2 2 ELECTROLYTES IN BIOLOGICAL SYSTEMS 



in the phenol-ammonia-water solvent. Chromatograms of the C'^-labeled glu- 

 tamic acid before its use in this experiment showed that the total radioactive 

 impurities were less than i %. Since no radioactive glutamic acid was observed 

 in the supernatant or in the wash solution, it was concluded that the original 

 material had been converted to metabolic products. 



Satisfactory results were obtained in a second experiment in which the 

 following modifications were made: a) the cells (0.34 ml) were harvested after 

 a four-day culture period in order to obtain cells in the stationary growth 

 phase, b) After being washed in saline solution, the cells were pretreated by 

 suspending for 15 minutes in 0.85% saline solution containing 10 mg sodium 

 glutamate per ml. c) The cells were centrifuged and resuspended in i ml of a 

 similar glutamate-saline solution containing C'^-glutamic acid. The pretreat- 

 ment with sodium glutamate was used to minimize effects which might occur 



Table 19. Metabolic uptake of 



C"-GLUTAMIC acid BY E. COLI 



Table 20. Nonmetabolic uptake of 

 c^^-glutamic acid by e. coli 



in a direct transfer of cells from one kind of medium to another. The results of 

 this experiment are shown in table 20. 



The metabolic uptake of C'^ was small. It is, therefore, possible to use the 

 loss of radioactivity observed in the supernatant fluid to calculate the water 

 space volume. The measurement gave a water space of 76%. Approximately 

 90% of the radioactivity removed from the medium by the cells was washed 

 out with the saline washes. Chromatograms of the first wash solution showed 

 that the radioactive material contained in the wash solution was indeed glu- 

 tamic acid. This positive identification of glutamic acid was carried out by 

 chromatographic 'fingerprinting', (24). Sufficient carrier glutamic acid was 

 added to this wash solution to demonstrate by the ninhydrin reaction the 

 presence of glutamic acid on the paper chromatogram. A radioautograph of 

 the same chromatogram revealed that the radioactivity matched in every 

 detail the pattern of the carrier glutamic acid obtained with ninhydrin (fig. 6). 

 The results indicate that glutamic acid is able to enter and emerge from the 

 cell as the intact amino acid, and that passive diffusion is the mode of transport. 



