212 GEORGE H. HOGEBOOM AND WALTER C. SCHNEIDER 



e. Isolation of Mitochondria and Microsomes from Liver 



(1) Preliminary Steps. It is often desirable to perfuse the livers in order to elim- 

 inate erythrocytes and other blood elements from the cell fractions. Reasonably 

 complete perfusion can be obtained through the hepatic-portal vein in rats and the 

 inferior vena cava in mice. A needle of appropriate size (22 to 25 gauge), fitted to a 

 three-way stopcock, is inserted into the vein and held in place by a hemostat. The 

 vena cava in the rat or the portal vein in the mouse is severed, and the liver is per- 

 fused first with cold isotonic saline until free of blood and subsequently with cold 

 0.25 or 0.88 M sucrose to remove the saline. Most of the connective tissue can then 

 be eliminated and the organ reduced to a pulp when it is forced, by means of a tissue 

 press, through a stainless steel or plastic disk, perforated by numerous 1-mm. holes. 

 Recent experiments*" have suggested, however, that the latter procedure results in 

 damage to an appreciable number of nuclei and is therefore of questionable value. 

 The whole liver or liver pulp is weighed and homogenized in 9 volumes of 0.25 or 0.88 

 M sucrose. In the authors' experience, the use of more concentrated tissue suspensions 

 has reduced considerably the yield of mitochondria, and it has thus been desirable 

 to prepare "10%" homogenates even when relatively large amounts of liver are frac- 

 tionated. 



The complete fractionation of liver homogenates prepared in 0.25 M sucrose will 

 now be described. If 0.88 M sucrose is used as the medium, the fractionation involves 

 similar steps, but higher centrifugal forces are required.** The entire procedure is 

 carried out at a temperature of 5° or less. 



(2) Removal of Nuclei and Intact Liver Cells. Ten milliliters of the homogenate 

 is pipetted into each of two 30-ml. graduated test tubes and centrifuged at 2000 r.p.m. 

 (700 g) in the International refrigerated centrifuge (horizontal head No. 269). An 

 alternative procedure permitting somewhat more efficient separation of nuclei from 

 mitochondria consists of layering 10 ml. of the homogenate over 10 ml. of 0.34 M 

 sucrose and centrifuging at the same speed. ^"'^^ The supernatant is withdrawn with 

 a capillary pipet, and the residue is resuspended by adding 4.0 ml. of 0.25 M sucrose 

 to each tube and homogenizing for 10 to 15 seconds with a loosely fitting Kel-F pestle. 

 The suspension is recentrifuged for 10 minutes at 2600 r.p.m. in the same head. The 

 supernatant is removed and combined with the first supernatant to form the cyto- 

 plasmic extract. The residue or "nuclear fraction" is made up to a known volume 

 by adding 0.25 M sucrose and rehomogenizing. It contains all the nuclei present in 

 the homogenate, the residual intact liver cells, erythrocytes, and, as determined by 

 actual count, about 10% of the original number of free mitochondria.*' Although 

 obviously inhomogeneous, this fraction is nevertheless useful in studies of the bio- 

 chemical properties of the nucleus, since a quantitative recovery of nuclei is obtained 

 and the contribution of the other cellular components present can be estimated from 

 additional measurements. The usefulness of the fraction is still further increased if 

 the homogenate is made in 0.25 M sucrose containing 0.0018 M calcium chloride and 

 the nuclei isolated by a modified procedure.'* Under these conditions, 70 to 90% of 

 the nuclei can be recovered in preparations containing less than 0.5% of the free 

 mitochondria of the homogenate and less than 1% of the cells of the original whole 

 tissue. Unfortunately, however, the calcium-containing sucrose solution is not a 

 suitable medium for the separation of mitochondria and microsomes. 



(3) Isolation of Mitochondria. The cytoplasmic extract remaining after removal 



60 G. H. Hogeboom, and W. C. Schneider, J. Biol. Chem. 197, 611 (1952). 

 6' G. H. Hogeboom and W. C. Schneider, J. Biol. Chem. 204, 233 (1953). 



