Subcellular localization of enzyme activities in chemostat-grown murine myeloma cells.

As part of the development of structured models for the metabolism of myeloma cells in suspension culture, a study was made of the subcellular localization of key enzymes of glucose and glutamine metabolism. Steady state chemostat cultures of the mouse myeloma SP2/0-Ag14 were used as a reproducible source of biomass. Homogenates of the cells, obtained via mechanical disruption, were separated into a mitochondrial and a cytosolic fraction via differential centrifugation. The following conclusions are drawn: (1) approximately one fifth of the hexokinase activity of cell-free homogenates is associated with the mitochondria; (2) a malate-aspartate shuttle may operate for oxidation of cytosolic NADH, as indicated by high levels of malate dehydrogenase and aspartate aminotransferase in both particulate and soluble fractions; (3) the pentose phosphate pathway and isocitrate dehydrogenase may contribute to the provision of cytosolic NADPH; (4) phosphoenolpyruvate carboxykinase and pyruvate kinase, which are present in high activities, are exclusively cytosolic and probably play a key role in glutamine metabolism; (5) oxidation of glutamine via these enzymes leads to the formation of pyruvate that enters the same pool as pyruvate generated by glycolysis. As a result, lactate and alanine formation can occur from both glucose and glutamine.

Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture.

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture.

Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h(-1), were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L(-1). Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L(-1), and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L(-1) glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism.