DAGLEY AND SYKES 63 



SEDIMENTATION OF ENZYMES 



E. coli cells were usually grown without aeration in media containing 0.13 M 

 KH2PO4 brought to pH 7 with NaOH; growth in 0.01 M glucose was then 

 limited entirely by exhaustion of the source of carbon, and changes in pH 

 were negligible. The medium was completed by addition, per liter, of 0.2 g 

 MgSOWHoO and either 1 g (NH 4 ) 2 S04 (for a "mineral salts" medium) or 

 10 g Difco bactopeptone. Extracts were prepared from cells disintegrated in 

 the Hughes [5] bacterial press without abrasive; soluble material was ex- 

 tracted by stirring with 0.066 M phosphate buffer, pH 7, and cell debris was re- 

 moved by centrifuging for 15 minutes at 102,000^. The protein content of the 

 extracts was then determined by the biuret colorimetric method and adjusted 

 to 10 mg protein/ml by addition of buffer. This solution could be stored at 

 0° C for 24 hours before examination, but freezing and thawing caused altera- 

 tions in ultracentrifuge patterns. When glucose was the source of carbon, 

 irrespective of whether it was limiting for growth or supplied in excess, or 

 whether (NH^SOi or peptone was the nitrogen source, cells in mid-logarith- 

 mic phase gave patterns with boundaries that sedimented at about 40, 29, 20, 

 and 8 S. 



It was only when cell division had ceased for several hours that modifica- 

 tions could be seen, namely, a reduction in the size of the 40 S peak and the 

 appearance of a small 13 S peak. By contrast, when cells grown on glucose 

 were incubated in a lactose growth medium, a 13 S boundary was observed 

 for actively dividing cells. When lactose was utilized as sole carbon source in 

 a mineral salts medium there was a lag of 80 minutes before cell division 

 began, but the (3-galactosidase activity of the culture was increasing in this 

 period and a trace of the 13 S component appeared in that time. When pep- 

 tone was the nitrogen source, 3-galactosidase was synthesized much faster and 

 the lag preceding cell division was only 20 minutes. 



Ultracentrifuge patterns for extracts from these cells are shown in figure 1, 

 where, since photographs were taken at 32 minutes, the 40 S boundaries have 

 already sedimented. After incubation with lactose for 100 minutes, peaks of 

 29, 20, and 13 S are well defined. Extracts from cells at this stage of growth 

 on glucose show only 29 and 20 S boundaries in addition to 8 S after centrifug- 

 ing for 32 minutes, the trace of 13 S component in the initial stationary phase 

 culture being lost during cell division. When cells were adapted to utilize galac- 

 tose and D-xylose as sources of carbon, boundaries that sedimented at about 

 13 S also appeared; and they were visible in extracts containing the induced 

 enzyme citratase (Dagley and Sykes [6]). 



A correlation may therefore be suggested between the appearance of 13 S 

 components and the induction of certain enzymes, including those that may 

 be developed by glucose-grown cells as they remain in the stationary phase in 

 the presence of accumulated products of metabolism. The significance of such 

 a correlation, however, is not evident. It is possible, for example, that 13 S 



