E. S. CANELLAKIS AND EDWARD HERBERT 



TABLE III 



The Effect of Pyrophosphate 

 ON THE Incorporation of [1'*C]-CMP into S-RNA 



Treatment 



['^C]-CMP incorporated 

 (c.p.m.) 



1. None 150 



2. Pyrophosphate included 



in incubation mixture 600 



3. S-RNA pretreated with 



pyrophosphate* 610 



2 -o Eoen units of S-RNA-^ and -y were used throughout this experiment. The 

 first tube contained [^*C]-CTP (30 m/umoles, 2 x lo^/c.p.m./jumole), S-RNA, 

 Mg + + 2 -o /Ltatoms, and ribonucleotide incorporating enzyme in o -08 m potassium 

 phosphate buffer. Final volume i o ml. The second tube contained in addition 

 to above, i o fimole pyrophosphate. The S-RNA used in the third tube had been 

 pretreated with the ribonucleotide-incorporating enzyme, Mg + + 2-0 /xatoms, 

 I -o /xmoles pyrophosphate in 008 M potassium phosphate buffer. Final volume 

 I -o ml. This was then isolated free of the ribonucleotide incorporating enzyme, 

 and re-incubated in an incubation mixture identical to that in the first tube. 



* Inorganic pyrophosphate does not enhance the incorporation of [^■*C]-CTP 

 into S-RNA if during the preincubation either the S-RNA or the ribonucleotide- 

 incorporating enzyme is omitted. 



TABLE IV 



Liberation of Ribonucleoside 5'-Triphosphates into the 

 Acid-Soluble Fraction by the Pyrophosphorolysis of S-RNA 



Radioactivity recovered in the 

 ribonucleoside 5'-triphosphates 

 (c.p.m.) 



ATP CTP UTP GTP 



Experiment i 7000 2600 1350 850 



Experiment 2 11 60 310 330 180 



Experiment 2A 6600 950 2070 1320 



In Experiment i, S-RNA-/3 and -y was pyrophosphorylyzed in the presence of 

 [^'-P]-inorganic pyrophosphate, the ribonucleotide-incorporating enzyme, 2-o 

 /Liatoms Mg + + per ml. and 20 mjumoles each of non-radioactive ATP, CTP, UTP 

 and GTP in 008 m potassium phosphate, pH 7-2. Final volume i -o ml. In 

 Experiments 2 and 2A, [^-P]-S-RNA (prepared by isolating S-RNA from rat 

 liver which had been labelled with ^'-P in vivo) was incubated with i -o /xmole of 

 non-radioactive inorganic pyrophosphate, 2-0 /xatoms Mg + +, 20 m/nmoles each 

 of non-radioactive ATP, CTP, UTP and GTP, in the absence (Experiment 2) 

 and in the presence (Experiment 2A) of added non-radioactive ribonucleotide- 

 incorporating enzyme. The small but definite liberation of ribonucleoside s'-tri- 

 phosphate in Experiment 2 may be due to contamination of the S-RNA preparation 

 with the ribonucleotide-incorporating enzyme. 



