GLUCOSE AND GLUTAMINE-METABOLISM OF A MURINE B-LYMPHOCYTE HYBRIDOMA GROWN IN BATCH CULTURE

The energy metabolism of a mammalian cell line grown in vitro was anal yzed by substrate consumption rates and metabolic flux measurements. T he data allowed the determination of the relative importance of the pa thways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture co ntributed to the changing catalytic activities of key enzymes, which w ere 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 durin g the exponential growth phase of 0.55 mg/10(6) cells. Glutamine was c ompletely depleted, but glucose only partially depleted to 50% of its original concentration when the cells reached a stationary phase follo wing exponential growth. 2. The specific rates of glutamine and glucos e utilization varied during culture and showed maximal values at the m idexponential 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 metabo lized by glycolysis, but only limited amounts by the pentose phosphate pathway (3.3%) and TCA cycle (0.21%). 4. The maximum catalytic activi ty of hexokinase approximates to the measured flux of glycolysis and i s suggested as a rate-limiting step. In the stationary phase, the hexo kinase activity reduced to 11% of its original value and may explain t he reduced glucose utilization at this stage. 5. The maximal activitie s of two TCA cycle enzymes were well above the measured metabolic flux and are unlikely to pose regulatory barriers. However, the activity o f 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 l ikely to occur via glutaminase and amino transaminase, which have sign ificantly higher activities than glutamate dehydrogenase.The calculate d potential ATP production suggests that, overall, glutamine is the ma jor contributor of cellular energy. However, at the midexponential pha se, the energy contribution from the catabolism of the two substrates was finely balanced-glutamine (55%) and glucose (45%).