Effects of Cycloheximide on Amino Acid Biosynthesis in Corn Roots

The antibiotic cycloheximide inhibits chain initiation and the extension of nascent protein chains (6). One might therefore expect cycloheximide to have a number of indirect effects depending on the duration of the treatment and the concentrations used. For example, in several in vivo systems cycloheximide has been shown to block protein synthesis completely at concentrations of 5 to 10 ,ug/ml within 15 min (1, 4, 10). Because protein synthesis has a high energy requirement, any compound which interferes with protein synthesis could have an indirect effect on respiration. Fletcher and Beevers (5) have used cycloheximide in studying the regulation of amino acid biosynthesis in suspension cultures of Paul's Scarlet Rose. Their premise was that if protein synthesis were blocked the amino acids should accumulate in the soluble fraction. Where there is no accumulation, endproduct inhibition should be in effect. In their system, the synthesis of a number of amino acids was inhibited. Those which were inhibited most strongly, lysine, threonine, isoleucine, and arginine, were the same ones which Dougall (2) had found to be most sensitive to exogenous additions of amino acids. In two cases, however, the methods do not agree: (a) leucine synthesis was not inhibited at all by cycloheximide, and (b) proline synthesis was inhibited more strongly in the cycloheximide experiments than in the endogenous feeding experiments (2, 3, 5). Similar experiments in our laboratory with corn roots showed that the relationship between cycloheximide inhibition of protein synthesis and end-product inhibition of amino acid biosynthesis was not straightforward. In the experiments illustrated in Tables I and II, the roots were exposed to cycloheximide for a total of 5 hr, a 3-hr pretreatment time and a 2-hr experimental time. Growth conditions and extraction procedures have been described previously (9, 10). Glutamic and aspartic acids and glutamine and asparagine were removed with Dowex-l-acetate and the remaining neutral and basic amino acids were separated on paper with butanol: acetic acid:water (3:1:1/v:v:v). Radioactivity in the individual amino acids was counted as described previously (10) with a Nuclear Chicago scintillation spectrometer. In Table I, we see that the total soluble radioactivity is not affected by the addition of the antibiotic. With concentrations of 0.4 and 1 ,ug/ml of cycloheximide, there is an increase in the recovery of radioactive-soluble neutral and basic amino acids, whereas at a concentration of 5 fg/ml almost normal amounts were recovered. At 0.4 and 1.0 ,ug/ml of inhibitor there is a higher recovery of radioactivity in the following amino acids: arginine-lysine,