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
- 91 Downloads
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.
KeywordsWood frog Freeze-tolerance Urea cycle Metabolism Post-translational modification Carbamoyl phosphate synthetase I
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.
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.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 4.Costanzo JP, Lee RE, Lortz PH (1993) Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica. J Exp Biol 181:245–255Google Scholar
- 6.Russell EL, Storey KB (1995) Glycogen-synthetase and the control of cryoprotectant clearance after thawing in the freeze-tolerant wood frog. Cryo-Letters 16:263–266Google Scholar
- 17.Tuchman M, Holzknecht RA (1990) N-acetylglutamate content in liver and gut of normal and fasted mice, normal human livers, and livers of individuals with carbamyl phosphate synthetase or ornithine transcarbamylase deficiency. Pediatr Res 27:408–412. https://doi.org/10.1203/00006450-199004000-00020 CrossRefGoogle Scholar
- 29.Kamau Machua S, Mukuria JC, Ngure RM, Gitu PM (2014) Modulation of partially purified rat liver mitochondrial carbamoyl phosphate synthetase I using two glutamic acid analogues. Unique Res J Biochem Biotechnol 1:1–10Google Scholar
- 31.Green SR, Storey KB (2016) Regulation of crayfish, Orconectes virilis, tail muscle lactate dehydrogenase (LDH) in response to anoxic conditions is associated with alterations in phosphorylation patterns. Comp Biochem Physiol Part B 202:67–74. https://doi.org/10.1016/j.cbpb.2016.08.004 CrossRefGoogle Scholar
- 32.Brooks SP (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13:906–911Google Scholar
- 33.Brooks SP (1994) A program for analyzing enzyme rate data obtained from a microplate reader. Biotechniques 17:1154–1161Google Scholar
- 37.Strauss KA, Brumbaugh J, Duffy A et al (2011) Safety, efficacy and physiological actions of a lysine-free, arginine-rich formula to treat glutaryl-CoA dehydrogenase deficiency: Focus on cerebral amino acid influx. Mol Genet Metab 104:93–106. https://doi.org/10.1016/j.ymgme.2011.07.003 CrossRefGoogle Scholar
- 42.García-España A, Alonso E, Rubio V (1991) Influence of anions on the activation of carbamoyl phosphate synthetase (ammonia) by acetylglutamate: Implications for the activation of the enzyme in the mitochondria. Arch Biochem Biophys 288:414–420. https://doi.org/10.1016/0003-9861(91)90214-4 CrossRefGoogle Scholar