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

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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 Km and I50 values with pyruvate and urea separately in foot muscle; however, hepatopancreas exhibited significant differences in Km and I50 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.

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Abbreviations

LDH:

Lactate dehydrogenase

PTMs:

Posttranslational modifications

PMSF:

Phenylmethylsulfonyl fluoride

NAD:

Nicotinamide adenine dinucleotide

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBST:

Tris-buffered saline and tween

TBS:

Tris-buffered saline

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Acknowledgements

The authors wish to thank C.L. Childers for editorial review of the manuscript. This research was supported by a discovery grant (6793) from the Natural Sciences and Engineering Research Council (NSERC) of Canada to KBS and the Canada Research Chairs program. A.M.S. Mattice and I.A. MacLean both hold NSERC Undergraduate Student Research Awards.

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    Authors and Affiliations

    1. 1125 Colonel By Dr, Ottawa, ON, K1S 5B6, Canada

      Isabelle A. MacLean, Amanda M. S. Mattice, Nadine J. Adam & Kenneth B. Storey

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    1. Isabelle A. MacLean
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    2. Amanda M. S. Mattice
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    3. Nadine J. Adam
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    4. Kenneth B. Storey
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    Correspondence to Kenneth B. Storey.

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    Isabelle A. MacLean and Amanda M. S. Mattice are the primary authors and have contributed equally.

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    MacLean, I.A., Mattice, A.M.S., Adam, N.J. et al. Purification and Characterization of Lactate Dehydrogenase in the Foot Muscle and Hepatopancreas of Otala lactea . Protein J 35, 467–480 (2016). https://doi.org/10.1007/s10930-016-9689-3

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