STUDIES ON THE INCORPORATION OF ARGININE 299 



1% casein hydrolysate, and once with 2 ml. of cold ethanol-ether (2 :i). 

 It was dissolved by adding o • 3 ml. of o ■ 2 m triethylamine and heating to 

 about 60° for 2 min. transferred to planchets, dried and counted with a 

 windowless gasflow counter. A sample in which RNA was omitted was used 

 as a blank except if otherwise stated. Correction for self absorption and 

 geometry was read from a standard curve. 



The large scale labelling of RNA with [^"^CJ-arginine was carried out 

 with the same reaction mixture as that used for the assay. The reaction 

 was terminated by addition of an equal volume of 88% phenol in water 

 and shaking for 2 min. After cooling to 4°, the phases were separated by 

 centrifugation. The water layer (at the top) was removed and alcohol was 

 added to it to a final concentration of 20% (to increase the solubility of the 

 remaining phenol). The arginine-RNA was then precipitated with lan- 

 thanum nitrate [8], redissolved in a small volume of 0-2 M potassium 

 ethylene diamine tetraacetic acid, pH 7, and finally dialysed for 15 20 hr. 

 with several changes of water. 



The protein and the nucleic acid contents were determined from the 

 extinctions at 280 and 260 m/x [13]. 



Chemicals used: Nitroarginine was synthesized according to Kossel 

 and Kennaway [14]. The melting-point was 248^ d. and it was found to be 

 homogeneous by paper electrophoresis at pH 6 and by chromatography in 

 isopropanol : ammonia : water (7:1 : 2) (R/= o • 56). The strong u.v. absorp- 

 tion of nitroarginine makes it convenient to observe it on paper by quench- 

 ing of fluorescence in the same way as nucleotides. Sulphaguanidine 

 (U.S. P.) was purchased from Amend Drug and Chem. Co., New York, 

 New York, L-canavanine sulphate from Nutritional Biochemical Corp., 

 Cleveland 28, Ohio; streptomycin sulphate (U.S. P.) from Ely Lilly and 

 Co., Indianapolis, Indiana; L-[^'*C]-arginine with 5*93 x 10^ c.p.m./m- 

 jumole from Nuclear Chicago Corp., Des Plaines, Illinois; L-[^'*C]-arginine 

 with 88-8 X 10^ c.p.m./m/Limole and [^*C]-alga protein hydrolysate with 

 1-4 X 10'' c.p.m./|U,g. were obtained from Volks Radiochemical Co., 

 Chicago 40, Illinois. 



Results 



PARTIAL PURIFICATION OF THE ARGININE-ACTIVATING ENZYME 



The frozen bacterial paste (about 11 g.) was placed in a temperature- 

 insulated mortar. Liquid nitrogen was poured into it until the paste was 

 well covered. As temperature equilibrium was approached the paste 

 became brittle and was then ground to a fine powder by gently tapping 

 with a pestle for 5-10 min. The liquid nitrogen was allowed to evaporate 

 and the frozen powder was spread over the bottom surface of a plastic 

 container. It was melted by dipping the container into water at 30° for 

 2-3 min., and cooled to 0°. It was suspended with stirring in about 4 ml. 



