PETERMANN ET AL. 



71 



We prepared such fractions by differential centrifugation at 78,000g in 0.79 

 M sucrose. The whole microsome fraction was centrifuged for 5 hours. The 

 large-microsome fraction was centrifuged for only 50 minutes, in order to sedi- 

 ment only large fragments of endoplasmic reticulum. From this supernatant 

 the small-microsome fraction was sedimented for 5 hours. Each fraction was 

 washed by resedimentation in 0.79 M sucrose containing 5 X 10~ 4 M K2HPO4 

 and KH2PO4, and 5 X 10" 4 M MgCl 2 to preserve the RNP [8], and finally was 

 suspended in this same buffer without the sucrose, or in water, to a RNP con- 

 centration of about 5 mg/ml. For ultracentrifugal analysis each sample was 

 diluted with 0.2 volume of a fivefold concentrated buffer. Two sets of analyses 

 carried out in 0.1 M KHCOs are shown in figure 1. When a mere trace of 

 magnesium is present, as in the upper row, the RNP from the large micro- 

 somes is chiefly B, with only small amounts of C and E. The small microsomes, 

 however, do show considerable amounts of C and E. With 0.0024 M magne- 

 sium, in the bottom row, all preparations show less C and E, but the same rela- 

 tionship is retained — there is more C and E in the small microsomes than in 

 the large ones. The whole microsome fraction falls in between, as one would 

 expect. Varying the pH or the concentrations of monobasic and dibasic ions 

 gives an assortment of patterns, but there is always more C and E in the ex- 

 tracts of the small microsomes. 



Whether any of the RNP in the small-microsome fraction is really "free" in 

 the liver cell, however, is difficult to determine. In extracts made in water or 

 the dilute phosphate-magnesium buffer, at pH 7, no RNP boundaries are seen 

 in the ultracentrifuge. They appear when the pH is raised to 8.0 or the ionic 

 strength is increased. Since the state of the particles is so dependent on their 

 ionic environment, they will have to be studied in buffers which approximate 



FRACTION 

 WHOLE LARGE 



BCE 



BCE 



SMALL 



BC E 



♦ i ♦ 



k^/_jLV ^J^J 



SOLVENT 



pH KHCO3 Phos. MgCI 2 

 M M M 



8.0 O.IO 



tr. 



tr. 



H 



■i 



^ 



8.0 O.IO 0.0008 0.0024 



Fig. 1. Ultracentrifugal patterns of RNP extracted from microsomal fractions. The 

 pictures were taken after 14 minutes at 37,020 rpm. 



