Characterization of Fructose-1,6-Bisphosphate Aldolase during Anoxia in the Tolerant Turtle, Trachemys scripta elegans: An Assessment of Enzyme Activity, Expression and Structure

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.

Reversible Enzyme Phosphorylation as a Mechanism for Metabolic Adaptation to Dehydration in the Skeletal Muscle of the African Clawed Frog, Xenopus laevis

“…laevis muscle aldolase was purified to apparent electrophoretic homogeneity by using a combination of ion-exchange and affinity chromatography (Figure 2.1). The apparent monomer subunit molecular weight (~45 kDa) determined by SDS-PAGE corresponded well with the rabbit skeletal muscle aldolase standard and the subunit molecular weight reported for aldolase from various other vertebrate species (MacDonald and Storey 2002;Dawson et al 2013). This purification scheme produced enzyme preparations that were apparently purified 13.9-fold with a specific activity of 5.11 U/mg ( Table 2.1).…”

“…Skeletal muscle is of particular interest because X. laevis is known to restrict blood circulation to this tissue under dehydrating conditions in order to conserve oxygen for delivery to higher order tissues like the brain (Hillman and Sommerfeldt 1981). Furthermore, the processes investigated in this thesis include pathways that have been shown previously to be regulated in other animal models in response to environmental stress (Horman et al 2005;Abnous and Storey 2007;Dieni and Storey 2009;Dawson et al 2013). However, these models commonly use whole animal metabolic rate depression as a major component of their strategy for dealing with worsening environmental conditions (Storey and Storey 2005;Storey 2010).…”

“…Fructose-1,6-bisphosphate aldolase (aldolase) is an intermediate step of glycolysis that cleaves fructose 1,6-bisphosphate (F1,6P2) to glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) as follows: F1,6P2 ⇌ DHAP + GAP It is thought that the continual depletion of GAP by later steps in glycolysis facilitates the flow of F1,6P2 toward GAP production and ultimately allows the forward flow of glycolysis to continue. Work on animals that face environmental stress has demonstrated that aldolase is typically regulated to alter substrate affinity during environmental stress (Storey 1980;Holden and Storey 1994;MacDonald and Storey 2002;Dawson et al 2013). However, changes to posttranslational modifications however have not been previously documented.…”

Severe pantothenic acid deficiency induces alterations in the intestinal mucosal proteome of starter Pekin ducks

“…PFKP catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6bisphosphate by ATP [39]. ALDOA and ALDOB are two isoforms of aldolase family which are located in skeletal muscle and liver tissue respectively, cleaves fructose-1,6-bisphosphate to triose phosphates [40].…”

Mouse hepatocytes and LSEC proteome reveal novel mechanisms of ischemia/reperfusion damage and protection by A2aR stimulation

“…Aldolase B activity was measured as described in [17], with minor modifications. a-enolase activity was measured accordingly to [18].…”