VIII. PROTEIN SYNTHESIS AND GENE ACTION 365 



and Lipmann (1960) have demonstrated the inhibitory effect of added 

 oxidized glutathione. A strong glutathione requirement was observed in 

 the rabbit reticulocyte system (Bishop and Schweet, 1961a) and shown 

 to be due to the reversible inactivation of an enzyme fraction in the 

 absence of sulfhydiyl compounds (Bishop and Schweet, 1961b). A fully 

 activated enzyme, freed of glutathione, could be maintained in the 

 activated state by the addition of Versene. Under these conditions, 

 transfer occurred in the absence of glutathione. 



In summaiy, there is general agreement on a GTP requirement in the 

 transfer reaction, although in two isolated cases ATP was required in 

 addition to GTP for maximum transfer. Whenever it has been studied, 

 a magnesium ion requirement has been found, although the optimum 

 varied from system to system. It is not known whether magnesium ion is 

 required in the transfer reaction itself, or to maintain integrity of the 

 ribosome. Glutathione was required in most cases; in one instance, the 

 requirement was absolute, and in one case glutathione was inhibitory. 



b. Enzymatic Requirements for Transfer. An enzymatic requirement 

 for the transfer process in the liver system was reported by Hoagland 

 et al. (1958) to be located in the pH 5 supernatant fraction. Hiilsman 

 and Lipmann (1960), however, have shown this to be almost completely 

 replaceable by glutathione and other sulfhydryl compounds. Subse- 

 quently, it appeared that a soluble enzyme fraction was, in fact, required 

 for transfer in the liver system, but that it was present in the microsomes 

 as they were then prepared (Nathans and Lipmann, 1960). By treating 

 their microsome preparation with 0.5% deoxycholate (and thus preparing 

 rihosomes) these authors were able to free their preparations of con- 

 taminating soluble enzyme and show an almost absolute requirement for 

 added enzyme, even in the presence of glutathione. Rabbit reticulocyte 

 ribosomes, when prepared according to conventional methods, without 

 resort to detergent treatment, are not associated with a lipoprotein 

 membrane. These ribosomes did show a stimulation of amino acid trans- 

 fer by added enzyme even in the presence of glutathione (Bishop et al., 

 1960a). However, rabbit reticulocyte ribosomes show an almost absolute 

 enzyme requirement following treatment with deoxycholate (Bishop 

 and Schweet, 1961b; von Ehrenstein and Lipmann, 1961). In the pea 

 seedling system (Webster, 1961) and in the E. coli system (Nathans and 

 Lipmann, 1961), an enzyme requirement has been observed with washed 

 ribosomes, in the absence of detergent treatment. Thus, it would appear 

 that the enzyme requirement is not an artifact of detergent treatment. 

 Other treatments employed to show an enzyme requirement in rat liver 

 microsomes have been a combination of two detergents (deoxycholate 

 and Lubrol W) and a high KCl level (von der Decken and Hultin. 



