Incorporation of radioactive amino acids into proteins of the microsome fraction of guinea-pig liver in a cell-free system.

Although the synthesis of specific soluble proteins has rarely, if ever, been unequivocally demonstrated to occur in cell-free preparations of mammalian cells, the capacity for the uptake of amino acids into peptide linkage in 'non-specific' protein is less readily destroyed by damage to the cell. Thus the incorporation of labelled amino acids into the acid-precipitable protein of tissue homogenates will continue for some time after the destruction of the cell (Winnick, Friedberg & Greenberg, 1948). In liver homogenates, amino acids are incorporated particularly rapidly into the protein of the microsome fraction (Siekevitz, 1952). In this respect the homogenate resembles the intact liver since, in the normal animal given isotope, the initial incorporation is mainly in the microsome fraction (e.g. Hultin, 1950). Zamecnik & Keller (1954) were able to obtain an active cell-free preparation from liver which consisted of only the microsome and soluble cell-sap fractions. In this preparation incorporation of [14C]amino acids into microsome protein would proceed under anaerobic conditions, provided that an adenosine triphosphate-generating system was present. The amino acids incorporated into microsome protein in vitro are undoubtedly linked to protein by peptide linkage since labelled peptides can be isolated by partial hydrolysis of microsome protein (Zamecnik, Keller, Littlefield, Hoagland & Loftfield, 1956). It has not, however, been clearly established whether the incorporation of amino acids into protein in such cell-free systems involves the same mechanism as occurs in the intact cell. The present paper provides some information concerning the difference between the incorporation process in the intact liver cell and in a cell-free system. The technique employed in this study was that of fractionation of the microsome material by successive extraction with solutions of varied ionic strength and pH. By this method a number of subfractions of distinct composition are obtained (Simkin, 1955; Simnkin & Work, 1957). We have previously reported the application of this method to a study of the incorporation of [14C]amino acids into the microsome fraction ofthe liver of the intact guinea pig, and it was established that amino acids were taken up into the proteins of the various subfractions at quite different rates (Sixkin& Work, 1957). We have now applied this method to microsome material isolated after incubation with [14C]_ amino acids in a cell-free system of guinea-pig liver similar to the type described by Zamecnik & Keller (1954). A preliminary report of this work has been made (Simkin, 1957).