Abstract
NMR can give more chemical information than any other non-invasive spectroscopic technique for investigating cell biochemistry (Gadian, 1995; Gillies, 1994). The technique can give information about pathway flux in vivo but rarely gives information about the control exerted by an individual enzyme within a pathway. One way of accessing this information is to titrate the activity of the enzyme of interest with a specific irreversible inhibitor. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of four glycolytic enzymes which together can catalyse the exchange of hydrogen between the C-2 position of lactate and water. We measured this flux in the human erythrocyte using 1H NMR to measure the rate of 1H/2H exchange at the C-2 position of L-[2-2H]lactate. By selectively reducing the active concentration of GAPDH in the cell with the irreversible inhibitor iodoacetate and then measuring the dependence of the exchange flux on the active concentration of the enzyme, measured in cell extracts, we were able to determine the specific exchange velocity of the enzyme in the cell (Brindle et al., 1982). This approach is limited, however, by its requirement for membrane permeable and specific irreversible, or quasi-irreversible, inhibitors. The use of molecular genetic techniques to change active enzyme concentration frees us from this restriction. Using these techniques we can, in principle, increase or decrease the concentration of any enzyme or membrane transporter in biological systems ranging from isolated cells to tissues in a whole animal. By measuring the effects of these interventions on an NMR-determined flux we can then measure the kinetic properties of these enzymes or transporters in a completely non-invasive, or at least minimally perturbing, way. For example, in the yeast Saccharomyces cerevisiae the glycolytic enzymes phosphoglycerate kinase (PGK) and GAPDH catalyse the coupled reaction {IE192-1} where GAP is glyceraldehyde 3-phosphate and 1,3-BPG is 1,3-bisphosphoglycerate. This reaction results in exchange of P i, with the γ-phosphate of ATP. The rate of this exchange, which is of the order of 1-3 mM s−1, can be measured in the intact cell using 31P NMR magnetization transfer techniques (Brindle, 1988a). We measured the flux control coefficients of PGK and GAPDH for this exchange by determining the effects of changes in their activity on the exchange flux. The activity of GAPDH was varied by titrating its activity with iodoacetate and the activity of PGK was varied by changing its concentration using molecular genetic techniques. At wild-type levels of the enzymes the flux control coefficient of PGK for the measured flux was about 1. Therefore this flux was a measure of PGK activity in the cell. The kinetic properties of the enzyme in vivo were shown to be similar to those displayed by the isolated enzyme in the test tube. There was no evidence for an alteration in its kinetic properties, as might result, for example, from substrate channelling.
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Keywords
- Human Erythrocyte
- Phosphoglycerate Kinase
- Exchange Flux
- Molecular Genetic Technique
- Flux Control Coefficient
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
References
Brindle, K. M. (1988a)3’P NMR magnetization-transfer measurements of flux between inorganic phosphate and adenosine 5’-triphosphate in yeast cells genetically modified to overproduce phosphoglycerate kinaseBiochemistry 276187–6196
Brindle, K. M. (1988b) NMR methods for measuring enzyme kineticsin vivo Prog. Nucl. Magn. Reson. Spectrosc.20, 257–293
Brindle, K. M., Campbell, I. D. & Simpson, R. J. (1982) A1H n.m.r. study of the kinetic properties expressed by glyderaldehyde phosphate dehydrogenase in the intact human erythrocyteBiochem. J.208, 583–592
Brindle, K. M., Williams, S.-P. & Boulton, M. (1989) ‘8F NMR detection of a fluorine-labelled enzymein vivo FEBS Lett.255, 121–124
Brown, F. F., Campbell, I. D., Kuchel, P. W. & Rabenstein, D. L. (1977) Human erythrocyte metabolism studied by ’ H spin echo NMRFEBS Lett.82, 12–16
Davies, S. E. C. & Brindle, K. M. (1992) Effects of overexpression of phosphofructokinase on glycolysis in the yeastSaccharomyces cerevisiae Biochemistry31,4729–4735
Fushimi, K. & Verkman, A. S. (1991) Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetryJ. Cell Biol. H2719–725
Gadian, D. G. (1995)NMR and its Applications to Living SystemsOxford University Press, Oxford
Gillies, R. J. (1994) NMRin Physiology and BiomedicineAcademic Press, San Diego
Haggie, P. M. & Brindle, K. M. (1999) Mitochondrial citrate synthase is immobilizedin vivo J. Biol. Chem.274, 3941–3945
Kreutzer, U., Wang, D. S. & Jue, T. (1992) Observing the ’ H NMR signal of the myoglobin Val-E1 1 in myocardium: an index of cellular oxygenationProc. Natl. Acad. Sci. USA89, 4731–4733
Lalloue, K., Jeffries, F. M. H. & Radda, G. K. (1986) Kinetic control of mitochondrial ATP synthesisBiochemistry25, 7667–7678
Lindon, J. C., Nicholson, J. K. & Wilson, I. D. (1996) Direct coupling of chromatographic separations to NMR spectroscopyProg. Nucl. Magn. Reson. Spectrosc.29, 1–49
Oliver, S. G., Winson, M. K., Kell, D. B. & Baganz, F. (1998) Systematic functional analysis of the yeast genomeTrends Biotechnol.16, 373–378
Randez-Gil, F., Sanz, P., Entian, K.-D. & Prieto, J. A. (1998) Carbon source-dependent phosphorylation of hexokinase PII and its role in the glucose-signaling response in yeastMol. Cell. Biol.18, 2940–2948
Ross, J. A. B., Senear, D. F., Waxman, E., Kombo, B. B.et al.(1992) Spectral enhancement of proteins: Biological incorporation and fluorescence characterisation of 5-hydroxytryptophan in bacteriophage X cI repressorProc. Natl. Acad. Sci. USA89, 12023–12027
Sheldon, J. G., Williams, S.-P., Fulton, A. M. & Brindle, K. M. (1996) 3’P NMR magnetization transfer study of the control of ATP turnover inSaccharomyces cerevisiae Proc. Natl. Acad. Sci. USA93, 6399–6404
Smith, B. R., Johnson, G. A., Croman, E. V. & Linney, E. (1994) Magnetic resonance microscopy of mouse embryosProc. Natl. Acad. Sci. USA91, 3530–3533
Williams, S.-P., Fulton, A. M. & Brindle, K. M. (1993) Estimation of the intracellular free ADP concentration by `9F NMR studies of fluorine-labeled yeast phosphoglycerate kinase invivo Biochemistry32, 4895-4902
Williams, S.-P., Haggie, P. M. & Brindle, K. M. (1997) ‘8F NMR measurements of the rotational mobility of proteinsin vivo Biophys. J. 72490–498
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Brindle, K.M., Haggie, P.M. (2000). Probing The Cell Interior With NMR Spectroscopy. In: Cornish-Bowden, A., Cárdenas, M.L. (eds) Technological and Medical Implications of Metabolic Control Analysis. NATO Science Series, vol 74. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-4072-0_21
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DOI: https://doi.org/10.1007/978-94-011-4072-0_21
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