THE LOCATION OF CYTOCHROMES IN 

 ESCHERICHIA COLI 



By A. TissiERES 



Biological Laboratories, Harvard University, 

 Cambridge, Mass. 



The cytochromes are attached to granules of widely different sizes in bacterial 

 extracts made by sonic vibration or by grinding the cells with abrasives 

 (Tissieres, 1952; Wilson and Wilson, and Smith and Kuby, cited by Smith, 

 1954; Alexander and Wilson, 1955; Marr and Cota-Robles, 1957; Tissieres, 

 Hovenkamp and Slater, 1957). A fraction containing cytochromes centrifuges 

 in a field of about 6000 x g, while small granules, also bearing the respiratory 

 catalysts, sediment at 100,000 X g, together with the ribonucleoprotein 

 (RNP) particles. The small granules, although usually more active than the 

 larger ones, are not quahtatively different : they all oxidize the same substrates 

 and contain the same cytochrome pigments. There is no evidence for a class 

 of respiratory granules homogeneous in size and shape. 



It is shown here that (a) the small respiratory granules are distinct from 

 RNP particles and can be separated from the latter, in agreement with the 

 experiments of Cota-Robles, Marr and Nilson (1958) on Azotobacter 

 preparations; (b) in E. coli, as in several bacterial species (Marr and Cota- 

 Robles, 1957; Cota-Robles e/«/., 1958; Mitchell, 1957; Storck and Wachs- 

 man, 1957), the cytochrome components are attached to the cell membrane. 

 The respiratory granules of varying sizes probably arise by disintegration of 

 the cell membrane. 



MATERIAL AND METHODS 

 E. coli, strain B, was grown, harvested, and the cell free extracts were made 

 by grinding with alumina powder, and extracting with 0-005 m 2-amino-2- 

 hydroxymethylpropane-1 :3-diol (tris) buffer pH 7-3 containing 0-01 m 

 magnesium acetate, as described previously (Tissieres, Watson, Schlessinger 

 and Hollingworth, 1959). The mixture of alumina, broken cells and buffer 

 was centrifuged at 6000 x g for 1 5 min, giving a sediment composed of two 

 layers: a lower layer of alumina and an upper brown layer of large cell 

 debris. The supernatant was centrifuged at 100,000 x g, yielding a pellet 

 composed of RNP particles and respiratory granules. The RNP fraction, still 

 containing respiratory granules, was freed of soluble proteins by differential 



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