EFFECTS ON AMINO ACID AND PROTEIN METABOLISM 155 



Effects on Protein Synthesis 



The intracellular synthesis of protein requires the simultaneous opera- 

 tion of many metabolic pathways and thus is susceptible to inhibition on a 

 variety of reactions. Some of the processes involved in protein synthesis 

 are: (1) the active uptake or accumulation of exogenous amino acids, (2) 

 the production of high-energy substances such as ATP from the oxidative 

 reactions of the cycle (except in anaerobes), (3) the formation of amino acid 

 precursors, again mainly by the operation of the cycle, and (4) all the com- 

 plex reactions for the activation and assemblage of the amino acids into 

 proteins. There are thus a multitude of possible sites for malonate action 

 but, at reasonable concentrations, the most important mechanism must 

 be a cycle block leading to both depletion of energy supplies and decrease 

 in amino acid precursors. There is no evidence that malonate can interfere 

 significantly either with the proteases or peptidases involved in the break- 

 down of proteins to amino acids or with the terminal assembling reactions 

 for the formation of protein. 



The effects of malonate on the uptake and accumulation of amino acids 

 by cells have been studied in three types of tissue. Excised diaphragm main- 

 tains the same tissue/medium ratio for glycine as in the whole animal, and 

 the marked effects of 2,4-dinitrophenol indicate that glycine is concentrated 

 actively (Christensen and Streicher, 1949). Malonate, however, at concen- 

 trations of 3-55 TaM does not uniformly alter the tissue/medium ratio. It 

 is possible that malonate does not penetrate adequately, because muscle 

 is often rather impermeable to anions. The situation is different in Ehrlich 

 mouse ascites carcinoma cells. Glycine is accumulated so that tissue/medium 

 ratios are often 10-15. In two experiments, malonate at 37 mM decreased 

 this ratio from 13.0 to 5.1 and at 40 mM from 13.9 to 4.0 (Christensen and 

 Riggs, 1952). This occurred despite the fact that malonate increased the 

 synthesis of glycine. In cell suspensions of Gardner lymphosarcoma the 

 uptake of labeled glycine is inhibited 73% and the uptake of alanine 56% 

 by 10 mM malonate (Kit and Greenberg, 1951). These studies demonstrate 

 that malonate can interfere with protein s>Tithesis, at least in some cells, 

 by inhibiting the initial process of amino acid uptake. 



The synthesis of protein is usually strongly inhibited by malonate, but 

 no analyses of the block have been made and the mechanisms are un- 

 known (see accompanying tabulation). The formation of adaptive enzymes 

 has often been taken as indicative of the synthesis of general cell proteins, 

 but this is not necessarily so, as pointed out by Mandelstam (1961). In E. 

 coli any substance acting as a substrate and source of energy represses en- 

 zyme synthesis, whereas inhibitors, such as malonate and 2,4-dinitrophenol, 

 counteract such effects and stimulate the synthesis. Furthermore, under 

 conditions in which /5-galactosidase synthesis is inhibited, the incorporation 

 of leucine-C^* into cell protein is not affected. The lack of inhibition in 



