14 M.MKirK J. nKSSMAN 



This would 1)(' (he only instance in the synthesis of deoxyi ihoiiuflco-idi 

 ti'iphospiiafcs in which a transfer of the intact pyropiiosphate supposedly 

 occurs, and the I'caction should \)r studied with the i)urifie(l enzyme. The 

 small amount of (ri'I)!' found in the cliromatogranis (Bianchi ct al., 

 19()1 I was attrihutcd to the hydrolysis of dTTP. The other possibility 

 (which 1 fa\()n is that dTDP is an intermediate hut does not accumulate 

 in the reaction mixture because another enzj'me, nucleoside diphosphate 

 kinase, is present in the crude extract which converts dTDP to the 

 triphosphate. This would be in keeping with their conditions of incuba- 

 tion which include an ATP-generating system. The continued conversion 

 of ADP to ATP by the generating system would tend to drive the reac- 

 tion to the right, lience favoring the foi-mation of dTTP and decreasing 

 the accumulation of dTDP: 



ATP generating system 



i 

 dTMF + ATP ^ dTDP + ADP — 

 + 

 ATP 



w 



(ITTP + ADP — 



We have evidence (^1. Hienhoif and AI. Bessman, in prejjaration ) that 

 partially purified extracts of regenerating rat liver catalyze the i:)lios- 

 |)liorylation of dTMP to the corresponding dii)hosphate. 



The interesting observation (Hiatt and Bojarski, 1960) that intra- 

 peritoneal injections of thymidine stimulate the formation of thy- 

 midylate kinase in rat liver was explained by these same authors 

 (Bojarski and Hiatt, 1960) as a stabilization of thymidylate kinase 

 during preparation of the extract. They showed that preincubation of a 

 liver extract resulted in a rapid decrease of thymidylate kinase activity 

 wiiich could be prevented by including thymidylate (or thymidine at 10 

 times the concentration) in the preincubation medium. This information 

 has been useful in preparing extracts for further fractionation. 



Romberg (1957) and Lehman et al. (1958a) described the phos- 

 phoiylation of dAMP, dCMP, dGMP, and dTMP by a partially purified 

 extract from E. coli. It became apparent that more than one enzyme was 

 responsible for the iihosphorylation of these four different nucleotides 

 when Lehman et nl. (1958a) observed that heated extracts lost their 

 ability to phosphorylate dCMP, dOMP, and dTMP while partially 

 retaining their ability to phosphorylate dAMP. An indication that the 

 other three activities w^re due to separate enzymes was the observation 

 of Hurwitz (1959) that acetone fractionation resulted in relative eni'ich- 

 ments of dCMP, dGMP, and dTMP kinases in different fractions. 



The only report of the study of a ])urified kinase to date is that of 



