VOL. 12 (1953) BIOSYNTHESIS OF NUCLEOSIDES AND NUCIEOTIDES 



259 



OH 



point of ottock of 

 urid^J tronsferose 



HOCH2 



point of otlock of 

 gli/cosy/ tronsferose 



Uridyl 



Fig. I. Uridyl-Phospho- 1 -Glucose (UDPG) 



TPN will dehydrogenate the latter ester (Warburg, Christian and Griese^^) the rate 



of liberation of glucose ester can be followed in the ultraviolet range at 340 m/x by 



reduction of TPN (Warburg, Christian and Griese^^) . 



^^P labelled inorganic pyrophos- 

 phate was utilized in the reaction and 

 for each mol radioactive pyrophosphate 

 used one mol a-glucose-i-phosphate was 

 liberated. If the cleavage took place at 

 arrow number i (Fig. i) the resulting 

 nucleotide should be uridine triphos- 

 phate (UTP). It is possible to isolate the 

 radioactive ester by barium precipita- 

 tion, by chromatography through a 

 column of Dowex CI", by paper-iono- 

 phoresis and by paper chromatogra- 

 phy (ethanol-ammonium acetate pH 7.0, 



(Paladini and Leloir^^). In all these procedures the isolated nucleotide behaves 



like a di- or tri-phosphate. In the chroma- Cs^o 



togram (see Fig. 2) it appears more charged 



than uridine diphosphate (UDP), moving, 



more slowly. Since it has one mol ^^P-pyro- 



phosphate per mol uracil it seems evident 



that it is UTP and a chemical analysis 



confirmed this. UTP seems to act slowly as a 



phosphoryl donor in the hexokinase test^^ ; 



interestingly enough, if a small amount 



of adenosine diphosphate (ADP) is present 



this reaction goes much f aster^^. UDP is not 



active in this test, which is a very sensitive 



assay for nucleoside triphosphates. 



It is of particular interest, however, to 



describe the enzymic reaction in which UTP 



plays the role of a uridyl donor. This is in 



the" back reaction" of thepyrophosphorol- 



ysis, i.e. UTP plus a-glucose-i -phosphate 



in the presence of Zwischenferment gives 



- test 

 PS JO [ control 



UTP UDP 



WOOr 



/SCO- 



1000- 



SOD 



--.»---!. 



...V-'- 





M ?» o -»^ T"»-9— o-?- 



-^- | ?— ?-?- » -<->-» 



iJOOO 



3000 



200c 



mc 



X cm 



Fig. 



. Paper chromatogram of norite eluate of 

 UDPG-pyrophosphcrylase digest, 

 (from A. Munch-Petersen et al.^^). 

 Reaction mixture: 0.2 /^mol UDPG, 2 ^mol in- 

 organic pyrophosphate (8- 10* cts/min), 50 ^1 

 phosphoglucomutase, 50 ^i\ Zwischenferment 

 (3.3 mg/ml), 3 ml Tris (Hydroxy methyl) amino 



methane HCl pH 8.0. 

 Control mixture: Same without pyrophosphate. 

 After 45 min. incubation, the digest were acidi- 

 fied, adsorbed on norite and eluted with 50% 

 ethanol. Chromatographed 44 hours in neutral 

 solvent. Chromatogram scanned in theBeckmann 

 260 m/< 



UDPGlucose (uridyl-phospho-glucose) plus inorganic pyrophosphate. If the latter is split 

 by a specific pyrophosphatase the reaction is pulled and the formation of UDPGlucose 

 from UTP and gluxose-i -phosphate is easy to demonstrate (see Fig. 3). The UDPG 

 pyrophosporylase could logically be classified as a "uridyl transferase". In extracts 

 from galactose adapted Saccharomyces fragilis it is simple to demonstrate that UTP 

 reacts with a-galactose-i-phosphate^''. In the final balance the presence of UTP bri ngs 

 about a conversion of a manyfold amount of galactose-i-phosphate to glucose-6-phos- 

 phate (see Fig. 4). The sequence of reactions is as follows: 



i. UTP -f a-galactose-i-phosphate ^ UDPGalactose + pyrophosphate. This is the 

 pyrophosphorylase reaction (uridyl transferase No. i) presumably specific for galactose 

 adapted yeast. 



References p. 263I264. 



