146 M. E. JONES, S. BLACK, R. M. FLYNN, F. LIPMANN VOL. 12 (1953) 



In the experiment shown in Table III a determination of adenosine-5-phosphate 

 (AMP) with the Kalckar-Schmidt^o procedure is included and, in addition, the acetyla- 

 tion of CoA is followed by determining the disappearance of SH-CoA^^ as well as the 

 formation of acetyl-S-CoA. A similar balance with pigeon liver extract is presented in 

 Table IV. 



TABLE III 



BALANCE FOR ACETYLATION OF COA 



* Hydroxamic acid method. 



**No sulfur bound PP could be demonstrated on treatment of the deproteinized samples with 

 mercuric acetate. 



*** Corrected for AMP formed in absence of CoA due to ATP-ase. 

 Concentration of factors per ml of reaction mixture: o.i ml (100 units) of yeast fraction 4; 5 jiiM 

 Pabst CoA, reduced with potassium borohydride; 5 /iM ATP; 20 jliM potassium acetate; 10 fxM 

 MgClg; 50 /<il/ KF; 100 juM tris (hydroxymethyl)aminomethane buffer, pH 7.5. The tubes were 

 incubated at 37° for 15 minutes. 



TABLE IV 



CATALYTIC BALANCE WITH CRUDE PIGEON LIVER EXTRACT 



* Assay by pyrophosphatase. 

 Each vessel contained, in a total volume of 3.2 ml: 47 /nAI ATP, of which 17 were added at o 

 minutes and 30 /uM at 30 minutes; 170 /_iM KF; 460 ^Af tris (hydroxymethyl)aminomethane buffer, 

 pH 8.2; 75 fiM MgCl^; 870 fiM NHgOH, pH 6.5; So fiM glutathione; 250 /i.U K acetate; and 0.96 ml 

 crude pigeon liver enzyme. Vessels were incubated at 30° for 120 minutes. 



TABLE V 



CONVERSION OF ACETYL COA, PYROPHOSPHATE, 

 AND ADENOSINE-5-PHOSPHATE TO ADENOSINE TRIPHOSPHATE 



ATP AMP Acetyl phosphate 



fxM fiM iJiM 



Complete system + 6.5 — 7.7 — 5.0* 



Replace pyrophosphate + 0.3 — 0.5 o 



with orthophosphate 



Omit CoA +0 — 0.2 — 0.7 



Each vessel contained: 25 jxM lithium acetyl phosphate; 0.25 /<M CoA; 100 jiM potassium 

 pyrophosphate; 10 ^M potassium adenylate; 10 1.1M MgClg; 10 /iM cysteine; and 50 fiM KF; o.i ml 

 transacetylase, Clostridium kluyvcri; 0.3 ml yeast enzyme preparation. Tlic pll was 7.1. The vessels 

 were incubated for 30 minutes at 37°. 



* These values are corrected for spontaneous breakdown of acetyl phosphate. 



References p. 149. 



