VOL. 12 (1953) REACTIONS OF ADENOSINE AND INOSINE PHOSPHATES 



173 



-flap attoched 

 to lid 



Paper clip 



Stapling clips 



the extension is fastened to A with two paper clips (see diagram). The end of A is then folded to 



provide a flap of about 2 cm to be attached to the lid of the glass tank. The exact measurements depend 



on the distance between the lid and the surface of the solvent. About 1.5 cm of the papers should be 



immersed in the solvent at the bottom of the tank. 



Glass tanks for ascending and descending chrom- 

 atography are of the shape described by Eggleston 

 AND Hems^; for descending chromatography they are 

 fitted with a framework of glass rods which carries two 

 glass troughs 28 cm long and holding 70-80 ml of solvent^. 

 Two solvents are used. The first consists of 90 ml 

 isopropyl ether/60 ml 90% (w/v) formic acid (Hanes and 

 TsHERWooD^) ; the second of 100 ml ?5obutyric acid, 60 

 ml iV ammonia, 1.6 ml o.i M ethylene diamine tetra- 

 acetic acid. This is a modification of the solvent described 

 bv Zetterstrom and Ljunggren^. 



The papers are developed first for 3-4 hrs by ascend- 

 ing chromatography in the first solvent. They are then 

 removed from the tank and, whilst still fastened to the lid, 

 dried in a current of cold air. This takes about 30 min. 

 When dry the papers are detached from the lid, and the 

 paper clips are removed so that the whole length of the 

 paper can be spread out flat. The position of the ortho- 

 phosphate is then located by scanning with a radiation 

 monitor (Alpha Beta Tj'pe 1021 B; Messrs McMichael 

 Radio Ltd., Slough, Bucks). When ^^P is not present in 

 the experimental solution it is added as a marker to make 

 location of the orthophosphate possible without chemical 

 treatment of the paper. The nucleotides are located by 

 their U.V. absorption with a "Hanovia Chromatolite" 

 (Hanovia Ltd., 3 Victoria St., London, S.W.i). The paper 

 carrying the orthophosphate is cut off from the remainder 

 of the chromatogram and kept for the analysis of or- 

 thophosphate. The nucleotides stay near the starting line, 

 and sufficient paper is left to form a "wick" for the descend- 

 ing chromatography with the second solvent. The direc- 

 tion of flow of this solvent is opposite to that of the first. 

 After 16-18 h the papers are removed and dried by heat- 

 The positions of the nucleotides are located with the U.V. light and ringed in 



pencil. The identity of the spots is confirmed by spraying with the acid molybdate according to 



Hanes and Isherwood^. 



The spots are cut out, wet-ashed and analysed for phosphorus and radioactivity as described 



by Eggleston .\nd Hems^. 



Enzyme material deproteinised with trichloroacetic acid is suitable for the above procedure. 



Samples of deproteinised solutions which could not be analysed immediately were stored at — 14°. 



Ipot of solution 



Fig. I. Folding of filter paper for chro- 

 matographic analysis. 



ing for 20 min at 80 



RESULTS 



Reactivity of ATP and ITP-phosphorus in pigeon breast muscle 



Pigeon breast muscle was minced in a Lapatie mill and disintegrated in a stainless 

 steel "homogeniser" of the Potter-Elvehjem type with 21.5 volumes of the medium 

 used by Krebs et al.^, all operations being carried out with ice-cooled containers and 

 reagents. Of this suspension 3 ml, together with i ml additional solution containing 

 substrates and cof actors, were shaken in Warburg manometers at 20°. The gas space 

 contained O2. The final concentration of the substrate (a-ketoglutarate) was 0.02 M, of 

 ATP about 0.0006 M, of ITP 0.0008 M. Inorganic phosphate containing ^^P was added 

 from a side arm at zero time. Enzymic activities were stopped by the addition of 0.5 ml 

 30% trichloroacetic acid. The phosphate fractions were separated by paper chromato- 

 graphic analysis of the filtrate. 

 References p. 180. 



