Neal Dawson, M. Sc. Biology, 2009

Regulation of tail muscle energetics during anoxia in the freshwater crayfish, Orconectes virilis.

Abstract: Metabolic rate depression is vital to the survival of many organisms in the face of low oxygen levels. This is achieved by a coordinated suppression of both ATP-consuming and ATP-producing metabolic pathways. The role of reversible protein phosphorylation in metabolic rate depression during anoxia was explored in the tail muscle of the anoxia-tolerant freshwater crayfish, Orconectes virilis. This study investigated glutamate dehydrogenase (GDH), the enzymatic bridge between amino acid and carbohydrate metabolism, arginine kinase (AK), an important enzyme involved in regulation of phosphagen reserves, and hexokinase (HK), the enzyme at the forefront of carbohydrate metabolism. The data obtained showed that GDH and AK are regulated by reversible phosphorylation during anoxia, resulting in less phosphorylated, less active forms of these enzymes. Experiments were performed under normoxic and anoxic conditions, and protein expression levels, susceptibility to urea denaturation, structural stability, response to specific protein kinase and phosphatase incubations as well as elution profiles from an ion-exchange column were explored. The data from GDH suggests that amino acid metabolism is left largely separate from carbohydrate metabolism by the reduction of this vital bridge point. AK results suggest that precious ATP is not involved in the regeneration of phosphagen reserves during anoxia. HK was also explored using similar experiments, and it seems that HK protein levels increase during anoxia, and reversible phosphorylation seems to increase protein stability and affect cellular localization. Overall, these studies suggest that reversible phosphorylation plays a key role in the regulation of muscle energetics in the freshwater crayfish, O. virilis.