220 A. TissiERES 



A difference spectrum (oxidized minus reduced), of a small granule fraction, 

 taken in the visible region with the Cary Spectrophotometer, is shown in 

 Fig. 1. 



2. Relative Amounts of Cytochrome b^ in the Large Cell Debris and in the 



Small Granule Fraction 



A rough estimation of the amount of cytochrome h^ present in the large 

 cell debris (sedimented in 15 min at 6000 x g) and in small granules (sedi- 

 mented in 120 min at 100,000 X g) was obtained as follows: the pellets from 

 the centrifuge tubes were resuspended in 3-4 vol. of 50 % (w/v) sucrose 

 solution containing 0-01 m phosphate buffer, pH 7-0, and 0-01 m sodium 

 succinate. These preparations were examined under the microspectroscope 

 with the light path adjusted so that the intensity of the a-absorption band 

 of reduced cytochrome b^ was the same for both large cell debris and small 

 granule fractions. Thus, knowing the hght path, and the volume for each 

 preparation, it was found that the large cell debris contained 75-85% of 

 cytochrome b^, and the small granules 15-25 %. The errors in these measure- 

 ments were probably as great as 20-30%. 



Grinding the cells with alumina for longer periods decreased the cytochrome 

 content of the large cell debris and increased the number of small particles 

 bearing cytochromes. This is consistent v^ith the finding that large cell 

 debris from Azotobacter can be broken down to yield small granules 

 (Tissieres et al., 1957). 



3. Separation of the DPNH Oxidase Activity from Ribonucleoprotein Particles 



The DPNH oxidase system includes the cytochrome components; thus its 

 activity, under some conditions, can be taken as a measure of the activity of 

 the cytochrome system (Slater, 1950; Tissieres et al., 1957). The DPNH 

 oxidase activity, the amounts of protein and RNA, and the area covered by 

 the RNP particle peaks on the schlieren centrifugation diagrams, were 

 measured on two RNP particle preparations isolated as described previously 

 (Tissieres et al, 1959). Preparation 1 contained RNP particles with sedi- 

 mentation coefficients of 305 and 505; preparation 2, particles of 505. 

 Preparations 1 and 2 were centrifuged, at 25,000 x g for 45 min and at 

 32,000 X g for 30 min respectively, as shown in Table 1 . The various measure- 

 ments were then repeated on the supernatant in order to find out whether the 

 RNP particles sedimented at the same rate as the DPNH oxidase system. 

 With both preparations, the DPNH oxidase activity in the supernatant after 

 centrifugation decreased 88-90 % from its original value whereas the amounts 

 of protein and RNA, and the area under the schlieren curve, decreased 25% 

 (Table 1). These results indicate that the oxidases are attached not to RNP 

 particles, but to granules which sediment faster. These granules are probably 

 not homogeneous in size, since they did not form a visible peak on the 



