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Glucose and glutamine metabolism of a murine B-lymphocyte hybridoma grown in batch culture

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Abstract

The energy metabolism of a mammalian cell line grown in vitro was analyzed by substrate consumption rates and metabolic flux measurements. The data allowed the determination of the relative importance of the pathways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture contributed to the changing catalytic activities of key enzymes, which were determined.

  1. 1.

    A murine B-lymphocyte hybridoma (PQXB1/2) was grown in batch culture to a maximum cell density of 1–2×106 cells/mL in 3–4 d. The intracellular protein content showed a maximum value during the exponential growth phase of 0.55 mg/106 cells. Glutamine was completely depleted, but glucose only partially depleted to 50% of its original concentration when the cells reached a stationary phase following exponential growth.

  2. 2.

    The specific rates of glutamine and glucose utilization varied during culture and showed maximal values at the midexponential phase of 2.4-nmol/min/106 cells and 4.3 nmol/min/106 cells, respectively.

  3. 3.

    A high proportion of glucose (96%) was metabolized by glycolysis, but only limited amounts by the pentose phosphate pathway (3.3%) and TCA cycle (0.21%).

  4. 4.

    The maximum catalytic activity of hexolinase approximates to the measured flux of glycolysis and is suggested as a rate-limiting step. In the stationary phase, the hexokinase activity reduced to 11% of its original value and may explain the reduced glucose utilization at this stage.

  5. 5.

    The maximal activities of two TCA cycle enzymes were well above the measured metabolic flux and are unlikely to pose regulatory barriers. However, the activity of pyruvate dehydrogenase was undetectable by spectrophotometric assay and explains the low level of flux of glycolytic metabolites into the TCA cycle.

  6. 6.

    A significant proportion of the glutamine (36%) utilized by the cells was completely oxidized to CO2.

  7. 7.

    The measured rate of glutamine transport into the cells approximated to the metabolic flux and is suggested as a rate-limiting step.

  8. 8.

    Glutamine metabolism is likely to occur via glutaminase and amino transaminase, which have significantly higher activities than glutamate dehydrogenase.

  9. 9.

    The calculated potential ATP production suggests that, overall, glutamine is the major contributor of cellular energy. However, at the midexponential phase, the energy contribution from the catabolism of the two substrates was finely balanced—glutamine (55%) and glucose (45%).

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References

  1. Seaver, S. S., ed. (1986), Commercial Production of Monoclonal Antibodies, Marcel Dekker, New York.

    Google Scholar 

  2. McCullough, K. and Spier, R. E. (1990),Monoclonal antibodies in Biotechnology: Theoretical and Practical Aspects, Cambridge Studies in Biotechnology, vol. 8, Cambridge University Press, Cambridge.

    Google Scholar 

  3. Glacken, M. W. (1988),Bio/Technology 6, 1041–1050.

    Article  CAS  Google Scholar 

  4. Butler, M. and Jenkins, H. A. (1989),J. Biotechnol. 12, 97–110.

    Article  CAS  Google Scholar 

  5. Morell, B. and Froesch, E. R. (1973),Eur. J. Clin. Invest. 3, 112–118.

    Article  CAS  Google Scholar 

  6. Reitzer, L. J., Wice, B. M., and Kennell, D. (1979),J. Biol. Chem. 254, 2669–2676.

    CAS  Google Scholar 

  7. Zielke, H. R., Ozand, P. T., Tildon, J. T., Sevdalian, D. A., and Cornblath, M. (1976),Proc. Natl. Acad. Sci. USA 73, 4110–4114.

    Article  CAS  Google Scholar 

  8. Wice, B. M., Reitzer, L. J., and Kennell, D. (1981),J. Biol. Chem. 256, 7812–7819.

    CAS  Google Scholar 

  9. McKeehan, W. L. (1986), Glutaminolysis in animals cells' inCarbohydrate Metabolism in Cultured Animal Cells, Morgan, M. J., ed., Plenum, New York, pp. 111–150.

    Google Scholar 

  10. Wu, G., Field, C. J., and Marliss, E. B. (1991),Am. J. Physiol. 260, E141-E147.

    CAS  Google Scholar 

  11. Wu, G. Y., Field, C. J., and Marliss, E. B. (1992),Metabolism-Clin. and Exp. 41, 982–988.

    CAS  Google Scholar 

  12. Zetterberg, A. and Engstrom, W. (1981),J. Cell Physiol. 108, 365–373.

    Article  CAS  Google Scholar 

  13. Ardawi, M. S. M. and Newsholme, E. A. (1983),Biochem. J. 212, 835–842.

    CAS  Google Scholar 

  14. Newsholme, E. A., Crabtree, B., and Parry-Billings, M. (1992), The energetic cost of regulation: An analysis based on the principles of metabolic-controllogic, inEnergy Metabolism: Tissue Determinants and Cellular Corollaries, Kinney, J. M., and Tucker, H. N., eds., Raven, New York, pp. 467–493.

    Google Scholar 

  15. Cooney, G. J., Taegtmeyer, H., and Newsholme, E. A. (1981),Biochem. J. 200, 701–703.

    CAS  Google Scholar 

  16. Ardawi, M. S. M. and Newsholme, E. A. (1982),Biochem. J. 208, 743–748.

    CAS  Google Scholar 

  17. Newsholme, P., Curi, R., Gordon, S., and Newsholme, E. A. (1986),Biochem. J. 239, 121–125.

    CAS  Google Scholar 

  18. Newsholme, P., Gordon, S., and Newsholme, E. A. (1987),Biochem. J. 242, 631–636.

    CAS  Google Scholar 

  19. Wright, A. F., Green, T. P., and Smith, L. L. (1985),Dev. Biol. Stand. 66, 495–501.

    Google Scholar 

  20. Patterson, M. K. (1979),Methods Enzymol. 58, 141–152.

    Google Scholar 

  21. Lund, P. (1974),Methods of Enzymatic Analysis, vol. 4, Bergmeyer, H. U., ed., Academic, London, pp. 1719–1722.

    Google Scholar 

  22. Fawcett, J. K. and Scott, J. E. (1960),J. Clin. Pathol. 13, 156–159.

    Article  CAS  Google Scholar 

  23. Gutmann, I. and Wahlefeld, A. W. (1974),Methods of Enzymatic Analysis, vol. 3, Bergmeyer, H. U., ed., Academic, London, pp. 1464–1468.

    Google Scholar 

  24. Bradford, M. M. (1976),Anal. Biochem. 72, 248–254.

    Article  CAS  Google Scholar 

  25. Bessell, E. M., Foster, A. B. and Westwood, J. H. (1972),Biochem. J. 128, 199–204.

    CAS  Google Scholar 

  26. Hamer, M. J. and Dickson, J. (1987),Biochem. J. 245, 35–40.

    CAS  Google Scholar 

  27. Kahn, A. and Marie, J. (1982),Methods Enzymol. 90, 131–140.

    CAS  Google Scholar 

  28. Zammit, V. A. and Newsholme, E. A. (1976),Biochem. J. 160, 447–462.

    CAS  Google Scholar 

  29. Schwartz, E. R. and Reed, L. J. (1970),Biochemistry 9, 1434–1439.

    Article  CAS  Google Scholar 

  30. Kvamme, E., Torgner, I. A., and Svenneby, G. (1985),Methods in Enzymology, vol. 113, Meister, A. ed., Academic, London, pp. 241–256.

    Google Scholar 

  31. Bergmeyer, H. U. and Bernt, E. (1974),Methods of Enzymatic Analysis, vol. 2, Bergmeyer, H. U., ed., Academic, London, pp. 727–733.

    Google Scholar 

  32. Fisher, H. F. (1985),Methods Enzymol. 113, 16–27.

    CAS  Google Scholar 

  33. Rowe, W. B., Ronzio, R. A., Wellner, V. P., and Meister, A. (1970),Methods in Enzymol. 17, 900–910.

    Article  Google Scholar 

  34. Srere, P. A., Brazil, H., and Goner, L. (1963),Acta Chem. Scand. 1, S129-S134.

    Google Scholar 

  35. Kleitzien, R. F. and Perdue, J. F. (1975),J. Biol. Chem. 250, 593–600.

    Google Scholar 

  36. Jenkins, H. A., Butler, M., and Dickson, A. J. (1992),J. Biotechnol. 23, 167–182.

    Article  CAS  Google Scholar 

  37. Joseph, S. K. and McGivan, J. D. (1978),Biochem. Biophys. Acta 543, 16–28.

    CAS  Google Scholar 

  38. Brand, K., Williams, J. F., and Weidemann, M. J. (1984),Biochem. J. 221, 471–475.

    CAS  Google Scholar 

  39. Katz, J. and Wood, H. G. (1963),J. Biol. Chem. 238, 517–523.

    CAS  Google Scholar 

  40. Bontemps, F., Hue, L., and Hers, H.-G. (1978),Biochem. J. 174, 603–612.

    CAS  Google Scholar 

  41. Haggstrom, L. (1991), Energetics of glutaminolysis—a theoretical evaluation, inProduction of Biologicals from Animal Cells in Culture, ESACT 10th Meet, Spier, R. E., Griffiths, J. B., and Meignier, B., eds., Butterworth-Heinmann, Oxford, pp. 79–81.

    Google Scholar 

  42. Schmid, G., Blanch, H. W., and Wilke, C. R. (1991), Hybridoma growth metabolism and product formation in HEPES-buffered medium: effect of passage number and pH, inProduction of Biologicals from Animal Cells in Culture, ESACT 10th Meet, Spier, R. E., Griffiths, J. B., and Meignier, B., eds., Butterworth-Heinmann, Oxford, pp. 73–75.

    Google Scholar 

  43. Hassell, T. and Butler, M. (1990),J. Cell Sci. 96, 501–508.

    CAS  Google Scholar 

  44. Zielke, H. R., Ozand, P. T., Tildon, J. T., Sevdalian, D. A., and Cornblath, M. (1978),J. Cell Physiol. 95, 41–48.

    Article  CAS  Google Scholar 

  45. Medina, M. A., Sancez-Jimenez, F., Quesada, A. R., Marquez, F. J., and Nunez de Castro, I. (1988),Biochemie 70, 833–834.

    Article  CAS  Google Scholar 

  46. O'Rourke, A. M. and Rider, C. C. (1989),Biochim. Biophys. Acta 1010, 342–345.

    Article  Google Scholar 

  47. Hassell, T., Gleave, S., and Butler, M. (1991),Appl. Biochem. Biotechnol. 30, 29–41.

    CAS  Google Scholar 

  48. Brand, K. (1985),Biochem. J. 228, 353–361.

    CAS  Google Scholar 

  49. Donnelly, M. and Scheffler, I. E. (1976),J. Cell Physiol. 89, 39–52.

    Article  CAS  Google Scholar 

  50. Zielke, H. R., Sumbilla, C. M., Sevdalian, D. A., Hawkins, R. L., and Ozand, P. T. (1980),J. Cell Physiol. 104, 433–441.

    Article  CAS  Google Scholar 

  51. Butler, M., Hassell, T. E., Doyle, C., Gleave, S., and Jennings, P. (1991), The effect of metabolic by-products on animal cells in culture, inProduction of Biologicals from Animal Cells in Culture, ESACT 10th Meet, Spier, R. E., Griffiths, J. B., and Meignier, B., eds., Butterworth-Heinmann, Oxford, pp. 226–228.

    Google Scholar 

  52. Reitzer, L. J., Wice, B. M., and Kennell, D. (1980),J. Biol. Chem. 255, 5616–5626.

    CAS  Google Scholar 

  53. Kalckar, H. M. and Ullrey, D. B. (1986), Studies of regulation of hexose transport into cultured fibroblasts, inCarbohydrate Metabolism of Cultured Cells, Morgan, M. J., ed., Plenum, New York, pp. 1–27.

    Google Scholar 

  54. Weernink, P. A. O., Rijksen, G., and Staal, G. E. J. (1991),Tumor Biol. 12, 339–352.

    Google Scholar 

  55. Newsholme, E. A. and Start, C. (1973), Regulation of carbohydrate metabolism in muscle, inRegulation in Metabolism, Wiley, London, pp. 88–145.

    Google Scholar 

  56. Wagner, A., Marc, A., Engasser, J. M., and Einsele, A. (1992),Biotech. Bioeng. 39, 320–326.

    Article  CAS  Google Scholar 

  57. McDermott, R. H. and Butler, M. (1993),J. Cell Sci. 104, 51–58.

    CAS  Google Scholar 

  58. Sri-Pathmanathan, R. M., Braddock, P., and Brindle, K. M. (1990),Biochim. Biophys. Acta 1051, 131–137.

    Article  CAS  Google Scholar 

  59. Sevdalian, D. A., Ozand, P. T., and Zielke, H. R. (1980),Enzyme 25, 142–144.

    CAS  Google Scholar 

  60. Moreadith, R. W. and Lehninger, A. L. (1984),J. Biol. Chem. 259, 6215–6221.

    CAS  Google Scholar 

  61. Feng, B., Shiber, S. K., and Max, R. (1990),J. Cell Physiol. 145, 376–380.

    Article  CAS  Google Scholar 

  62. Ardawi, M. S. M. and Newsholme, E. A. (1985),Essays in Biochemistry 21, 1–44.

    CAS  Google Scholar 

  63. Stoner, G. D. and Merchant, D. J. (1972),In Vitro 5, 330–343.

    Google Scholar 

  64. Murray, K., Dickson, A. J., and Gull, K. (1992), Metabolic management of a hybridoma cell line, inProduction of Biologicals from Animal Cells in Culture, ESACT 11th Meet, Spier, R. E., Griffiths, J. B., and MacDonald, C., eds., Butterworth-Heinmann, Oxford, pp. 261–263.

    Google Scholar 

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Authors and Affiliations

  1. Department of Biological Sciences, Manchester Metropolitan University, M1 5GD, Manchester, UK

    L. Fitzpatrick

  2. School of Science and Technology (Biomolecular Science), Liverpool John Moores University, Byrom Street, L3 3AF, Liverpool, UK

    H. A. Jenkins

  3. Department of Microbiology, University of Manitoba, R3T 2N2, Winnipeg, Manitoba, Canada

    M. Butler

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Fitzpatrick, L., Jenkins, H.A. & Butler, M. Glucose and glutamine metabolism of a murine B-lymphocyte hybridoma grown in batch culture. Appl Biochem Biotechnol 43, 93–116 (1993). https://doi.org/10.1007/BF02916435

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