The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 188.8.131.52) from foot muscle of aerobic control versus 24？h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. 1. Introduction Lactate dehydrogenase catalyzes the reversible conversion of pyruvate to lactate, with the concomitant oxidation of NADH to NAD+. Under anaerobic conditions, LDH becomes an important enzyme due to its ability to regenerate NAD+ and allows for continued carbon flow through the glycolytic pathway to support anaerobic ATP synthesis . This process can be especially important in those organisms that are exposed to hypoxic or anoxic conditions for extended periods of time and require energy balance to be maintained solely through the functioning of glycolysis. Littorina littorea are marine molluscs that are native to the intertidal zones of the Atlantic coast of Europe (from Scandinavia to Spain) and have been introduced to the east coast of North America as well as several other locations around the world. Changing tides frequently expose these gill-breathing snails to prolonged oxygen deprivation at low tide . Moreover, environmental conditions, such as high salinity, predation, or water pollutants can cause the snails to shut their shell openings, which over an extended period of time can also generate an anoxic exposure [3, 4]. In order to survive
A. de Zwaan and V. Putzer, “Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia to exercise anoxia),” Symposia of the Society for Experimental Biology, vol. 39, pp. 33–62, 1985.
E. L. Russell and K. B. Storey, “Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea,” Journal of Comparative Physiology B, vol. 165, no. 2, pp. 132–142, 1995.
Z. J. Xiong and K. B. Storey, “Regulation of liver lactate dehydrogenase by reversible phosphorylation in response to anoxia in a freshwater turtle,” Comparative Biochemistry and Physiology B, vol. 163, no. 2, pp. 221–228, 2012.
N. J. Dawson, R. A. V. Bell, and K. B. Storey, “Purification and properties of white muscle lactate dehydrogenase from the anoxia-tolerant turtle, the red-eared slider, Trachemys scripta elegans,” Enzyme Research, vol. 2013, Article ID 784973, 8 pages, 2013.
F. H. Niesen, H. Berglund, and M. Vedadi, “The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability,” Nature Protocols, vol. 2, no. 9, pp. 2212–2221, 2007.
K. K. Biggar, N. J. Dawson, and K. B. Storey, “Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler,” Biotechniques, vol. 53, no. 4, 2011.
E. M. Tarmy and N. O. Kaplan, “Kinetics of Escherichia coli B D-lactate dehydrogenase and evidence for pyruvate-controlled change in conformation,” The Journal of Biological Chemistry, vol. 243, no. 10, pp. 2587–2596, 1968.
I. Beis and E. A. Newsholme, “The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates,” Biochemical Journal, vol. 152, no. 1, pp. 23–32, 1975.
H. O. P？rtner, M. K. Grieshaber, and N. Heisler, “Anaerobiosis and acid-base status in marine invertebrates: effect of environmental hypoxia on extracellular and intracellular pH in Sipunculus nudus L,” Journal of Comparative Physiology B, vol. 155, no. 1, pp. 13–20, 1984.
K. B. Storey, D. C. Miller, W. C. Plaxton, and J. M. Storey, “Gas-liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates,” Analytical Biochemistry, vol. 125, no. 1, pp. 50–58, 1982.
S. P. J. Brooks and K. B. Storey, “Glycolytic controls in estivation and anoxia: a comparison of metabolic arrest in land and marine molluscs,” Comparative Biochemistry and Physiology A, vol. 118, no. 4, pp. 1103–1114, 1997.
K. B. Storey, B. Lant, O. O. Anozie, and J. M. Storey, “Metabolic mechanisms for anoxia tolerance and freezing survival in the intertidal gastropod, Littorina littorea,” Comparative Biochemistry and Physiology A, vol. 165, no. 4, pp. 448–459, 2013.
J. Fan, T. Hitosugi, T.-W. Chung et al., “Tyrosine phosphorylation of lactate dehydrogenase a is important for NADH/NAD+ redox homeostasis in cancer cells,” Molecular and Cellular Biology, vol. 31, no. 24, pp. 4938–4950, 2011.