GARFINKEL 23 



70,000^ for 25 minutes. In addition to the bulk microsomal pellet, the light 

 fraction is obtained as a suspension at the bottom of the centrifuge tube. In 

 order to permit spectroscopic study, both fractions are washed with Ringer's 

 solution, which removes the hemoglobin. It also washes out about 80 per cent 

 of the RNA (although the ribonucleoprotein granules do not disintegrate and 

 may still be isolated [3]), so that there are no accurate values for RNA con- 

 centration. It is found, however, that the bulk fraction contains appreciable non- 

 cytochrome heme, somewhat more than the cytochrome heme, whereas the light 

 fraction contains very little. A detailed description will be published elsewhere. 



We have seen that the microsomes that centrifuge down last, the light frac- 

 tion, are rich in cytochrome b 5 . Palade and Siekevitz [1] found that they were 

 likely to be smooth-surfaced. This finding suggests a correlation between 

 smooth-surfaced appearance and the presence of cytochrome b 5 . So does the 

 fact that studies [4] in various tissues indicate that much of the cytochrome 

 is present only where the electron microscope shows smooth-surfaced endo- 

 plasmic reticulum. 



Drs. Ian R. Gibbons and T. F. Anderson kindly took photographs of one 

 of these microsomal preparations with the electron microscope. Figure 1 shows 

 views of the light and bulk fractions. It is seen that there is much more ma- 

 terial of smooth-surfaced origin (free of the ribonucleoprotein granules) in the 

 light fraction than in the bulk fraction. Counts of the numbers of smooth- and 

 rough-surfaced microsomes, using unfixed preparations to avoid any enrich- 

 ment of one microsomal type in the process of fixation, indicated that the per- 

 centage of smooth-surfaced microsomes was proportional to the cytochrome con- 

 centration (a light fraction which was twice as rich in cytochrome as the bulk 

 fraction contained twice as many smooth-surfaced microsomes). Apparently 

 cytochrome b 5 is localized in the smooth-surfaced microsomes and the rough- 

 surfaced ones contain little of it. This observation is also in agreement with 

 the fact that predominantly rough-surfaced microsomes can be prepared (from 

 pancreas, for instance [4, 5]), and they contain little or no cytochrome. It 

 would be desirable to confirm this by preparing pure smooth- or rough-surfaced 

 microsomes and finding their cytochrome content (since the preparative method 

 used enriches the smooth-surfaced microsomes only twofold, the resulting con- 

 clusions regarding their properties should not be considered final). 



We are not limited to the electron microscope, but can also study the micro- 

 somes by biochemical methods. The procedure used here is digestion with 

 pancreatin, a mixture of digestive enzymes from the pancreas. After digestion, 

 which is not complete, what is left of the microsomes is centrifuged down and 

 examined. 



Initially, two suspensions of light and bulk microsomes have equal con- 

 centrations of cytochrome. Assuming that the smooth-surfaced microsomes 

 contain nearly all the cytochrome, then their concentration is equal in the two 

 suspensions. The protein concentrations are shown in table 1. The digestion 



