title: Glucose and glutamine metabolism of a murine B-lymphocyte hybridoma grown in batch culture.
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. A murine B-lymphocyte hybridoma (PQXB1/2) was grown in batch culture to a maximum cell density of 1-2 x 10(6) cells/mL in 3-4 d. The intracellular protein content showed a maximum value during the exponential growth phase of 0.55 mg/10(6) 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. The specific rates of glutamine and glucose utilization varied during culture and showed maximal values at the midexponential phase of 2.4 nmol/min/10(6) cells and 4.3 nmol/min/10(6) cells, respectively. 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. The maximum catalytic activity of hexokinase 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. 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. A significant proportion of the glutamine (36%) utilized by the cells was completely oxidized to CO2. 7. The measured rate of glutamine transport into the cells approximated to the metabolic flux and is suggested as a rate-limiting step. 8. Glutamine metabolism is likely to occur via glutaminase and amino transaminase, which have significantly higher activities than glutamate dehydrogenase. 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%).
authors.affiliation: Department of Biological Sciences, Manchester Metropolitan University, UK.
authors.forename: L
authors.initials: L
authors.lastname: Fitzpatrick
authors.forename: H A
authors.initials: HA
authors.lastname: Jenkins
authors.forename: M
authors.initials: M
authors.lastname: Butler
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004734:Energy Metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006825:Hybridomas / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D051379:Mice
chemical_list: D005973:Glutamine; D000255:Adenosine Triphosphate; D007655:Ketoglutarate Dehydrogenase Complex; D002950:Citrate (si)-Synthase; D005947:Glucose