Discussion and Conclusion
Regulation of enzyme activity for animals enduring long periods of anoxia is crucial for metabolic rate control and maintenance of fuel levels during anaerobic conditions. The ability of these freshwater crayfish to survive extended periods of time without oxygen relies on several biochemical changes in metabolic pathways, including the regulation of key metabolic enzymes by post-translational modification. Control over glycolysis is required to manipulate ATP levels during conditions of extreme stress. Earlier studies have shown that both glycolytic enzymes PK and PFK in O. virilis tail muscle and hepatopancreas are regulated by reversible phosphorylation during anoxia (Cowan and Storey, 1999). A cAMP-dependent protein kinase was shown to be responsible for this phosphorylation. Once activated by cAMP, the catalytic subunit of PKA is free to phosphorylate substrate proteins and also to migrate into the nucleus. Its role in regulating metabolic flux in O. virilis was further elucidated in thi s investigation through in vivo studies, which involved the assessment of percent active PKA and the distribution of the catalytic subunit in the subcellular fractions.
These studies clearly showed that anaerobic conditions had an effect on the activity and placement of the enzyme within the cell. Both tissues showed a decrease in percent active PKA after 20 hours of anoxia, which corresponded to a drop in the amount of catalytic subunit in the plasma membrane fraction. A general pattern of response has been shown in facultative anaerobes during extended exposure times to anoxia. This two-phase response includes an initial rise in anaerobic glycolysis followed by a dramatic drop in metabolic rate overall when critical levels of hypoxia are reached. PKA was almost fully active between 1-2 hours of anoxia in both tail muscle and hepatopancreas. The subcellular fractionation showing the distribution of PKA indicated an increase in the amount of PKA in the cytosol with the introduction of anaerobic conditions. It has been generally accepted that the compartmentalization of activated PKA is involved in cellular regulation, and the migration of PKA back into the cytosol after 20 hrs of anoxia, in both the tail muscle and the hepatopancreas, corresponds to a significant drop in PKAc activity. Changes in protein phosphorylation patterns corresponding to a reduction in enzyme phosphorylation during this stress have also been shown in the anoxia-tolerant O. lactea (Brooks and Storey 1994). This implies a less stimulated adenylate cyclase for the production of cAMP. Studies (Scott et al. 1993) have indicated that compartmentalization and therefore colocalization of the kinase with preferred substrates could be a means to adapt PKA for regulation of cAMP-responsive events through due to changes in physiological conditions.
The cAMP-dependent protein kinase catalytic subunit molecular weight of 43.8 +/- 0.4 kDa is well within the range of subunit size found for many species, including turtle liver (Mehrani and Storey, 1995), bovine liver (Sugden et al. 1976), bivalve mollusc mantle tissue (Cao et al. 1995), and snail foot muscle (Brooks and Storey 1996). Kinetic characterization of the purified form of PKAc revealed very strong inhibitory properties by some synthetic inhibitors (H89, H7, PKA-I) These are artificial inhibitors of mammalian PKAc, some of which have also been shown to inhibit R. sylvatica and T. scripta elegans PKAc (Holden and Storey, 1997, Mehrani and Storey, 1995, respectively). Utilizing these inhibitors helps to determine whether the invertebrate species of PKAc respond in a similar manner as the vertebrate species, and in fact the I50 values for PKA-I, H7, and H89 were as low as those found for many mammalian forms of the enzyme as well as that of turtles and frogs. These results signify an integral conservation of the inhibitor-binding site of PKAc. Only very high KCl concentrations are capable of inhibition, even higher than that for land snails and turtles. The Km value for Mg.ATP was roughly 3-4-fold higher than that characteristic for mammalian PKAc (Sugden et al 1976) but significantly lower than that of O. lactea and T. scripta elegans. Temperature also had a significant effect on PKAc activity, with a 2.5-fold increase in activation energy for the temperature range below 15°C versus the temperature range above 15°C. A break in the Arrhenious plot has also been seen in other stress-tolerant species, including T. scripta elegans (Mehrani and Storey 1995) and R. sylvatica (Holden and Storey, 1997). This drop in activation energy at low temperatures might result in a reduction in PKAc-regulated responses in the crayfish during colder conditions under water. The catalytic subunit also had a very narrow range in terms of pH optimum, a range characteristic for most purified PKAc subunits.
In conclusion, the purified, free catalytic subunit of PKA in O. virilis tail muscle is kinetically very similar to other vertebrate forms of the enzyme. Additionally, the enzyme shares kinetic and physical properties with invertebrate forms of PKAc, and further substantiates the belief that the structure and function of PKAc has been highly conserved throughout evolution (Taylor et al. 1990). Protein phosphorylation by PKAc is an important biochemical control mechanism used to regulate many different metabolic pathways at the cellular level. O. virilis tail muscle PKAc is responsible for the phosphorylation of and subsequently is involved in the control of metabolic enzymes such as PK and PFK in response to a low oxygen stress. The post-translational modification of key metabolic enzymes by reversible phosphorylation is critical for metabolic rate depression under anaerobic conditions, and therefore the survival of this anoxia-tolerant crustacean.
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|Cowan, K.J.; Storey, K.B.; (1998). Purification And Characterization Of A Camp-Dependent Protein Kinase >From The Tail Muscle Of The Crayfish, Orconectes Virilis.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/|
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