Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

Abstract

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo (r) protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications.

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

“…In CHO and BHK cell cultures, GOT activity was approx. eight times higher than GDH and ten times higher than GPT (Yallop et al 2003). However, in CHSE cell culture, GOT activity and GPT activity were almost equal and approx.…”

“…Enzymes were extracted according to the method of Yallop et al (2003) and kept on ice during the experiments. In assays involving NAD + /NADH, measurements were taken at 340 nm and the extinction coefficient E 340 nm of 6.2 · 10 )3 mol )1 l cm )1 was used.…”

Towards dynamic metabolic flux analysis in CHO cell cultures

“…To reduce ammonium accumulation by cell engineering efforts, glutamine synthetase (GS) gene has been overexpressed in CHO (GS-CHO) and NS0 (GS-NS0) cells [95,96]. GS activity is normally relatively low in CHO, hybridoma, BHK and HeLa cell lines [7,54,78,97,98]. Cell lines overexpressing GS can be successfully cultured in glutamine-free media [4].…”

Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes

“…It is evident from several studies on metabolism of animal cells that changes in enzyme activities highlight metabolic changes (Fitzpatrick et al, 1993;Leveille and Hanson, 1966;Neermann and Wagner, 1996;Segner and Verreth, 1995;Vriezen and van Dijken, 1998;Yallop et al, 2003). It is, however, worth mentioning that although change in enzyme activity is mostly represented by change in the total amount of an enzyme, catalytic activity can also be altered by allosteric effectors that, for instance, change the substrate affinity (K m ) of the enzyme.…”