164 METABOLISM AND PHYSIOLOGY 



Pi into mannose-6-P in the presence of PMS was subtracted from the 

 corresponding value obtained in the absence of PMS. Previous experi- 

 ments have established that aerobic phosphorylation is strongly in- 

 hibited by PMS (1). The phosphorylation assay thus was an estimation 

 of PMS-sensitive incorporation of Pi into mannose-6-P. The PMS 

 control was not used in studies of the properties of the purified 

 aerobic phosphorylation system (which was completely inhibited by 

 PMS). 



Biochemicals were obtained from Sigma Chemical Corporation. 

 PPO and POPOP were obtained from Packard Instrument Company. 



RESULTS 



Separation of enzymatic activity by sucrose density gradient centri- 

 fugation 



Initial centrifugation experiments were carried out with crude 

 extracts derived iromR. yuhnim cells grown in darkness under aero- 

 bic conditions. Succinic dehydrogenase activity was associated with a 

 collection of particles which sedimented more rapidly than the bulk 

 of the DPNH oxidase (Fig, 1), This was even more evident when the 

 centrifugation time was increased from 40 to 60 minutes (Fig. 2), In 

 both examples, the incorporation of inorganic phosphate into mannose- 

 6-P was associated with succinic dehydrogenase activity. 



With crude extracts derived from cells grown anaerobically in the 

 light (Fig. 3), succinic dehydrogenase activity paralleled chlorophyll 

 content. The rate of sedimentation of the dark succinoxidase particles 

 (Fig. 1) appeared to be similar to that of the "chromatophore fraction" 

 (Fig. 3). However, the patterns of DPNH oxidase activity differed 

 strikingly; most of the apparent DPNH oxidase in the photosynthetic 

 extract remained in the top "soluble" fraction (Fig. 3), whereas much 

 of the DPNH oxidase activity of the dark extract was found in particu- 

 late form (Fig. 1), 



In other experiments it has been found that the specific DPNH 

 oxidase activities of crude "photosynthetic" and "dark" extracts 

 differ considerably, whereas the succinic dehydrogenase activities 

 do not. For example, the specific activity of the DPNH oxidase (at 

 30°C) of a crude photosynthetic extract was 9.6 m/«moles DPNH 

 oxidized/mg protein/min. This was one-fifth of the value shown by a 

 crude dark extract, 49 m^wmoles. On the other hand, the specific 

 activity of succinic dehydrogenase (at 3 0°C) of the crude photosynthetic 

 extract was 26 m/i moles indophenol dye reduced/mg protein/min, only 

 one and one-half times the value for the crude dark extract, 18 

 m/i moles. 



The aerobic phosphorylation system of the photosynthetic extract, 

 however, appeared to sediment in the same zone as the chromatophore 



