42 THE BIOSYNTHESIS OF PROTEINS 



that high sucrose concentrations in the extracting medium protect osmotic- 

 ally sensitive structures which would swell and be disrupted if transferred 

 into dilute salt solutions (Claude, 1946; Hogeboom et al, 1948). Under 

 appropriate conditions it was possible to isolate from liver homogenates 

 beside the nuclei, large particles which were easily identified as the 

 mitochondria by their shape and their staining properties. From the 

 supernatant, a clearly difi"erent particulate fraction was obtained; it was 

 made of smaller particles or vesicles (Porter et ah, 1945) of about 200 m/x 

 in diameter which were called microsomes. From then on, it became a 

 common usage to fractionate tissue homogenates by centrifugation into 

 four fractions, the nuclear, mitochondrial and microsomal fractions, and a 

 supernatant containing the substances which are not sedimentable in one 

 hour in the Spinco centrifuge.* 



The microsomal fraction contained most of the RNA of the homogenate. 

 As histological studies had shown that the bulk of cellular RNA is in the 

 ergastoplasm or cytoplasmic ground substance, it was clear that the 

 microsomal fraction was derived, for a large part at least, from the cyto- 

 plasm. High speed centrifugation of intact liver tissue indeed confirmed 

 that RNA was bound to a sedimentable structure in the cell (Chantrenne, 

 1943; Claude, 1943 ; Brachet and Jeener, 1944; Brenner, 1947). 



Fractionation of cell particulates and the availability of labelled amino 

 acids opened a new approach to the study of the sites of protein formation 

 within the living cell. 



Several laboratories undertook kinetic studies on the incorporation of 

 labelled amino acids into the proteins of fractions isolated by centrifuga- 

 tion. Labelled amino acids were injected into rats ; the animals were killed 

 15-60 min after the injection, the liver was rapidly removed, chilled, 

 homogenized and the suspension was separated into nuclear, mitochondrial, 

 microsomal and supernatant fractions. Labelled amino acids were looked 

 for and determined in the proteins of these fractions. In such experiments, 

 Borsook et al (1950), Hultin (1950), Lee et al. (1951, 1953), Tyner et al. 

 (1953), Khesin (1954) established that all the fractions incorporate amino 

 acids, but that liver microsomes are labelled more intensively than any 

 of the other fractions. Allfrey et al. (1953) obtained similar results with 

 mouse pancreas. 



More striking were later experiments in which the liver was chilled and 

 fractionated a few minutes after the injection of labelled amino acids 

 (Keller et al, 1954; Hultin, 1955 ; Loftfield, 1957). For the first ten minutes 



* It should not be overlooked that these are extremely crude fractions which are 

 not made of pure nuclei, pure mitochondria or pure microsomes. Moreover, con- 

 ditions of fractionation which have been devised for rat liver, for instance, do not 

 necessarily apply to other tissues. (For a discussion of fractionation procedures, see 

 de Duve and Berthet, 1954.) 



