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.
Vriezen
N
N
Department of Microbiology and Enzymology, Kluyver Laboratory for Biotechnology, Delft University of Technology, The Netherlands. N.Vriezen@stm.tudelft.nl
van Dijken
J P
JP
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D002458:Cell Fractionation; D003600:Cytosol / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D009101:Multiple Myeloma / Q000201:enzymology*; D010427:Pentose Phosphate Pathway; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005973:Glutamine; D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D001219:Aspartate Aminotransferases; D006593:Hexokinase; D011770:Pyruvate Kinase; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005947:Glucose