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Purification of carbamoyl phosphate synthetase 1 (CPS1) from wood frog (Rana sylvatica) liver and its regulation in response to ice-nucleation and subsequent whole-body freezing

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Abstract

Carbamoyl phosphate synthetase I (CPS1) represents an important regulatory enzyme of the urea cycle that mediates the ATP-driven reaction ligating ammonium, carbonate, and phosphate to form carbamoyl phosphate. The freeze-tolerant wood frog (Rana sylvatica or Lithobates sylvaticus) accumulates high concentrations of urea during bouts of freezing to detoxify any ammonia generated and to contribute as a cryoprotectant thereby helping to avoid freeze damage to cells. Purification of CPS1 to homogeneity from wood frog liver was performed in control and frozen wood frogs by a three-step chromatographic process. The affinity of CPS1 for its three substrates was tested in the purified control and freeze-exposed enzyme under a variety of conditions including the presence and absence of the natural cryoprotectants urea and glucose. The results demonstrated that affinity for ammonium was higher in the freeze-exposed CPS1 (1.26-fold) and that with the addition of 400 mM glucose it displayed higher affinity for ATP (1.30-fold) and the obligate activator N-acetylglutamate (1.24-fold). Denaturation studies demonstrated the freeze-exposed enzyme was less thermally stable than the control with an unfolding temperature approximately 1.5 °C lower (52.9 °C for frozen and 54.4 °C for control). The control form of CPS1 had a significantly higher degree of glutarylated lysine residues (1.42-fold increase) relative to the frozen. The results suggest that CPS1 activation and maintenance of urea cycle activity despite the hypometabolic conditions associated with freezing are important aspects in the metabolic survival strategies of the wood frog.

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Acknowledgements

The authors would like to thank J.M. Storey and C.L. Childers for their help in editing the manuscript. The research was funded by a Discovery Grant (Number: 6793) through the Natural Sciences and Engineering Research Council of Canada (NSERC). K.B. Storey currently holds the Canada Research Chair in Molecular Physiology and S.R. Green was a recipient of a Queen Elizabeth II Graduate Scholarship in Science and Technology during the time of the study.

Funding

The research was funded by a Discovery Grant (Number: 6793) through the Natural Sciences and Engineering Research Council of Canada (NSERC). K.B. Storey currently holds the Canada Research Chair in Molecular Physiology and S.R. Green was a recipient of a Queen Elizabeth II Graduate Scholarship in Science and Technology during the time of the study.

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

    Stuart R. Green & Kenneth B. Storey

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Correspondence to Kenneth B. Storey.

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Green, S.R., Storey, K.B. Purification of carbamoyl phosphate synthetase 1 (CPS1) from wood frog (Rana sylvatica) liver and its regulation in response to ice-nucleation and subsequent whole-body freezing. Mol Cell Biochem 455, 29–39 (2019). https://doi.org/10.1007/s11010-018-3468-8

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