Protein kinase and phosphatase responses to anoxia in crayfish, Orconectes virilis: Purification and characterization of cAMP-dependent protein kinase
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.
|Keywords||Anoxia tolerance, Crayfish, Hepatopancreas, Metabolic control, Muscle, Protein kinase A, Protein phosphatase, Reversible phosphorylation|
|Journal||Comparative Biochemistry and Physiology - B Biochemistry and Molecular 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