Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Adaptation to anoxia, an environmental condition where there is no oxygen available, is a key factor for the survival of Orconectes virilis, a small freshwater crayfish inhabiting the lakes and streams of eastern Canada. These crayfish are able to survive circumstances of extreme hypoxia due to a rapid increase in respiratory water flow and potentially due to the scope of energy produced during anaerobic metabolism (Hochachka and Somero 1984). The metabolic pathways of O. virilis are influenced by changes in the availability of oxygen, in particular glycolysis. Key metabolic enzymes involved in this pathway, pyruvate kinase (PK) and phosphofructokinase (PFK), have been shown to be regulated by phosphorylation in several species (Storey 1984, Michaelidis and Storey 1990, Whitwam and Storey 1990, Fernandez et al. 1994, Benoit et al. 1994, Holwerda et al. 1983, Brooks and Storey 1990, Walsh et al. 1991) as is the case for this species (Cowan and Storey 1999).

The phosphorylation of proteins has long been established as key in the regulation of many critical enzymes (Walsh and Van Patten 1994). PKA has been shown to be involved in the regulation of metabolic enzymes, such as pyruvate kinase and phosphofructokinase of the glycolytic pathway. The enzyme consists of two regulatory and two catalytic subunits, and upon binding of the second messenger, cyclic 3',5'-adenosine monophosphate, to the regulatory subunits, the enzyme dissociates and releases the active catalytic subunits. The free catalytic subunits catalyze the ATP-dependent addition of phosphate groups onto proteins, this covalent modification typically changing a number of properties of the target protein which could include kinetic constants, susceptibility to different activators or inhibitors, and binding to other proteins or to subcellular sites. The catalytic subunit of PKA (termed PKAc) has been purified and characterized from several animal sources, including vertebrates (Mehrani and Storey 1995, Sugden et al. 1976, Yamamura et al. 1973) and invertebrates (Bishoff et al. 1990, Brooks and Storey 1996, Cao et al. 1995), for all of which the molecular weight of PKAc was within the range of 38-47 kDa but each with dramatically different kinetic properties. The Km of bovine liver PKAc for ATP determined in the presence of histone was 7.6 然 +/- 0.7 然 (Sugden et al. 1976), for the turtle Trachemys scripta elegans liver it was determined to be 83 然 +/- 6.5 M when assayed in the presence of kemptide (Mehrani and Storey 1995), for Mytillus galloprovincialis mantle tissue the Km for ATP was 43 然 +/- 7 然 in the presence of histone (Cao et al. 1995), and for the land snail Otala lactea foot muscle the Km for ATP was 176 然 +/- 25 然 when measured in the presence of kemptide (Brooks and Storey 1996). A catalytic subunit of PKA has been shown to be involved in the regulation of metabolism through reversible phosphorylation in O. virilis (Cowan and Storey 1999). Several other studies have focused on the ability of PKA to translocate from the cytosol to other compartments in the cell (Combest and Gilbert 1989, Scott and Carr 1992).

To further explore the metabolic responses to anoxia by a freshwater crayfish and to assess the connection between tolerance of this stress and the regulation of metabolism by reversible phosphorylation by this species, we focused on the responses of PKA to anoxia, and in particular on isolating and characterizing the catalytic subunit of PKA and its distribution in the cell. Responses by PKA varied with increased exposure to anoxia. PKA itself clearly is more prevalent in the microsomal/cytosolic fraction following several hours of exposure, as its activity drops significantly and its role in modifying key metabolic enzymes is reduced. Overall, the results link a primary role for PKA in regulating organ metabolism in response to anoxia.

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