C;()LC;I MIMBRANES 



iry 



Figure 3 illustrates both the method of fractionation and some of the bio- 

 chemical results that we obtained in our first study (23). The homogenates were 

 prepared with a loosely fitting plastic homogcnizer of the Potter-Elvehjem type 

 in a medium consisting of 0.25 m sucrose containing 0.34 m sodium chloride, 

 The high salt concentration was employed because it providetl optimum preserva- 

 tion of the CJolgi structures as judged by phase microscopy. After centrifugation 

 of the gradient tubes, bands of light-scattering material were present at each in- 

 terface between solutions of differing density (fig. 3). Phase microscopic ex- 

 amination of the material in the bands revealed that characteristic Golgi struc- 

 tures were concentrated at the interface between densities of 1.09 and 1.13 grams 

 per centimeter^ (referred to as interface 1.09/1.13). The light band at interface 

 1.05/1.09 appeared to be optically empty under phase and was collected with 

 the supernatant fluid. The band at interface 1.13/1.15 consisted almost entirely 

 of very small particulate material, generally below the resolving power of the 

 phase microscope. Sperm, nuclei, mitochondria and residual whole cells were 

 found in the sediment. 



The results of the biochemical analyses of the various fractions are presented 

 in figure 3 in a manner introduced by de Duve and co-workers (8). Without 



[H] 

 1.04 



1.05 



.09 



13 



1.15 



G 

 Sed. 



Fig. 3. Densitv gradient frac- DPNH- 



ti.mation of rat' epididvmis. Left, TOTAL LIPID ALK. CYTO. C 



,,.,'., N P P ASE RED. 



diagram of a lu.steroid centrifuge 



tube with actual dimensions of i .5 

 X 4-8 cm, volume about 5.2 mi. 

 A homogenate of epididymis (H, 

 see text) was placed above a dis- 

 continuous sucrose gradient freshly 

 prepared by layering solutions of 

 the densities indicated. All solu- 

 tions containctl 0.34 m sodium chlo- 

 ride. Centrifugation at 35,600 rj)m 

 (145,000 g, max.) was carried out 

 in the SW-39 rotor of a Spinco 

 Model K ultracentrifuge for i hour 

 at 0-4°, after which the fluid col- 

 umn was sampled to give the frac- 

 tions indicated to the right of the 

 tube. The results of the biochemical 



analyses are presented in the graph, the left hand column of which indicates the percentage 

 of the total nitrogen of the homogenate that was found in each fraction. The vertical dimen- 

 sions of the bars in the other columns are proportional to these nitrogen values, while the 

 horizontal dimensions indicate the specific activities or C(jncentrations of the hiochemical com- 

 ponents in the fractions relative to those in the whole homogenate. Abbreviations: N, nitrogen; 

 P, phosphorus; Alk. Pase, alkaline phosphatase; RNA, ribonucleic acid; DPNH-cyto.c- red., 

 DPNH -cytochrome c reductase. All data, except those relating to the latter enzyme, were taken 

 from Schneider and Kuff (21,). 



246 24 246 246 



RELATIVE CONCENTRATION 



