VOL. 12 (1953) REACTIONS OF ADENOSINE AND INOSINE PHOSPHATES I77 



activity of the ITP phosphorus rose rapidly even during the first 6 min when its con- 

 centration hardly changed. The specific activity reached a maximum when all ATP 

 had broken down and then remained constant. These findings can be explained by the 

 assumption that both ATP and ITP are hydrolysed by the muscle preparation and that 

 the IDP formed is rephosphorylated by ATP; in other words that the muscle suspension 

 contains an enzyme capable of transferring a phosphate group of ATP to IDP. 



To test whether this transfer is brought about by one of the known enzymes a similar 

 experiment was carried out where the myosin ATPase of Bailey^** replaced the pigeon 

 breast muscle suspension. The incubated solution contained /S-y-labelled ATP, non-isotopic 

 ITP and IDP, 0.002 M CaClg, 0.02 M triethanolamine buffer pH 7.4, and 2 ml myosin in 

 a total volume of 10 ml. Both ATP and ITP were hydrolysed but there was no change in 

 the specific activity of the two substances. The enzyme preparation used therefore did 

 not transfer ATP phosphorus. Another experiment with the soluble ATPase of Kielley 

 AND Me\'e;rhof^^, where CaClg was replaced by MgCl2, also failed to indicate a transfer. 



On the other hand a dialysed crude extract of rat intestinal mucosa effected a rapid 

 transfer of radiophosphorus from ATP to ITP. When added separate^ to extracts the two 

 substrates were hydrolysed at about the same rate (70 micrograms P formed permgdry/wt 

 per h at 38° and pH 8.y). As expected^- ^^ all three phosphate groups were split off. 



The phosphate transfer is shown in the following experiment. The mucosa was 

 scraped off the rat ileum and disintegrated with 10 volumes of water. The suspension 

 was filtered through glass wool and the filtrate was dialysed against water for i h to 

 reduce the concentration of inorganic phosphate. 2 ml dialysed enzyme solution, 2 ml 

 j8-y-labelled ATP (containing some ADP; ATP + ADP phosphorus about o.oi il/), 2 ml 

 non-isotopic ITP (containing some IDP; ITP + IDP phosphorus about o.oi M), 0.4ml 

 0.2 M triethanolamine buffer pH 7.4 and 3.6 ml water were incubated at 30°. At inter- 

 vals I ml samples were removed and deproteinised with o.i ml 30% trichloroacetic acid. 

 The results of the analyses are shown in Table VI. The concentration of ATP progressively 

 decreased and unlike in previous experiments the specific activity of ATP phosphorus also 

 fell. The concentration of ITP changed little in the earlier stages of incubation, but as 

 before decreased more rapidly when the concentration of ATP had fallen to a low value. 

 The specific activity of ITP rose rapidly as long as ATP was present. The concentration 

 and radioactivity of inorganic phosphate rose as expected. There was also an increase 

 in the quantities of IMP and AMP, but this was less than expected on the assumption 

 that these substances were the only end products. No transfer occurred under the same 

 conditions when ITP was replaced by IMP or glucose. 



The results can be explained by the assumption that in addition to the hydrolysis 

 of the pyrophosphates a reversible transfer of phosphorus takes place between ATP and 

 ITP. The fall of the specific activity of ATP indicates that ATP must be formed during 

 the incubation. Under the given conditions the transfer of phosphate of ITP to ADP is the 

 only feasible mechanism of ATP formation. 



Crude extracts of the intestine are known to contain relatively large amounts of 

 alkaline phosphatases and the preceding experiment was therefore repeated with a com- 

 mercial preparation (Armour Laboratories) of purified calf intestinal phosphatase. This 

 was prepared according to Schmidt and Thannhauser^^ and is stated to contain 15 

 units/mg. Both ATP and ITP were hydrolysed but the rate of decomposition was slow 

 compared with that of the hydrolysis of glycerophosphate^^. At 30° at pH 7.4, 170 /xg P 

 were released from ATP per mg dry/wt per hr. No transfer of phosphate took place. 



References p. 180. 



