Abstract
One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.
Highlights
Living animals are constantly faced with various environmental stresses that challenge normal life, such as oxygen and water limitation, very low or high temperatures and food restriction [1]
Mammalian tissues are notoriously sensitive to even brief episodes of anoxia, whereas turtle tissues are able to survive by: (1) rapidly decreasing their metabolic rates to,10% of normoxic resting rates, (2) increasing metabolic fuel by utilizing stores of glycogen loaded into all organs and (3) buffering and storing the lactic acid produced by anaerobic glycolysis in their bony shell [4,5]
The ratio between FBP/F1P aldolase activity was found to be 3.68 for normoxic turtles and 2.86 for anoxic turtles; these values were not significantly different (P,0.05). This suggests that turtle liver aldolase favors FBP as a substrate and is unchanged between normoxia and anoxia
Summary
Living animals are constantly faced with various environmental stresses that challenge normal life, such as oxygen and water limitation, very low or high temperatures and food restriction [1]. One of most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. These turtles are able to survive without oxygen for up to 18 weeks when submerged in cold water (3uC) [3,4]. Mammalian tissues are notoriously sensitive to even brief episodes of anoxia, whereas turtle tissues are able to survive by: (1) rapidly decreasing their metabolic rates to ,10% of normoxic resting rates, (2) increasing metabolic fuel by utilizing stores of glycogen loaded into all organs and (3) buffering and storing the lactic acid produced by anaerobic glycolysis in their bony shell [4,5]
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