776 



BIOCHEMISTRY: KORNBERG ET AL. 



Proc. N. a. S. 



corporated into viral DNA (see below) and was essentially similar in extracts pre- 

 pared by either Method I or Method II. 



The product of the dHMC-5-P kinase action (with the presumed participation 

 of nucleoside diphosphate kinase in the preparation) was shown to be the triphos- 

 phate. Using a 20-fold purified kinase preparation/^ 25 /unioles of dHMC-TP 

 were prepared and isolated by ion-exchange chromatography, a yield of 92 per cent 

 based on the starting dHMC-5-P. Theoretical specific radioactivity values were 

 found in the isolated dHMC-TP when P32- or C '^-labeled dHMC-5-P was the 



10 20 30 



TIME- minutes 



Fig. 3. — dCTPase levels before and after infection with phage 

 T2 or T5. The arrow indicates the start of infection ("zero 

 minutes," see Method I). The incubation mixtures (0.25 ml) 

 contained : 6.0 m/imoles of dCTP labeled with P'^ in the terminal 

 pyrophosphate group (10' cpm per m/umole), glycine buffer, pH 

 9.2 (0.04 M), MgCl2 (0.008 M), 2-mercaptoethanol (0.01 M) and 

 a quantity of extract containing about 0.3 ng of protein. After 

 20 min of incubation at 37°, the reaction was terminated by add- 

 ing 0.5 ml of 0.1 A/^ HCl, followed by 0.2 ml of a mixture (contain- 

 ing per ml: 5 mg of crystaUine bovine serum albumin, 25 Mmoles 

 of sodium pyrophosphate and 25 ^nioles of potassium phosphate, 

 pH 7), and 0.1 ml of a Norit suspension (20 per cent packed 

 volume). The Norit was removed by centrifugation and the 

 Supernatant fluid was assayed for radioactivity. 



starting material. After concentration with Norit, analysis revealed a ratio of 

 HMC: total P: acid-labile P of 1.0: 3.2: 2.2, using an E„, value of 13.5 X 10^ 

 at 284 mju at pH 1 . 



Kinase Levels for the Other Deoxynucleotides and Levels of the Hydroxymethylating 

 Enzyme. — It is noteworthy that after T2 infection, the kinase levels for dT-5-P 

 and dG-5-P were increased approximately 20 and 45 times, respectively, while 

 that for dA-5-P was essentially unaltered (Fig. 2 B, C, D). Bessman has made 

 similar observations independently.^' As mentioned above, the kinase levels for 



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