in ATP AND RELATED NUCLEOTIDES 45 



with a rat liver homogenate, the highest specific activity in the homogenate pro- 

 tein, was to be found in the microsome fraction (Siekevitz, 1952). Significant 

 incorporation did not occur when either isolated microsomes or mitochondria 

 were incubated with radioactive alanine but when mitochondria and microsomes 

 were incubated together, active incorporation was observed. When mitochondria 

 were incubated with a-ketoglutarate or with succinate and cofactors, a soluble 

 factor was formed which permitted the anaerobic incorporation of labelled alanine 

 into microsome protein. Amino acid incorporation into microsomal protein in the 

 absence of mitochondria was studied by Zamecnik and Keller (1954). Incor- 

 poration proceeded under anaerobic conditions provided that a soluble, non-dialyzable 

 liver fraction, ATP, and an ATP generating system such as ATP and phospho- 

 creatine were added. The formation of labelled microsomal protein thus seemed 

 to involve two main steps. The first step, catalyzed by the soluble cytoplasmic 

 proteins, constituted the carboxyl-activation of the amino acids. The second 

 step involved the condensation of the carboxyl activated amino acids to form 

 polypeptide chains and took place most actively on ribonucleoprotein par- 

 ticulates of the microsomes. With regard to the second step, it was found that 

 after incorporation of labelled amino acids into the cell free liver system, the 

 ribonucleoprotein particles contained a large part of the total labelled protein. 

 The same was found for the incorporation into rat liver proteins in vivo at two to 

 three minutes after the intravenous injection of a labelled amino acid. In addition 

 to the activating enzymes, the microsomes, ATP, and the ATP generating system, 

 GTP was required (Keller and Zamecnik, 1956). The role of GTP has not as yet 

 been clarified. 



The amino acid activating system could be precipitated from the soluble 

 cytoplasmic proteins by the addition of acetic acid to pH 5.2. The precipitated, 

 "pH 5.2 proteins", catalyzed an amino acid dependent exchange of 32p_32p 

 with ATP (Hoagland, 1955). A similar amino acid dependent exchange of 32p_ 

 ^^P has been described in bacterial extracts (De Moss and Novelli, 1955). 



The exchange catalyzed by the liver enzyme was dependent both on the total 

 concentration and number of amino acids present. The microsome fraction of 

 the cell also catalyzed a pyrophosphate exchange reaction but in this case, the 

 exchange was not influenced by amino acids. AMP failed to inhibit the amino acid 

 dependent exchange; and ^"^G-AMP did not itself exchange with ATP. These re- 

 sults suggested that the amino acids were activated as amino acid-AMP com- 

 pounds. This possibility was given further support by the finding that a-amino 

 hydroxamic acids were formed in the presence of high hydroxylamine concen- 

 trations, with concomitant loss of ATP. One mole of pyrophosphate was formed 

 per mole of hydroxamate but no orthophosphate. Since the amino acids did not 

 cause a net splitting of ATP unless hydroxylamine was present, it was proposed 

 that AMP was bound on the enzyme surface and dissociated from the enzyme to 

 only a small extent (Hoagland et al., 1956). The L-amino acid effect on exchange 

 and on hydroxamic acid formation was additive with different amino acids while 

 D-amino acids were inert in this system^ : 



^ See Addendum, Note 2, p. 123. 



Lileralure p. 412 



