222 



TORE HULTIN et ol . 



One might therefore assume that after the break-down of the gross 

 structural organization of the cells the activity of the particles rapidly 

 reaches a uniform level, independently of the previous functional state. 

 If this were always the case, it would seem doubtful whether the effects 

 of rate-regulating factors would ever become accessible to experimental 

 study by cell-free systems. 



In the following some experimental evidence will be discussed, indi- 

 cating that under certain conditions the actual rate of amino acid incorpora- 

 tion in vitro is determined not only by the concentration of potentially active 

 RNP-particles. The incorporation activity may also be influenced by other 

 factors, including some which depend on physiological (or pathological) 

 conditions in the living tissues before the preparation of the homogenates. 



Microsomes and RNP-particles in incorporation systems 



According to the picture obtained by electron microscopy RNP- 

 particles may occur in the cytoplasm either in a free state, or associated 

 with the membranes of the endoplasmic reticulum [23, 27, 31]. Especially 

 in the cells of highly differentiated animal tissues which have become 

 specialized for protein secretion or protein accumulation, the majority of 

 RNP-particles may appear in a membrane-bound form. It has been 

 suggested that in such cells the membranes are directly or indirectly con- 

 cerned with the anabolic functions of the particles [11, 29]. 



In rat liver a high proportion of the RNP-particles are firmly attached 

 to the endoplasmic membranes. Membrane fragments with adhering 

 particles can be isolated from liver homogenates by differential centri- 

 fugation, accumulating in the microsome fraction. Rat liver microsomes, 

 as is well known, has become a classical material in the study of protein 

 metabolism by cell-free systems [28, 33]. 



It has recently been observed that the membrane components of micro- 

 somes are not necessarily required for the function of the particles in 

 amino acid incorporation [4, 17, 26]. Liver systems containing purified 

 enzyme preparations in combination with isolated RNP-particles have, for 

 instance, the same ability as systems with whole microsomes to incorporate 

 labelled amino acids into interior sites of protein molecules [22]. Neverthe- 

 less, there are certain potential differences between incorporation systems 

 with whole liver microsomes and such ones with isolated RNP-particles. 



This fact is illustrated by the experiment shown in Fig. i. The particles 

 used in this experiment were prepared by treating rat liver microsomes with 

 a mixture of detergents (deoxycholate and Lubrol W) in a solution of 

 fairly high ionic strength [26]. The detached particles were then separated 

 from the digestion mixture by centrifugation through a density gradient 

 [6, 26], The experiment shows a characteristic difference between the 



