300 H. G. BOMAN, I. A. BOMAN 



of 0-02 M MgClo in 0-02 M tris-HCl, pH 7-3 until a viscous but homo- 

 geneous mixture was obtained. This was centrifuged at 100 000 g for i hr. 

 The pellet (about one-third of the volume) was discarded and the clear 

 supernatant was dialyzed at 4° for 12-15 hr. against 0-04 m tris-HCl, 

 pH 7-4. The resulting solution (Fraction I) contains about 9-9 mg. of 

 protein and 0-9 mg. of nucleic acid per ml. It was used as a general 

 source of all activating enzymes as well as for the further purification of 

 the arginine-activating enzyme. 



For this further purification the extract was chromatographed on DEAE 

 cellulose [15]. A column containing a 100 ml. of absorbent was equilibrated 

 with 0-04 M tris-HCl, pH 7-4, and was then loaded with a volume of 

 Fraction I corresponding to 30 mg. of protein. The chromatogram was 

 developed by stepwise elution, using the following amounts of tris-HCl 

 bufl^ers, pH 7-4: («) 65 ml. of 0-23 m; (b) 45 ml. of 0-38 M, and (r) 100 ml. 

 of I M. Step (a) elutes a protein fraction with none or very little arginine- 

 activating enzyme ; step (b) contains most of this activity ; step (c) elutes a 

 fraction consisting mainly of nucleic acid. It was observed that the first use 

 of a batch of DEAE cellulose caused a considerable loss of enzyme activity. 

 Repeated use of the same column gave, however, satisfactory results. 



The fractions containing the arginine-activating enzyme were pooled 

 and concentrated about twenty times using negative pressure dialysis with 

 I cm. wide dialysis tubing. The concentrated solution (and also Fraction I) 

 were stored at — 15° with 30-40% ethylene glycol as antifreeze because it 

 was found that repeated freezing and thawing results in marked loss of 

 activity. A typical concentrated solution of the arginine-activating enzyme, 

 including the ethylene glycol, had a protein content of 2 -9 mg. per ml. and 

 a nucleic acid content of 002 mg. per ml. 



LEVEL OF ACTIVATING ENZYMES UNDER DIFFERENT CONDITIONS OF GROWTH 



As mentioned previously the addition of arginine to the growth medium 

 represses the formation of enzymes in its own biosynthesis. It was therefore 

 of interest to examine the effect of arginine on the formation of its activating 

 enzyme. For comparison a similar test was carried out for leucine, although 

 a corresponding repression by leucine has not yet been investigated. 



Two cultures of strain 3oSq were grown, one in medium A + l- 

 arginine (200 /ig./ml.), the other in medium A + L-leucine (200 yug./ml.). 

 In the latter, L-isoleucine (100 /xg./ml.) and L-valine (100 /xg./ml.) were 

 added to counteract the slight inhibition of growth produced by L-leucine. 



Fraction I was prepared from the harvested cells as described in the 

 preceding section and designated I^ for the arginine-grown and I^ for the 

 leucine-grown cells. The levels of the activating enzymes were determined 

 by following the formation of arginine- and leucine-RNA. In all tests the 



