26o 



H. M. KALCKAE 



VOL. 12 (1953) 



800- 



600- 



'iOO 



200^ 



ii. Pyrophosphate + HgO -» 2 orthophosphates. This is catalyzed by inorganic 

 pyrophosphatase which is also present in crude 5. fragilis extracts. 



iii. UDPGalactose ^ UDPGlucose. This f^eo 

 is catalyzed by the inversion enzyme 

 ("Galacto Waldenase") specific for galactose 

 adapted yeast. 



iv. UDPGlucose + a-galactose-i-phos- 

 phate ^ UDPGalactose + a-glucose-i- 

 phosphate. This is catalyzed by a non- 

 pyrophosphorolytic uridyl transferase (No. 

 2) presumably specific for galactose adap- 

 ted yeast. 



V. a-glucose-i-phosphate ^ glucose-6- 

 phosphate catalyzed by phosphoglucomu- 

 tase which is also present in unfractionated 

 5. fragilis extracts. 



vi. Glucose-6-phosphate + TPN+ ^ 6- 

 phospho-gluconic acid -f reduced TPN. This 

 is catalyzed by glucose-6-phosphate de- 

 hydrogenase, also present in crude S. fragilis 

 extracts. 



- SOD 



- 600 



^00 



!CC 



ID 



IS 



?0 



30 



35 err 



Fig. 



3. Paper chromatogram of norite eluate 

 of UTP-pyrophosphorylase digest, 

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

 Reaction mixture: 0.2 ^imol UTP, 10 /tmol 

 glucose-phosphate, 100 ^tl inorganic pyro- 

 phosphatase, 50 lA Zwischenferment, i ml 

 Tris (hydroxy methyl) amino methane HCl 



pH 8.0 

 Control mixture: Same without glucose-i- 

 phosphate. After 50 min. incubation the 

 gigests were acidified, adsorbed on norite and 

 eluted with 50% ethanol. Chromatographed 

 44 hours in neutral solvent. Chromatogram 

 scanned in the Beckmann at 260 m^. 



4«« 



«a» 



noo 



1200 



woo 



800 



600 



' I Stoichiometr.c 

 utth^Ot'Or^ Of 

 0<//mo/ UTP 



100 



Since all the six step enzymes are present 

 in dialyzed S. fragilis extract the addition of TPN 30 minutes after a-galactose-i- 



phosphate and UTP have been added 

 to a dialyzed S. fragilis extract will 

 bring about a rapid density increase at 

 340 m/x due to the reduction of TPN 

 by the accumulated glucose-6-phosphate. 

 Of the six step enzymes mentioned 

 number iv deserves a few comments. 

 This enzyme which can best be dem- 

 onstrated in diluted extracts of S. fra- 

 gilis manifests itself b}' a formation 

 of I mol glucose-i-phosphate per mol 

 UDPG and galactose-i-phosphate con- 

 sumed. Since addition of inorganic 

 pyrophosphate is superfluous (occasional- 

 ly inhibitory) and the presence of pyro- 

 phosphatase apparently does not inter- 

 fere, it is clearly a non-pyrophosphoroly- 

 tic uridyl transferase where the uridyl ac- 

 ceptors are a-galactose-i-phosphate and 

 a-glucose-i-phosphate"', resp. It is pre- 

 sumably the same enzyme which cataly- 

 zes the following reaction : 



loaHf-x 



leoo 



tVOD 



mo 



tooo 



eoD 



SCO 



100 



203 



'iSmin 



Fig. 4. Spectrophotometric analysis (TPN reduc- 

 tion) of glucose-6-phosphate from UTP and 

 gaalctose- 1 -phosphate in 5. fragilis extract, 

 (from H. M. Kalckar, A. Munch-Petersen 



AND B. BrAGANZa". 



Reaction mixture: o.i /tmol UTP, 0.6 /imo] 

 Gal-i-P*, 50 ^1 dialyzed S. fragilis extract 

 (approx. 250 fig protein), 50 fj,l o.i M Tris 

 (hydroxy methyl) amino methane HCl, pH 8.0. 

 Control mixture: Same without UTP. After 30 

 min incubation excess of TPN was added (time 

 zero on the graph). At 5 min 0.1 ^mol UTP was 

 added to the control. 



* Courtesy Leloir and Reissig. Ba-salt, subjected to reprecipitation with ethanol acid labile P 

 estimated. 



References p. 263I264. 



