canada)

Contact Person: Dr. Kyra J. Cowan (kcowan@corr.com)


Many invertebrates tolerate oxygen deprivation by profoundly reducing metabolic rate. Reversible phosphorylation of enzymes is key to coordinating metabolic suppression and hence regulation of protein kinases is central to anoxia survival. We analyzed effects of anoxia exposure in vivo on cAMP-dependent protein kinase (PKA) in tail muscle and hepatopancreas of crayfish, Orconectes virilis. A sharp increase in % PKA present as the active catalytic subunit (PKAc) occurred within the first two hours of anoxia, followed by a gradual decrease in active enzyme with longer exposures. Subcellular fractionation showed a drop in PKA activity associated with plasma membrane fractions under anoxia whereas cytosolic PKA increased. To assess adaptations that aid PKA function in anoxia, PKAc was purified from muscle, using negative affinity chromatography through Matrix Gel Red and hydroxylapatite and affinity chromatography on protamine agarose; final specific activity was 52.4 U/mg protein. The pure enzyme showed a molecular weight of 43.8 +/- 0.4 kDa (n = 3), a pH optimum of 6.8, weak inhibition by salts (I50 = 739.3 +/- 14.5 mM), and high affinity for Mg.ATP (Km = 131.0 +/- 14.4 microM) and Kemptide (Km 31.6 +/- 5.2 microM). These studies substantiate the importance of PKA and of reversible phosphorylation as a mechanism for regulating metabolism during anoxia.

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Poster Number PAcowan0409
Keywords: phosphorylation, metabolic, arrest, crustacean, protein

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Cowan, K.J.; Storey, K.B.; (1998). Purification And Characterization Of A Camp-Dependent Protein Kinase >From The Tail Muscle Of The Crayfish, Orconectes Virilis.. 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/oxidative/cowan0409/index.html
© 1998 Author(s) Hold Copyright