gS Cytoplasmic Localization o£ Lipids 



centrifugation of suspensions in saline solutions opened up a new line of 

 approach to the problem of the composition of intracellular structures, and 

 in particular the distribution of lipids in cytoplasm. The possibilities of this 

 method were further extended when Claude" announced the successful re- 

 covery, by high speed centrifugation, of a submicroscopic particle from 

 filtered saline extracts of chick embryo. This fundamental discovery, that 

 protoplasm contained particles of complex composition of a size too small to 

 be seen under the microscope except by the use of the dark field, not only 

 clears the way for the investigation of the chemical nature of these constituents 

 of protoplasm but also reveals the nature of the pitfalls which await the bio- 

 chemist in his attempts to separate the constituent proteins of cells. These 

 particles, both microscopic and submicroscopic, have considerable stability 

 within certain pH ranges and electrolyte content, and the answer to the ques- 

 tion as to whether the extract contains only the mobile protein dissolved in 

 the cell water or, in addition, in a state of suspension, the special proteins 

 of submicroscopic particles, mitochondria, nuclei, and other structural ele- 

 ments, can be achieved only by meticulous study at each stage of differential 

 centrifugation with microscopic and chemical examination of each precipitate 

 obtained. The extracts of liver examined by Plosz^° and Haliburton" un- 

 doubtedly contained both mitochondria and submicroscopic particulates. The 

 extracts of the liver made by Luck" using 5 per cent saline at a pH of 5.0 were 

 probably free from particulates but contained some of the structural proteins 

 and nucleoproteins which are insoluble in 0.85 NaCl at pH 6.8, but are soluble 

 in higher concentrations of salt even at relatively low pH. 



It is unfortunate that Claude, misled by an incorrect estimate in the litera- 

 ture of the size of mitochondria should have identified the submicroscopic 

 particles with mitochondria. Mitochondria maintain a certain uniformity of 

 size and type for a given species and physiological state and do not grade in 

 size down to invisibility. On the other hand, the possibility of fragmentation 

 of mitochondria into submicroscopic elements in saline solution, foreign in 

 composition to the cell water, should not be overlooked. These relations will 

 be fully discussed in papers now in preparation dealing with the composition 

 of the particulates. For the purposes of this article it is sufficient to mention 

 that both mitochondria and the submicroscopic particles have a higher per- 

 centage of fat than that found in the intact cell. The writer's report on the 

 fat content of mitochondria (Bensley") must now be revised, since the dis- 

 covery of the submicroscopic particles and of the particulate nature of intra- 

 cellular glycogen (Lazarow") makes it certain that the original preparations 

 were contaminated to a varying degree with the other two products. Further- 

 more, the phospholipid determinations were far too low, because of the use 

 of the unreliable acetone precipitation method. 



Mitochondria, separated at centrifuge speeds too low to permit the pre- 

 cipitation of either glycogen or submicroscopics, have a narrower range of 

 variability in fat content than previously reported. The total fat amounts to 



