The inhibitory effect of glutamate on the growth of a murine hybridoma is caused by competitive inhibition of the x(C)(-) transport system required for cystine utilization

Glutamic acid was found to be growth inhibitory to a murine lymphocyte hybr idoma in a concentration-dependent manner from 3 to 12 mM glutamate. At 12 mM glutamate there was a 70% decrease in the specific growth rate of the ce lls. Attempts to alleviate inhibition or adapt cells to growth in glutamate -based media were unsuccessful. It is proposed that elevated glutamate leve ls impair adequate uptake of cystine, a critical amino acid for the synthes is of glutathione. Glutathione is required by cells to prevent intracellula r oxidative stress. The measured rate of uptake of U-C-14 L-cystine into th e cells was found to have the following parameters: K-m = 0.87 mM, V-max = 0.9 nmole/mg cell protein per min. The uptake was sodium independent and re sembled the previously described x(c)(-) transport system, with elevated gl utamate levels causing extensive inhibition. Glutamate at a concentration o f 1.4 mM caused a 50% decrease in cystine uptake from the serum-free growth medium. Glutamate was taken up from the external medium (K-m = 20 mM and V -max = 12.5 nmole/mg cell protein per min) by the same transport system in a stereo specific, sodium independent manner. Of the amino acids examined, it was found that cystine and homocysteic acid were the most extensive inhi bitors of glutamate uptake and that inhibition was competitive. Metabolic p rofiles of the cells grown in cultures containing enhanced glutamate levels revealed an overall increase in net production of alanine, serine, asparag ine and aspartate. A substantially increased specific consumption of glutam ate was accompanied by a decreased consumption of cystine, valine and pheny lalanine. The combined kinetic and metabolic results indicate that glutamate and cyst ine are taken up by the anionic transport system x(c)(-). The increasing le vels of glutamate in the medium result in a decreased transport of cystine by this system due to competitive inhibition by glutamate.