Intermediate Reactions in Protein Synthesis^"'^ 



MARY L. STEPHENSON, LISELOTTE I. HECHT, 

 JOHN W. LITTLEFIELD, ROBERT B. LOFTFIELD 



AND PAUL C. ZAMECNIK 



Massachusetts General Hospital 

 Boston, Massachusetts 



SINCE ACCEPTING THE PLEASANT INVITATION to participate in a symposium on 

 subcellular particles we wondered what to present in the time allotted to 

 us. Recently several reviews have covered the field of protein synthesis and the 

 critical role of the ribonucleoprotein particle as the initial site of incorporation of 

 amino acid into protein has been well established (32, 8, 20, 10). For these 

 reasons we decided that a presentation of our current studies dealing primarily 

 with the non-particulate enzymatic reactions preceding peptide bond formation 

 would be timely and not out of place. 



The present discussion of the problem is not on the whole a historical ap- 

 proach, but rather is designed to point out the essential factors and possible 

 steps involved. Most of the experiments described herein have been carried out 

 with mammalian cell systems and deal predominantly with the mechanism of the 

 incorporation of amino acids into uncharacterized proteins rather than with the 

 net synthesis of one or more specific proteins. 



The initial site of the incorporation of amino acid into protein has been shown 

 by several independent investigators to be in the microsome fraction (5, 25, 28, 

 30, I, 36). In a representative experiment the amino acid was administered to 

 the whole animal, after which, at various time intervals, the livers or other organs 

 were removed, homogenized, and separated into fractions by dififerential cen- 

 trifugation. The proteins in the microsomal fraction were the most rapidly labeled. 



As is well known, the microsomal fraction refers to the pellet obtained after 

 centrifugation of the 15,000 g supernatant of a homogenate for one or two hours 

 at 15,000 g. The bulk of the cytoplasmic ribonucleic acid (RNA) is concentrated 

 in this fraction (9, 7, 2). Two fractions can be obtained after treatment of micro- 

 somes with sodium deoxycholate (37). One is an insoluble fraction consisting of 

 dense spherical particles with a diameter of about 150-250 A, which contain 



1 This investigation was supported by grants-in-aid from the U. S. Attjinic Energy Com- 

 mission, the Public Health Service and the American Cancer Society. 



- Publication no. 926 of the Harvard Cancer Commission. 



3 From the John Collins Warren Laboratories of the Collis P. Huntington Memorial Hospital 

 of Harvard University at the Massachusetts General Hospital. 



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