Vriezen N, van Dijken J P
Department of Microbiology and Enzymology, Kluyver Laboratory for Biotechnology, Delft University of Technology, The Netherlands.
J Biotechnol. 1998 Mar 26;61(1):43-56. doi: 10.1016/s0168-1656(98)00015-7.
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.
作为悬浮培养骨髓瘤细胞代谢结构化模型开发的一部分，对葡萄糖和谷氨酰胺代谢关键酶的亚细胞定位进行了研究。小鼠骨髓瘤SP2/0-Ag14的稳态恒化器培养物被用作可重复的生物质来源。通过机械破碎获得的细胞匀浆，通过差速离心分离成线粒体和胞质部分。得出以下结论：（1）无细胞匀浆中约五分之一的己糖激酶活性与线粒体相关；（2）苹果酸-天冬氨酸穿梭可能参与胞质NADH的氧化，这可从颗粒部分和可溶性部分中高水平的苹果酸脱氢酶和天冬氨酸转氨酶看出；（3）磷酸戊糖途径和异柠檬酸脱氢酶可能有助于提供胞质NADPH；（4）高活性的磷酸烯醇式丙酮酸羧激酶和丙酮酸激酶仅存在于胞质中，可能在谷氨酰胺代谢中起关键作用；（5）通过这些酶氧化谷氨酰胺会导致丙酮酸的形成，该丙酮酸进入与糖酵解产生的丙酮酸相同的代谢池。因此，葡萄糖和谷氨酰胺都可以生成乳酸和丙氨酸。
