168 METABOLISM AND PHYSIOLOGY 



TABLE 1 



Substrate Requirement for Aerobic Phosphorylation by a Sucrose 

 Gradient Fraction of Dark Extract 



The incubation volume was reduced to 200 |Ul, with proportionate reduction 

 of all components (see experimental procedure). Each tube contained an aliquot 

 of the sucrose gradient fraction used in Fig. 5 (29 /ig protein). The inorganic 

 phosphate content was 0.37 //moles (107 cpm)/200 /il. All values given have 

 been corrected for the zero time control (960 cpm). 



Additions Mannose-6-Phosphate formed (cpm) 



1. None 1,600 



2. DPNH (0.05 //mole) 163,000 



3. DPN (0.05 //mole) 2,200 



4. Succinate (0.5 //mole) 21,900 



5. DPNH (Extract omitted) 80 



fraction. It must be noted that the association of this phosphorylation 

 system with either succinic dehydrogenase or chlorophyll does not 

 appear to be as evident as the parallel distribution of succinic dehydro- 

 genase and chlorophyll seen in Fig. 3, or succinic dehydrogenase and 

 the phosphorylation system of dark extracts (Figs, 1 and 2), The phos- 

 phorylation exhibited by the peak fraction in Fig, 3 (fraction 4) has 

 been shown to have the same substrate requirements as the corre- 

 sponding fraction from sucrose gradient experiments with crude dark 

 extracts (see Table 1), 



Preliminary attempts to show a possible interaction between the 

 "nonphosphorylating DPNH oxidase" fractions and the phosphorylation 

 system have met with negative results (Fig. 4), The usual sucrose 

 density gradient experiment (using a crude dark extract) was per- 

 formed, the assays yielding the usual curves for DPNH oxidase, suc- 

 cinic dehydrogenase, and phosphorylation (curve M-6-P-I). Phos- 

 phorylation assays were also made using fraction 5 in combination 

 with each sucrose fraction; the curve obtained (M-6-P-II) is identical 

 within experimental error to the dotted curve calculated by a simple 

 arithmetic addition of activities (expected if there were no interaction 

 of fractions). 



Some properties of sucrose gradient fractions having maximum 

 aerobic phosphorylation activity 



DPNH was required for aerobic phosphorylation by a sucrose 

 gradient fraction of dark extract (Table 1), Little phosphorylation was 

 seen with succinate. The same substrate requirement has been ob- 

 served in similar experiments with the corresponding fraction from 



