178 METABOLISM AND PHYSIOLOGY 



strate a distinction between these two electron pathways in photo- 

 phosphorylation. The results confirm and extend the evidence for non- 

 cyclic photophosphorylation which we reported earlier (22) and do not 

 support the recent contention of Bose and Gest (23) that the ATP formed 

 concurrently with the photoreduction of DPN by the ascorbate-DPIP 

 couple is the result of cyclic photophosphorylation. 



METHODS 



R. ruhrum was grown as previously described (22), at a tempera- 

 ture of about 27°C in 4,5 1, Pyrex glass-stoppered bottles, completely 

 filled to exclude air. A blower was used to prevent overheating of the 

 culture bottles. An inoculum of approximately 1 per cent was used for 

 each bottle. 



The inoculum was grown first for about 30 hours as a stab culture 

 in illuminated 100 ml screw-capped tubes, using nutrient agar con- 

 taining the components of the nutrient solution (22). At the end of this 

 period some cells were transferred to another stab culture tube. The 

 old stab culture tube was filled with a nutrient solution of the same 

 composition, incubated again for 30 hours, and the liquid portion was 

 used as the inoculum, (This technique was based on a suggestion by 

 Dr, D, M. Geller.) At intervals of several weeks the purity of the cul- 

 tures was checked by groAving the cells in Petri dishes on nutrient 

 agar. 



The cells were harvested 40-45 hours after inoculation, the cultures 

 yielding then about 4 g of wet packed cells per liter of nutrient solu- 

 tion. The cells were sedimented by centrifugation for 5 min. at 4000 

 X ^ at 1°C, The supernatant solution was discarded and the sedimented 

 cells were washed once by suspending them in 0,1 M tris/HCl buffer 

 at pH 7,8. The washed cells were collected by centrifugation. 



The method of preparing chromatophores under anaerobic conditions 

 was the same as that previously described (22), Washed chromatophore 

 preparations were suspended in 0.1 M tris buffer, pH 7.8, and either 

 used directly or stored at 4°C for several days under argon prior to 

 use, Bacteriochlorophyll, ATP and DPNH2, and lactate were deter- 

 mined as previously described (22). The experiments were carried 

 out in Thunberg-type cuvettes or in Warburg vessels, which were 

 illuminated by incandescent reflector spot lights. All experiments 

 were carried out under argon unless otherwise specified. 



When aged chromatophores were used, hexokinase, d-glucose, and 

 a catalytic amount of ADP were added to the reaction mixture as a 

 trapping system for ATP, to prevent loss of ATP by ATPase activity, 

 (The ATPase activity was negligible in fresh chromatophores but 

 gradually increased upon aging,) The "aged" chromatophores were 

 prepared by storage of fresh preparations at 2°C for 30 days under 



