172 BIOCHIMICA ET BIOPHYSICA ACTA VOL. 12 (1953) 



SOME REACTIONS OF ADENOSINE AND INOSINE PHOSPHATES IN 



ANIMAL TISSUES* 



by 



H. A. KREBS AND R. HEMS 



Medical Research Council Unit for Research in Cell Metabolism, 

 Department of Biochemistry, The University of Sheffield {England) 



The starting point of the present investigations was the observation that inosine 

 triphosphate (ITP), on addition to respiring pigeon breast muscle suspensions, becomes 

 radioactive when ^^pQ^ is present. Such suspensions readily synthesise adenosine tri- 

 phosphate (ATP) from adenosine diphosphate (ADP) and inorganic orthophosphate, 

 and the formation of isotopic ITP raised the question whether inosine diphosphate 

 (IDP) can replace ADP in the enzyme system forming the triphosphate. The experiments 

 reported in this paper show that this is not the case. ITP does not arise by synthesis 

 from IDP and inorganic phosphate but by transfer of isotopic phosphate from ATP to 

 IDP. 



MATERIALS AND METHODS 



The ATP preparations were the same as those used in previous work from this laboratory 

 (Eggleston and Hems^; Krebs, Ruffo, Johnson, Eggleston and Hems-. ITP was prepared 

 according to Kleinzeller^. Both preparations contained some diphosphates ; the quantities of these 

 are given in the Tables as the zero values. Radioactive ATP, containing ^^P in the two terminal 

 phosphate groups of ATP, was prepared according to Hems and Bartley*. 



The quantities of the various adenosine and inosine phosphates in the enzyme solution were 

 determined paper chromatographically by a modification of the method of Eggleston and Hems^. 

 The original method separates the three adenosine phosphates from other phosphates, including 

 ITP, the Rp value of the latter being lower than that of ATP. But the Rp value of IDP and ATP, 

 and of IMP and ADP, are about the same in the solvents used by Eggleston and Hems^.TIic follow- 

 ing modification satisfactorily separates the three inosine and adenosine phosphates. At a first ascen- 

 ding stage the inorganic orthophosphate, having the highest Rp value, is separated from all other 

 phosphates and the paper containing it is cut off. The chromatography is then continued in the opposite 

 direction with a different solvent. The new procedure is quicker than the earlier one. It can handle 

 larger amounts of material and is less affected by the presence of salts and by variations of temperature. 

 The relative Rp values of the adenoside and inosine phosphates arc as follows: AMP, i.o; ADP, 0.80; 

 ATP, 0.66; IMP, 0.53; IDP, 0.39; ITP, 0.26. 



Whatman No. i filter papers (20x60 cm) are washed in aqueous ethylenediamine tctra-acetic 

 acid by the method described by Eggleston and Hems^ and, after drying, the papers are folded along 

 a line 18 cm from one end, dividing the paper into two parts, a longer one subsequently called A and 

 a shorter one B. 



Solutions to be analysed and standard marker solutions, including KHj^^pQ^, are placed in 5 ^1 

 quantities at intervals of al)out 3 cm on a line 3 cm from the fold on B. The spots are dried in a current 

 of warm air from a hair drier, at a temperature not higher than 30 '. By placing up to ten 5 ji\ quanti- 

 ties of solution on one spot with an "Agla" micrometer syringe, between 2 and 10 /<g total P are 

 accumulated on each spot. 



The paper required has to be longer than the commercially available size** and an extension piece, 

 with a pad, is made as follows: an (unwashed) paper (20 x 40 cm) is folded over half its length to form 

 a pad 2 cm wide and this is attached to the end of B with a stapling machine, whilst the other end of 



* This work was aided by a grant from the Rockefeller Eoundation. 

 100 ft lengths of tliis paper have now become commercially available. 



References p. 180. 



