458 ANDRE T, JAGENDORF AND JOSEPH S. KAHN 



o-i6or 2-0 m/xmoles of p-P]-ATP, the increase due to pre-illumination 

 followed by ADP in the dark is i m/xmole//xmole of chlorophyll. 



The high-energy phosphate intermediate suggested by these experi- 

 ments is rather unstable. Our measurements show it has a half-life of 

 about 4 min. in the reaction mixture at 5°. 



Isolating and identifying an unstable intermediate may be a formidable 

 job. We have not yet attempted to do so. Instead, we have discovered, 

 solubilized and partially purified an enzyme which might either be, or 

 function close to, our theoretical X ~ P. 



The enzyme is one which causes an exchange of the third phosphate 

 from ATP to ADP. It would appear to be analogous to the ADP-ATP 



6 8 10 

 Time (mm) 



14 16 



Fig. 2. Time course of ADP-ATP exchange reaction. Reaction mixture was 

 o-i ml. total volume, containing [i*C]-ADP at 5 x lo"^ m, ATP at i x 10^=^ M, 

 MgCU at 3 X lo"^ M, tris at 2 x 10"^ M, and solubilized enzyme, the whole brought 

 to pH 8-0. Reactions were run at 30" for the length of time shown. 



exchanging enzyme isolated by Wadkins and Lehninger [9]. Activity is 

 measured by incubating labelled [^^CJ-ADP, unlabelled ATP, the enzyme 

 and Mg for 5 to 10 min. The reaction products are separated chromato- 

 graphically and counted separately. Figure 2 shows that the label in ATP 

 comes to equilibrium with that in ADP, in proportion to their relative 

 concentrations. The high value in ATP at zero time is due to a 20% 

 contamination of p^CJ-ADP (Schwartz Biochem. Co.) with [i^CJ-ATP. 



Purification of the enzyme (to be reported elsewhere) involved extrac- 

 tion from chloroplasts by blending with water, followed by two acetone 

 fractionations and several cycles of freezing and thawing. This results in a 

 solution which has 80" „ of its protein under one peak in electrophoresis, 

 with two minor components. Upon analysis of various fractions in a 

 preparative electrophoresis apparatus (designed and constructed by 



