The freshwater crayfish, Orconectes virilis, shows good anoxia tolerance, enduring 20 h in N2-bubbled water at 15°C. Metabolic responses to anoxia by tolerant species often include reversible phosphorylation control over selected enzymes. To analyze the role of serine/threonine kinases and phosphatases in signal transduction during anoxia in O. virilis, changes in the activities of cAMP-dependent protein kinase (PKA) and protein phosphatases 1, 2A, and 2C were measured in tail muscle and hepatopancreas over a time course of exposure to N2-bubbled water. A strong increase in the percentage of PKA present as the free catalytic subunit (% PKAc) occurred between 1 and 2 h of anoxia exposure whereas phosphatase activities were strongly reduced. This suggests that PKA-mediated events are important in the initial response by tissues to declining oxygen availability. As oxygen deprivation became severe and prolonged (5-20 h) these changes reversed; the % PKAc fell to below control values and activities of phosphatases returned to or rose above control values. Subcellular fractionation also showed a decrease in PKA associated with the plasma membrane after 20 h anoxia whereas cytosolic PKA content increased. PKAc purified from tail muscle showed a molecular weight of 43.8 ± 0.4 kDa, a pH optimum of 6.8, a high affinity for Mg ATP (Km = 131.0 ± 14.4 μM) and Kemptide (Km = 31.6 ± 5.2 μM). Crayfish PKAc was sensitive to temperature change; a break in the Arrhenius plot occurred at approximately 15°C with a 2.5-fold rise in activation energy at temperatures < 15°C. These studies demonstrate a role for serine/threonine protein kinases and phosphatases in the metabolic adjustments to oxygen depletion by crayfish organs.

Anoxia tolerance, Crayfish, Hepatopancreas, Metabolic control, Muscle, Protein kinase A, Protein phosphatase, Reversible phosphorylation
Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Department of Biology

Cowan, K.J. (Kyra J.), & Storey, K. (2001). Protein kinase and phosphatase responses to anoxia in crayfish, Orconectes virilis: Purification and characterization of cAMP-dependent protein kinase. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 130(4), 565–577. doi:10.1016/S1096-4959(01)00467-5