214 GEORGE H. HOGEBOOM AND WALTER C. SCHNEDIER 



isolation of mitochondria are combined, and 35 ml. is centrifuged for 60 minutes at 

 25,980 r.p.m. (57,000 g) in the type D rotor of the Spinco Model E ultracentrifuge, 

 (Specialized Instruments Corp., Belmont, Cal.). The pellet, which is a transparent 

 amber to red color, depending on whether the livers have been perfused or not, is 

 dispersed by homogenization in 12.5 ml. of 0.25 M sucrose, and the suspension is 

 recentrifuged for 30 minutes at 50,740 r.p.m. (148,0(K) g) in the type A preparative 

 rotor. The washed sediment, consisting of microsomes together with about 7% of the 

 original number of mitochondria, is made up to a definite volume by rehomogeniza- 

 tion in 0.25 M sucrose, and the supernatant is combined with that of the first cen- 

 trifugation to form the final supernatant or soluble fraction of the cytoplasm. 



If the Model L Spinco centrifuge is used in the isolation of microsomes, rotors 

 No. 30 and 40 can be emploj'ed, or the entire procedure can be carried out with rotor 

 No. 40. Microsomes can also be sedimented at maximum speed in the multispeed 

 attachment of the International centrifuge. In this case, the time of centrifugation 

 must be prolonged (2 hours or more), and the tubes should have a small diameter 

 i}4 to ^ inch) to shorten the path of sedimentation. It may also be noted that the 

 initial sedimentation of microsomes results in a firmly packed pellet containing little 

 of the soluble fraction. It is therefore probably not essential to wash the particles by 

 resuspension and resedimentation. 



/. Isolation of Pigment Granules 



Melanin granules have been isolated by differential centrifugation from amphiuma 

 liver," from the ciliary processes of beef eyes,** from frog eggs,^* and from rat and 

 mouse melanomas.*^ •'° The granules have also been obtained recently from mela- 

 nomas" by means of the chromatography of tissue suspensions on Celite columns. 

 Conclusions relating to the biochemical properties of melanin granules must be 

 weighed in terms of possible contamination of the preparations by mitochondria, 

 which are of a similar size. In the case of melanin granules isolated from amphiuma 

 liver, electron micrographs have conclusively demonstrated the absence of mito- 

 chondria or other particulate material." Effective separation of the mitochondria 

 and melanin granules of frog eggs was also obtained by Recknagle,*' who observed 

 striking differences in the cytochrome oxidase activity of the two particulates. On 

 the other hand, the question of mitochondrial contamination of the melanin granules 

 isolated from the other tissues has not been adequately resolved.^' ''^ Although elec- 

 tron micrographs of melanin granules isolated by chromotography from pigmented 

 tumors do not disclose the presence of many extraneous elements,'^ the view that the 

 granules carry most of the enzyme systems associated with the mitochondria of other 

 cell types (in contrast to Recknagel's finding^') is open to some question. Thus, the 

 specific activities of the mitochondrial enzymes in the pigmented tumors are not 



" A. Claude, Harvey Lectures 43, 121 (1947-48). 



68 H. Herrmann and M. B. Boss, J. Cellular Comp. Physiol. 26, 131 (1945). 



6' R. O. Recknagel, J. Cellular Comp. Physiol. 35, 111 (1950). 



7" M. W. Woods, H. G. duBuy, D. Burk, and M. L. Hesselbach, J. Natl. Cancer 



Inst. 9, 311 (1949); H. G. duBuy, M. W. Woods, D. Burk, and M. D. Lackey, J. 



Natl. Cancer Inst. 9, 324 (1949). 

 71V. Riley, G. Hobby, and D. Burk, in "Pigment Cell Growth" (Gordon, ed.), 



p. 231. Academic Press, New York, 1953. 

 " A. J. Dalton and M. D. Felix, in "Pigment Cell Growth," (Gordon, ed.), p. 267. 



Academic Press, New York, 1953. 



