loo Cytoplasmic Localization o£ Lipids 



were therefore undertaken to determine what proportion of the total pro- 

 teins and Hpids respectively were tied up in those little packets which we 

 call the submicroscopic particulates, in the mitochondria, and in the struc- 

 tural proteins of the cell, and also what portion might be considered to be 

 free in the cell water. 



At this point I wish to make it clear that we have no assurance at present 

 that the submicroscopic particulates constitute a group uniform in composi- 

 tion. Until all the arts of the biochemist are exhausted in the effort to sepa- 

 rate them into uniform categories we shall suspect that many different kinds 

 of particles may be included in this group. 



The cell emulsions for this study were prepared by the methods previously 

 described. The animals were stunned, quickly bled, and perfused with 0.85 per 

 cent solution of sodium chloride buffered to pH 6.8 with phosphate buffer and 

 cooled to 2° C. Then the livers are removed and homogenized with three times 

 their weight of the same saline, by forcing through gauze and then through 

 bolting silk. The resulting emulsion was centrifuged at 1,900 r.p.m. for 15 

 minutes to remove intact cells and cell debris. Most of the free fat rises to the 

 surface of the centrifuge tube and may be skimmed off, but since the fat 

 globtiles in the suspension become surrounded with a membrane of surface- 

 denatured protein some may be carried down with the mitochondria and 

 cell debris. For this reason livers that contained few visible fat globules were 

 selected for these studies. The supernatant from the first centrifugation con- 

 tains mitochondria, submicroscopic particulates, and various substances in 

 solution. A second centrifugation for 15 minutes at the same speed removed 

 most but not all the mitochondria. The supernatant was next submitted to a 

 15-minute centrifugation at 6,000 r.p.m. A new copious precipitate appeared, 

 which is a mixture of mitochondria and submicroscopic particulates. 



A final centrifugation of the supernatant at 12,000 r.p.m. yielded a clear 

 supernatant and a transparent cherry-red pellet (see also Claude^^) at the 

 bottom of the tube. If the liver contains particulate-glycogen, there will be 

 below the red pellet a transparent colorless layer of glycogen. Three and one- 

 half hours at 12,000 r.p.m. usually suffices to clarify the solution completely, 

 although one must keep in mind that there may be particulates of even smaller 

 size in suspension which could be sedimented by higher rotational speeds. 

 However, at the speed used we are well below the gravitational field at which 

 Wyckoff and his associates recovered their macromolecular substances. 



Equivalent volumes of the original suspension, before and after centrifuga- 

 tion at the speeds indicated, were analyzed for total protein, total fat, and 

 lecithin content as follows". 



The suspension was precipitated by the addition of about three volumes 

 of absolute alcohol. The precipitate, recovered by centrifugation, was trans- 

 ferred to a weighed pyrex extraction thimble and extracted for 48 hours with 

 boiling alcohol, then an additional 24 hours with ether. The extracts were 

 added to the original supernatant from alcohol precipitation, and the whole 



