Purification and Characterization of Lactate Dehydrogenase in the Foot Muscle and Hepatopancreas of Otala lactea

Abstract

Lactate dehydrogenase (LDH) has a crucial role in maintaining ATP production as the terminal enzyme in anaerobic glycolysis. This study will determine the effect of posttranslational modifications (PTMs) on the activity of LDH in the foot muscle and hepatopancreas of an estivating snail, Otala lactea. LDH in foot muscle of O. lactea was purified to homogeneity and partially purified in hepatopancreas in a two-step and three-step process, respectively. The kinetic properties and stability of these isoforms were determined where there was a significant difference in K and I values with pyruvate and urea separately in foot muscle; however, hepatopancreas exhibited significant differences in K and I in salt between control and stress. Interestingly, hepatopancreas has a higher affinity for pyruvate in the control state whereas foot muscle has a higher affinity for its substrate in the estivated state. PTMs of each isoform were identified using immunoblotting and dot blots, which prove to be significantly higher in the control state. Overall, foot muscle LDH enters a low phosphorylation state during estivation allowing more efficiency in consuming pyruvate with higher thermal stability but less structural stability. Hepatopancreas LDH becomes dephosphorylated in the estivating snail that decreases the efficiency of the enzyme in the forward direction; however, the snail has an increased tolerance to the presence of salt when water becomes scarce. Such tissue-specific regulations indicate the organism's ability to reduce energy consumption when undergoing metabolic depression.

­Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression

“…This would suggest that sustained PK activity and glycolytic function occurs even in the face of a high accumulation of products, suggesting that red muscle PK is well suited for supporting glycolytic flux under anoxic conditions. A differential regulation of glycolytic enzymes according to tissue type has also been noted recently in the snail, Otala lactea , during estivation, another form of hypometabolism ( MacLean et al, 2016 ). In snail foot muscle, LDH function was increased as supported by a reduced pyruvate K m value.…”

“…In snail foot muscle, LDH function was increased as supported by a reduced pyruvate K m value. However, LDH function in the hepatopancreas decreased in the pyruvate-consuming direction during estivation ( MacLean et al, 2016 ). These tissue-specific responses were proposed to be the result of a differential prioritization of metabolic action in each tissue, with foot muscle LDH favouring forward flux through glycolysis whereas the hepatopancreas enzyme may be primed for a role in gluconeogenesis ( MacLean et al, 2016 ).…”

Estivation-responsive microRNAs in a hypometabolic terrestrial snail

“…The roles of transcriptional, translational, and post-translational modifications in supporting MRD have been extensively analyzed in multiple animal systems over the years (including in hibernation, daily torpor, anaerobiosis, and estivation), whereas post-transcriptional miRNA-mediated metabolic regulation has only recently become evident (Storey & Storey, 2004; Biggar & Storey, 2011; Storey, 2015). Indeed, various post-translational regulatory controls on O. lactea signal transduction and metabolic enzymes have been reported (Brooks & Storey, 1990; MacLean et al., 2016; Ramnanan et al., 2009; Ramnanan et al., 2010; Ramnanan & Storey, 2006a; Ramnanan & Storey, 2006b; Whitwam & Storey, 1990) but, to date, the role of miRNA had not been explored. This study evaluates this level of post-transcriptional control.…”