216 GEORGE H. HOGEBOOM AND WALTER C. SCHNEIDER 



periphery of the cell, and the other, 2 to 3 ^ in diameter, located at the interior of the 

 cell. The former were considered to correspond to secretory granules and the latter 

 to lipid droplets that accumulated as the result of fasting. These and other studies*"'** 

 make it evident that the status of the Golgi apparatus in liver is unsettled — a situa- 

 tion that is likely to persist unless the material can be identified with certainty in the 

 unfixed liver cell. 



The existence of a Golgi apparatus in cells of the epididymis, however, appears 

 to be more firmly established. Thus, in investigations reported by Dalton and Felix,** 

 examination of unfixed, unstained epididymal cells with phase -contrast illumination 

 revealed a cytoplasmic structure that was as large as the nucleus and in morphological 

 characteristics closely resembled the classical Golgi apparatus visualized in prepa- 

 rations fixed and stained by accepted cytological methods. The structure was re- 

 fractory to vital staining with neutral red and methylene blue, although numer- 

 ous granules, staining with these dyes, surrounded it. When epididymal cells were 

 crushed, it was noted that the Golgi substance maintained its characteristic morph- 

 ology after release from its intracellular environment. Since its morphological 

 integrity was also preserved after the epididymis was homogenized in the apparatus 

 of Potter and Elvehjem,^' it thus became possible to attempt to isolate the material. 

 Isolation of the Golgi bodies was accomplished*^ by layering 1.4 ml. of a 20 or 25% 

 homogenate of rat epididymis in 0.25 M sucrose over a sucrose density gradient and 

 centrifuging for 60 minutes at 35600 r.p.m. (108,000 g) in the Spinco SW 39 horizontal 

 rotor. The density gradient was made by pipetting 1.0-ml. layers of 1.11,0.957,0.636, 

 and 0.335 M sucrose solutions into the centrifuge tube. Each of the sucrose layers 

 contained 0.34 M NaCl in addition. After centrifugation, it was found*^ that a layer 

 of fat had migrated centripetally and formed a cap at the top of the tube sur- 

 mounting a layer containing the soluble proteins of the homogenate. Below the latter, 

 bands of particulate material were seen to occur at the positions corresponding to the 

 junction of the various sucrose layers. The band at the junction of the 0.636 and 

 0.957 M sucrose layers was found to contain most of the Golgi bodies of the original 

 homogenate and, in addition, some submicroscopic particulate material, which was 

 considered to be derived from the Golgi apparatus since it possessed osmication 

 properties characteristic of the Golgi substance. The main mass of submicroscopic 

 particles was found at the bottom of the 0.957 M sucrose layer. A sediment at the 

 bottom of the centrifuge tube contained mitochondria, nuclei, sperm, and un- 

 broken cells. 



III. Biochemical Properties of Isolated Cell Structures 



1. The Nuclear Fraction 



As indicated above, the interpretation of analyses of isolated cytoplasmic 

 components is dependent on the analysis of the whole tissue as well as that 

 of all fractions obtainable from the whole tissue. In the initial step in the 

 centrifugal fractionation of homogenates, essentially all of the cell nuclei, 

 together with residual unbroken cells, connective tissue, and some mito- 



*» G. E. Palade and A. Claude, /. Morphol. 85, 35 (1949). 

 81 G. E. Palade and A. Claude, J. Morphol. 85, 71 (1949). 

 *2 A. J. Dalton and M. D. Felix, Atn. J. Anat. 92, 277 (1953). 



*3 W. C. Schneider, A. J. Dalton, E. L. Kuff, and M. D. Felix, Nature 172, 161 (1953); 

 W. C. Schneider, and E. L. Kuff, Am. J. Anat. 94, 209 (1954). 



