176 H. A. KREBS, R. HEMS VOL. 12 (1953) 



be taken to indicate that the two nucleotide triphosphates are hydrolysed by the same 

 enzymes and that they competitively inhibit the decomposition. 



TABLE IV 



HYDROLYSIS OF ATP AND ITP BY SOLUBLE ATIase 



Each test tube contained i ml enzyme solution, 0.15 ml o.i M MgClj, 0.2 ml 0.5 M triethanolamine 

 buffer pH 7.4, and substrate as indicated below. Water to 5.5 ml. 30°. Inorganic P determined accor- 

 ding to LowRY AND LoPEZ^^. Substrate (i ml each o.oi M) I: ATP; II: ITP; III: ATP and ITP 



Anaerobic transfer of phosphate from ATP to ITP 



The synthesis of ITP from IDP and inorganic phosphate could be a direct reversal 

 of the hydrolysis ; if this were the case ITP should become radioactive when incubated 

 with ^2P04 anaerobically in the presence of ITPases. Experiments however show that 

 this is not the case. Alternatively, inorganic phosphate might first be incorporated into 

 ATP, e.g. by oxidative phosphorylation, and the radioactivity of ITP could then arise 

 by transfer of ATP phosphorus to IDP. The following experiment is evidence in support 

 of this mechanism. Pigeon breast muscle was disintegrated as described before and sus- 

 pended in 73 ml saline medium and i ml o.i 71/ MgClg; 1.6 ml of this suspension was 

 incubated at 22° anaerobically with 0.2 ml isotopic ATP (about 0.0055 ^^> ^"P i^^ ^-and 

 y-phosphate) and 0.2 ml non-isotopic ITP (0.009 ^^)- Nine parallel tubes were set up 

 and the incubation was stopped at various intervals by the addition of 0.2 ml 30% (w/v) 

 trichloroacetic acid. The results of the analyses are shown in Table V. The amounts of 

 ATP gradually fell and reached zero value after about 10 min. The specific activity of 

 the ATP phosphorus remained virtually constant, indicating that no ATP was synthe- 

 sized. There was some initial radioactivity in the ITP phosphorus and inorganic phos- 

 phorus which was due to the presence of these substances in the sample of radioactive 

 ATP. The amounts of ITP decreased slowly only as long as appreciable quantities of 

 ATP were present, i.e. during the first 6 min. Afterwards it decreased rapidly. The specific 



TABLE V 



ANAEROBIC TRANSFER OF PHOSPHATE FROM ATP TO ITP IN PIGEON BREAST MUSCLE SUSPENSIONS 



(For experimental conditions see text) 



References p. 180. 



