72 MICROSOMAL PARTICLES 



AFTER 3 WASHES AFTER BARIUM PRECIPITATION 



Fig. 2. Electrophoretic patterns of purified RNP in 0.10 M KHC0 3 , pH 8.2, contain- 

 ing 0.001 M MgCl 2 . Pictures taken after 60 minutes at 5.2 volts/cm. The left-hand pat- 

 terns show RNP that had been washed three times. The right-hand patterns show the 

 same material after removal of the ferritin. (See text.) 



the ionic composition of the liver cell before any conclusions can be drawn. 3 

 Most of our recent work has been carried out on purified RNP [9]. The 

 microsomes are disrupted with deoxycholate, and the RNP is purified by alter- 

 nate cycles of high- and low-speed centrifugation. Recently we have made some 

 improvements in the procedure. The livers do not have to be perfused. The 

 addition of penicillin (100 units/ml) to all the solutions increases the stability. 4 

 Instead of washing with calf-liver dialysate we now use the potassium phos- 

 phate-magnesium chloride buffer; its pW is about 7. The washed RNP still 

 shows about 5 per cent of ferritin on electrophoretic analysis (fig. 2). This can 

 be removed by precipitating the RNP with 0.005 M barium acetate and redis- 

 solving it by dialysis against 5 X 10" 4 M K2HPO4, KH 2 P0 4 , and MgS0 4 (fig. 2) . 

 We have carried out extensive studies of the stability of purified RNP. Like 

 the crude microsomal extracts, the purified RNP is very sensitive to pH, ionic 

 strength, and dibasic and monobasic cations. Figure 3 shows the effect of mag- 

 nesium in the presence of 0.1 M NaHCOs. Fresh RNP looks like the third 



3 Recently we have prepared large- and small-microsome fractions by sedimentation in 

 0.20 M sucrose, and resuspended them in water at pH 7.1 without washing. On ultracen- 

 trifugal analysis sizable C and E boundaries were observed in the small-microsome frac- 

 tion, although none were apparent in the large-microsome fraction. 



4 The purification of the RNP requires about 3 days; although the preparation is kept 

 cold, unless penicillin or sterile technique is used the RNP has an odor like that of spoiled 

 meat. This may be due to cadaverine produced from N-terminal lysine by bacterial lysine 

 decarboxylase; after treatment of the RNP with dinitrofluorobenzene and acid hydrolysis, 

 the only DNP amino acid detectable is lysine. 



