DKAX H. COWIK AND RICHARD B. ROBERTS 21 



lion (.onlaining (''^-glutamic acid as the only supplement. When the cell 

 \-olume is large (approximately i.o ml) and the available glutamic acid of the 

 medium relatively small (o.i mg total), all the glutamic acid is metabolized 

 before the cells can be separated from the medium by centrifugation. As a 

 result, water space determinations cannot be made under these conditions. 

 l'\ir e.xample, when 0.68 ml of washed E. coli was suspended in i ml of a saline, 

 C'^-glutamic acid mixture (60 /xg glutamic acid) at 4°C and immediately cen- 

 trifuged, 6g% of the original radioactivity was taken up by the cells (table 19). 

 Eighty-live per cent of this radioactivity was metabolically bound by the 

 cells and could not be removed by two saline washes. The remainder (15%) 

 was diffusible and was recovered in the wash solution. Calculations of the water 

 space of these cells from measurement of the supernatant radioactivity gave 



T.\BLE 17. Immediate uptake 

 OF s'*-labeled cystine* 



* Sulfur concentration per ml of medium 

 was 0.025 nig. 



Table 18. Immediate uptake 



or S'*-LABELED METHIONINE* 



* Sulfur concentration per ml of medium 



was 0.02=; mg. 



an apparent value of 327%. Use of the radioactivity of the wash solution as a 

 measure of water space does not take into account residual metabolism occur- 

 ring during the wash period (time elapsed during the suspension of cells in the 

 wash solution and their centrifugation). Furthermore, in view of the large 

 metabolic use of the C'''-glutamic acid by the cells there is no assurance that 

 the washed-out material is still glutamic acid. 



Two-dimensional chromatograms were made of aliquots of the supernatant 

 solution and the first wash solution. Carrier glutamic acid was added to the 

 wash solution before chromatographic analysis. The chromatogram shown in 

 figure 5 indicates that the radioactivity of these solutions was not in glutamic 

 acid. The nonradioactive carrier glutamic acid added to the wash solution 

 moved with an Rf characteristic of that amino acid, as was shown when the 

 chromatogram was sprayed with ninhydrin. A radioautograph of the paper 

 chromatogram showed that all the radioactivity moved with the front in the 

 sec-butanol-formic acid-water solvent and very little movement was observed 



