title: Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture.
abstract: 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.
authors: Department of Microbiology and Enzymology, Kluyver Laboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. N.Vriezen@stm.tudelft.nl
authors: N
authors: N
authors: Vriezen
authors: J P
authors: JP
authors: van Dijken
mesh_terms: D000818:Animals; D018533:Biomass; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival; D002474:Cell-Free System; D003470:Culture Media; D004798:Enzymes / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D009101:Multiple Myeloma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: D003470:Culture Media; D004798:Enzymes; D005973:Glutamine