DEAN B. COWIE AND RICHARD B. ROBERTS 19 



radioaclivily from the cells. It can be seen lliat the radioactivity appearing 

 in the wasli solutions becomes progressively less even though the quantity of 

 cells per ml of medium remains constant. This wash-solution radioactivity 

 may be used as a measure of the water space of metabolizing cells. As we have 

 pointed out before, the decrease in wash-solution radioactivity and hence the 

 apparent decrease in water space is due to the reduction in the concentration 

 of dififusible radioactive materials in the medium caused by metabolic incor- 

 poration. It is therefore evident that at the concentrations of radiorubidium 

 and cesium used, most of the labeled salts are incorporated. The bound mate- 

 rial can be removed by metabolic exchange but not by exchange with sodium, 

 as shown by Roberts, Roberts and Cowie (27). It is concluded that E. coli 

 is highly permeable to all members of this group of ions and that approxi- 

 mately 75% of the cell volume is always available for passive diffusion of these 

 materials. In addition to the ions contained in the water space, potassium, 

 rubidium and cesium ions are bound in the cell by compounds associated with 

 some of the early steps of glucose metabolism (5, 23, 26). Such compounds 

 provide an adequate basis for explaining the ability of the cell to concentrate 

 these ions. 



Permeability of Escherichia Coli to Amino Acids. The previous paragraphs 

 have emphasized the necessity of recognizing and correcting for metabolically 

 bound or altered material in any attempt to measure the water space of the 

 cells. Such procedures are particularly important when permeability to such 

 metabolically active compounds as amino acids is investigated. The quantity 

 of material altered by metabolism is usually limited by the enzymatic capacity 

 or the metabolic activity of the cells rather than by the concentration of the 

 material in the medium. On the other hand, the quantity of material contained 

 in the water space is directly proportional to the concentration in the medium. 

 When the concentration is high, the water space content is larger in comparison 

 with the metabolically altered material. It is also advisable to make chromato- 

 graphic identification of the water space material washed out of the cells in 

 order to determine whether it has been altered during the course of the experi- 

 ment. In some cases these precautions may not be sufficient to yield an un- 

 equivocal result, but at least the sources of error will be recognized. 



a) Uptake of S^^-labeled cystine. Investigation of washed E. coli cells in a 

 glucose-free, nitrogen-free medium reveals an immediate uptake of S^^ from 

 exogenous radiocystine. This uptake is in excess of that anticipated for non- 

 metabolizing cells. Additional uptake occurs in time even though no apparent 

 growth occurs. Table 16 shows the results of such an experiment. Four mg of 

 S^^-labeled cystine were added to 40 ml of buffered saline solution. This mixture 

 was kept in an ice bath during the entire experiment and aerated by bubbling 

 air through it. Immediately after the addition of 1.5 ml of washed cells, a 5-ml 

 sample of the culture fluid was removed and centrifuged. The radioactivity in 



