Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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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