OBLIGATE PHOTOAUTOTROPHY OF GREEN BACTERIA 153 



isocitrate is also required for the assimilation of acetate via the 

 glyoxylate cycle. The condensing enzyme was not directly assayed in 

 extracts of C. thiosulfatophilum , but its existence was indicated by 

 experiments with labeled acetate, Chlorobium extract, acetate- l-C^"^, 

 Coenzyme A, MgCl2, and oxalacetate were incubated for various time 

 intervals and the products chromatographed on paper in two dimen- 

 sions. Autoradiography revealed a radioactive compound which had 

 migrated to the same region of the chromatogram as citrate. This 

 compound cochromatographed with citrate in two different solvent 

 systems. Its appearance was dependent upon the presence of oxalace- 

 tate in the reaction mixture. These experiments indicated that C. 

 thiosulfatophilum extracts contained condensing enzyme and aceto- 

 CoA-kinase, The presence of the latter enzyme was verified by direct 

 assay using the procedure of Jones and Lipmann (19), 



As shown in Table 1, aconitate hydratase activity could not be 

 demonstrated in C. thiosulfatophilum extracts. An increase inabsorb- 

 ancy at 240 m/i with either citrate or isocitrate or a decrease with 

 aconitate was not detected. On the other hand, the extracts contained 

 a very active TPN-linked isocitrate dehydrogenase, TPN reduction 

 was not observed in this assay if isocitrate was replaced by either 

 aconitate or citrate even in the presence of an excess of purified pig 

 heart isocitrate dehydrogenase. The same results were obtained with 

 cells grown in the presence of sodium acetate (0,005 M), 



In another experiment, a cell-free homogenate of C. thiosulfato- 

 philum and the supernatant fluid and sediment from a centrifuged 

 portion of the homogenate (144,000 x g for 60 minutes) were incubated 

 separately with aconitate. After two hours, enzymic assay failed to 



TABLE 1 

 Enzymic assays ivith extracts of C. thiosulfatophilum 



Enzyme 



Assay 



Substrates 



Activity 



Aconitate 

 hydratase 



Aconitate 

 hydratase 



Isocitrate 

 dehydrogenase 



Isocitrate 

 dehydrogenase 



Absorbancy 

 change at 

 240 m// 



Coupled to 

 isocitrate 

 dehydrogenase 



TPN reduction 

 DPN reduction 



Citrate, aconitate 

 or isocitrate 



Citrate or 

 aconitate 



jumoles/min/g protein 



Isocitrate 



Isocitrate 



1940 



