Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider,Trachemys scripta elegans

Neal J Dawson,Ryan A V Bell,Kenneth B Storey

doi:10.1155/2013/784973

Abstract

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

Highlights

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a critical enzyme involved in anaerobic metabolism
The initial purification step using blue agarose only resulted in a 1.1-fold purification of normoxic LDH, oxamate agarose removed all other proteins leaving normoxic turtle muscle LDH electrophoretically pure with an overall yield of 23% (Table 1)
The Vmax for anoxic LDH was nearly 4-fold higher in the lactate-oxidizing direction and over 7-fold higher in the pyruvate-reducing direction, as compared to LDH from the control condition (Table 2; Supplementary Figure 1). These results suggest that turtle muscle LDH may function more efficiently during anoxia

Summary

Introduction

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a critical enzyme involved in anaerobic metabolism. In this capacity, LDH favors the pyruvate reducing direction in skeletal muscle tissue, converting the glycolytic end product to lactate and regenerating the NAD+ pools to maintain a prolonged glycolytic flux [1]. LDH favors the pyruvate reducing direction in skeletal muscle tissue, converting the glycolytic end product to lactate and regenerating the NAD+ pools to maintain a prolonged glycolytic flux [1] This process is especially critical to those organisms that enter periodically into hypoxic/anoxic environments, where maintaining NAD+/NADH balance is essential for ATP production. The greatly reduced production of ATP via glycolysis, as compared to that of oxidative phosphorylation, results in difficult challenges for anoxia-tolerant organisms to overcome Several of these organisms employ alternate anaerobic pathways to increase

Methods

Results

Conclusion