Abstract
BackgroundPyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate.MethodsTo explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (Trachemys scripta elegans). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia.ResultsBoth muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia.DiscussionAnoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia.
Highlights
The present study analyzes enriched pyruvate kinase (PK; 2.7.1.40) from the red-eared slider turtle, Trachemys scripta elegans, red muscle and liver tissues
As Pyruvate kinase (PK) is a glycolytic control point that turtles rely on during the anaerobic catabolism of glycogen stores, analysis of PK kinetic changes helps to elucidate how reorganization of energy metabolism is coordinated throughout pathways and tissues to enable survival during oxygen restriction
This study evaluated the effects of 20-hr whole-animal anoxia exposures on PK properties and assessed the potential involvement of methylation and phosphorylation in contributing to stress-responsive PK control
Summary
The present study analyzes enriched pyruvate kinase (PK; 2.7.1.40) from the red-eared slider turtle, Trachemys scripta elegans, red muscle and liver tissues. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 L-alanine was only reduced in the liver Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia
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