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Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis

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Abstract

The African clawed frog, Xenopus laevis, is able to withstand extremely arid conditions by estivating, in conjunction with dehydration tolerance and urea accumulation. Estivating X. laevis reduce their metabolic rate and recruit anaerobic glycolysis, driven by lactate dehydrogenase (LDH; E.C. 1.1.1.27) enzymes that reversibly convert pyruvate and NADH to lactate and NAD+, to meet newly established ATP demands. The present study investigated purified LDH from the liver of dehydrated and control X. laevis. LDH from dehydrated liver showed a significantly higher K m for l-lactate (1.74 fold), NAD+ (2.41 fold), and pyruvate (1.78 fold) in comparison to LDH from the liver of control frogs. In the presence of physiological levels of urea found in dehydrated animals, the K m values obtained for dehydrated LDH all returned to control LDH K m values. Dot blot analysis showed post-translational modifications may be responsible for the kinetic modification as the dehydrated form of LDH showed more phosphorylated serine residues (1.54 fold), less methylated lysine residues (0.43 fold), and a higher level of ubiquitination (1.90 fold) in comparison to control LDH. The physiological consequence of dehydration-induced LDH modification appears to adjust LDH function in conjunction with urea levels in dehydrated frogs. When urea levels are high during dehydration, LDH retains its normal function. Yet, as urea levels drop during rehydration, LDH function is reduced, possibly shunting pyruvate to the TCA cycle.

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Abbreviations

ANOVA:

Analysis of variance

ATP:

Adenosine triphosphate

BWCi :

Initial body water content

DEAE+ :

Diethylaminoethanol

DSF:

Differential scanning fluorimetry

EDTA:

Ethylene diamine tetraacetic acid

EGTA:

Ethylene glycol tetraacetic acid

IgG:

Immunoglobulin G

K m :

Michaelis constant

LDH:

Lactate dehydrogenase

M d :

Measured mass

M i :

Initial mass

MW:

Molecular weight

NAD+ :

Nicotinamide adenine dinucleotide

NADH:

Reduced form of nicotinamide adenine dinucleotide

PEG:

Polyethylene glycol

PMSF:

Phenylmethanesulfonyl fluoride

PVA:

Polyvinyl alcohol

Rf:

Distance migrated over the gel length

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEM:

Standard error of the mean

TBS:

Tris-buffered saline

TBST:

Tris-buffered saline with 0.05 % Tween-20

TCA cycle:

Tricarboxylic acid cycle

T m :

Melting point

V max :

Maximum initial velocity of the enzyme catalyzed reaction

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Acknowledgments

The authors thank JM Storey for helpful discussions during the preparation of this manuscript. This research was supported by a discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (6793 to KBS). BAK was supported by a NSERC Post Doctoral Fellowship and NJD was supported by an Ontario Graduate Scholarship.

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Authors and Affiliations

  1. Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada

    Barbara A. Katzenback, Neal J. Dawson & Kenneth B. Storey

Authors
  1. Barbara A. Katzenback
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  2. Neal J. Dawson
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  3. Kenneth B. Storey
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Corresponding author

Correspondence to Barbara A. Katzenback.

Additional information

Communicated by I. D. Hume.

B. A. Katzenback and N. J. Dawson contributed equally.

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Katzenback, B.A., Dawson, N.J. & Storey, K.B. Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis . J Comp Physiol B 184, 601–611 (2014). https://doi.org/10.1007/s00360-014-0824-1

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