GLUCOSE AND GLUTAMINE-METABOLISM OF A MURINE B-LYMPHOCYTE HYBRIDOMA GROWN IN BATCH CULTURE
GLUCOSE AND GLUTAMINE-METABOLISM OF A MURINE B-LYMPHOCYTE HYBRIDOMA GROWN IN BATCH CULTURE
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DOI：
10.1007/bf02916435
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发表时间：
1993-11-01
影响因子：
3
通讯作者：
BUTLER, M
中科院分区：
文献类型：
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作者：
FITZPATRICK, L;JENKINS, HA;BUTLER, M
通过底物消耗速率和代谢通量测量分析体外生长的哺乳动物细胞系的能量代谢。这些数据允许确定葡萄糖和谷氨酰胺代谢途径对细胞能量需求的相对重要性。研究表明，培养过程中底物浓度的变化导致了关键酶催化活性的变化.小鼠B淋巴细胞杂交瘤（PQXB 1 - 2）分批培养3-4 d，细胞密度达1-2 × 10（6）个/mL。细胞内蛋白质含量在对数生长期出现最大值，为0.55 mg/10（6）个细胞。谷氨酰胺完全耗尽，但葡萄糖仅部分耗尽到其初始浓度的50%，当细胞达到指数生长的稳定期.谷氨酰胺和葡萄糖利用率在培养过程中变化，在中期出现最大值，分别为2.4 nmol/min/10（6）个细胞和4.3 nmol/min/10（6）个细胞。3. 高比例的葡萄糖（96%）通过糖酵解代谢，但只有有限的量通过戊糖磷酸途径（3.3%）和TCA循环（0.21%）。 己糖激酶的最大催化活性接近糖酵解的测量通量，并建议作为限速步骤。在稳定期，己糖激酶的活性降低到原来的11%，这可能是葡萄糖利用率降低的原因. 两个TCA循环酶的最大活性远高于测得的代谢通量，不太可能构成监管障碍。然而，丙酮酸脱氢酶的活性通过分光光度法检测不到，这解释了糖酵解代谢物进入TCA循环的低水平通量。 细胞利用的谷氨酰胺的显著比例（36%）被完全氧化为CO2.7。 谷氨酰胺转运到细胞中的测量速率近似于代谢通量，并被认为是限速步骤。 谷氨酰胺的代谢可能通过谷氨酰胺酶和氨基转移酶进行，这两种酶的活性明显高于谷氨酸脱氢酶。计算出的潜在ATP产量表明，总的来说，谷氨酰胺是细胞能量的主要贡献者。然而，在中间指数阶段，两种底物的催化剂的能量贡献很好地平衡-谷氨酰胺（55%）和葡萄糖（45%）。
The energy metabolism of a mammalian cell line grown in vitro was analyzed by substrate consumption rates and metabolic flux measurements. The data allowed the determination of the relative importance of the pathways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture contributed to the changing catalytic activities of key enzymes, which were 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 during the exponential growth phase of 0.55 mg/10(6) cells. Glutamine was completely depleted, but glucose only partially depleted to 50% of its original concentration when the cells reached a stationary phase following exponential growth.2. The specific rates of glutamine and glucose utilization varied during culture and showed maximal values at the midexponential 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 metabolized by glycolysis, but only limited amounts by the pentose phosphate pathway (3.3%) and TCA cycle (0.21%).4. The maximum catalytic activity of hexokinase approximates to the measured flux of glycolysis and is suggested as a rate-limiting step. In the stationary phase, the hexokinase activity reduced to 11% of its original value and may explain the reduced glucose utilization at this stage.5. The maximal activities of two TCA cycle enzymes were well above the measured metabolic flux and are unlikely to pose regulatory barriers. However, the activity of 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 likely to occur via glutaminase and amino transaminase, which have significantly higher activities than glutamate dehydrogenase.The calculated potential ATP production suggests that, overall, glutamine is the major contributor of cellular energy. However, at the midexponential phase, the energy contribution from the catabolism of the two substrates was finely balanced-glutamine (55%) and glucose (45%).