366 MAHLON B. HOAGLAND 



The first success with cell-free mammalian systems was reported about 

 7 years ago, and although these systems were less active than the in vivo 

 incorporating systems, they did appear, qualitatively, to be carrying out 

 the identical process. 30 Thus the energy-dependent nature of the proc- 

 ess, and the initial incorporation into microsomal protein was confirmed. 

 Siekevitz and Zamecnik, 89 Siekevitz, 90 and Peterson and Greenberg 91 ob- 

 tained some C 14 -amino acid incorporation into protein which was dependent 

 on addition of actively phosphorylating mitochondria. In the absence of 

 mitochondria, glycolysis was also found to serve as an equally effective 

 energy source. 92, 93 Success with the latter cell-free system seemed chiefly 

 to derive from the use of gentle homogenization of the tissues 94 and the use 

 of Mg ++ in the homogenizing medium. 93 The latter empirical observation is 

 of interest in the fight of recent advances in our knowledge of the require- 

 ments for maintaining the structural integrity of ribonucleoprotein particles 

 which we have discussed above. 



In agreement with the in vivo incorporation studies, it was found, as we 

 have said, that the protein of the ribosomes was the initial site of incorpora- 

 tion of the C 14 -amino acid. The rate of incorporation was considerably slower 

 than that observed in vivo; the initial rate of incorporation of leucine into the 

 protein of rat liver, for example, being 0.4 % uptake per hour, as compared 

 to a rate of about 2% in vivo. 9b Calculations based on the rate and total 

 amount of labeling of microsomal protein in vivo indicated that only a small 

 fraction (~1 %) of the total protein of the ribonucleoprotein particles was 

 actually participating in the synthetic process. 30 



Littlefield and Keller 35 were successful in obtaining ribosomes from as- 

 cites tumor cells by the use of 0.5 M NaCl to separate them from associ- 

 ated protein. These ribosomes, when supplemented with the required solu- 

 ble enzymes and nucleotides, incorporated amino acids into their protein 

 moiety at an appreciable rate, thus indicating the ability of these particles 

 to synthesize protein in the absence of associated lipoprotein material. 

 Similar observations were made by Simkin and Work. 48 



In company with the development of our understanding of the role of 

 the ribosomes in the mammalian incorporation system, elucidation of the 

 role of the soluble components was also advanced. Thus, as we see from 

 Fig. 1, the participation of GTP in the incorporation reaction, amino acid 



89 P. Siekevitz and P. C. Zamecnik, Federation Proc. 10, 246 (1951). 



90 P. Siekevitz, J. Biol. Chem. 195, 549 (1952). 



91 E. A. Peterson and D. M. Greenberg, J. Biol. Chem. 194, 359 (1952). 



92 P. C. Zamecnik, Federation Proc. 12, 976 (1953). 



93 P. C. Zamecnik, and E. B. Keller, J. Biol. Chem. 209, 337 (1954). 



94 N. L. R. Bucher, J. Am. Chem. Soc. 75, 498 (1953). 



95 E. B. Keller, P. C. Zamecnik, and R. B. Loftfield, J. Histochem. and Cytochem. 2, 

 378 (1954). 



