The effect of L-glutamate on growth of CC9C10 hybridomas

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Broadhurst, Edward Ross
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In attempts to grow CC9C10 hybridoma cells on less ammoniagenic media by substituting glutamate for glutamine, it was found that elevated glutamate levels were inhibitory to cell growth. Attempts to alleviate inhibition or adapt cells to growth were unsuccessful. It is proposed that elevated glutamate levels impair adequate uptake of cystine, a critical amino acid for the synthesis of glutathione. Glutathione is required by cells to prevent oxidative stress. It was found that CC9C10 hybridomas can take up glutamate from the external medium (k$\rm\sb{m}$ 20 mM and V$\rm\sb{max}$ 5 nmoles/10$\sp6$ cells/min.) in a stereo specific, sodium independent manner. Of the amino acids examined, it was found that cystine and homocysteic acid were th most extensive inhibitors of glutamate uptake and that inhibition was competitive. The uptake of cystine was also found to be (k$\rm\sb{m}$ 0.87 mM, V$\rm\sb{max}$ 0.36 nmoles/10$\sp6$ cells/min.) sodium independent, with elevated glutamate levels causing extensive inhibition. Metabolic profiles of the cells in elevated glutamate levels revealed an overall increase in net production of amino acids, with consumption remaining unchanged. Specifically, production of alanine, serine, asparagine and aspartate increased. Consumption of cystine, arginine, lysine, valine, isoleucine, and phenylalanine decreased, while the consumption of glutamate increased. The combined kinetic and metabolic results indicate that glutamate and cystine are taken up by uptake system $\rm x\sp-\sb{c}.$ The increasing levels of glutamate in the medium result in decreased consumption of cystine due to competitive inhibition by glutamate.