144 



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



in the following manner: for each fiM of CoA (310 units = i fxM), 75 ixM of KBH4 in 

 0.002 M KOH and 10 fxM of tris (hydroxymethyl) aminomethane buffer, pH 9, were 

 added. The vessel was incubated for 15 minutes at 37°, at which time the contents were 



//M mg/ml 



Fig. 2. Effect of magnesium con- 

 centration of the rate of acetate- 

 ATP-CoA reaction. Each tube con- 

 tained, in I ml. of reaction mixture: 

 25 units COA; 10 /j,M ATP; 10 

 /nM potassium acetate; 200 /<M tris 

 (hydroxymethyl) - aminomethane 

 buffer, pH 7.5; 50 /nM KF; 10 /nM 

 glutathione; and aoo/iMNHjOH, 

 neutralized to pH 7.4 with KOH; 

 0.0 1 ml (10 units) of yeast fraction 

 4 and MgClg as indicated. Final pH 

 was 7.4-7.2. The tubes were incu- 

 bated at 37°, and 0.2 ml. samples 

 were taken at 13, 30, and 60 mi- 

 nutes to determine the rate of 

 the reaction. 



Fig. 3. Effect of pH on the rate of 

 acetate-ATP-CoA reaction. Each 

 vessel contained, in i ml reaction 

 mixture: 25 units CoA; 10 /j,M 

 ATP; 10 /iiM potassium acetate; 

 50 jiiM KF; 10 f^iM MgClj; 10 /nM 

 glutathione; 200 juM NHjOH (pH 

 adjusted with KOH to that of 

 buffer) ; 50 fiM each tris (hydroxy- 

 methyl) aminomethane and potas- 

 sium phosphate buffer adjusted to 

 the desired pH; and o.oi ml (10 

 units) of yeast fraction 4. The 

 tubes were incubated at 37°, and 

 0.2 ml samples were taken at 20, 

 40, and 60 minutes to determine 

 the rate of the reaction. 



neutralized with HCl to pH 7.2 and the volume adjusted with water to give a CoA solu- 

 tion of the desired concentration. Although solutions store fairly well on freezing, we 

 have routinely prepared fresh solutions each day. 



CoA concentration. It is a characteristic of the hydroxamicacid system that relatively 

 high concentrations of CoA and hydroxylamine^^ are required for saturation. Under 

 our conditions, as shown in Fig. 4, the maximum activity is approached at about 100 

 units of CoA. Since the reaction between acetyl mercapto CoA and hydro.xylaraine is non- 

 enzymatic, this relatively high saturation concentration is not surprising. 



The effect of inorganic phosphate. The addition of 50 to 100 fj.M of phosphate per 

 ml generally increases the rate of the hydroxamic acid reaction considerably. For this 

 reason phosphate was included in our assay system. Phosphate, however, is not indis- 

 pensable. The mechanism of the phosphate effect is not clear. 



References p. I4g. 



