Sequential Induction of Phenylalanine Ammonia-lyase and a Lyase-inactivating System in Potato Tuber Disks

Abstract

The light induced synthesis of phenylalanine ammonia-lyase in disks cut from potato tubers is very sensitive to cycloheximide. Synthesis is inhibited 50% in disks cultured on 5 mum cycloheximide instead of water and almost completely in disks aged in the presence of 10 mum inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 mum cycloheximide do not synthesize enzyme during the subsequent 24 hours.Normally a maximal enzyme activity develops in disks about 24 hours after being cut from the tuber. Thereafter enzyme activity declines. The disappearance of enzyme is not affected by concentrations of cycloheximide sufficient to inhibit the synthesis of enzyme initially. No disappearance of enzyme is noted during the initial phase of induction if enzyme synthesis is inhibited by cycloheximide. However, enzyme does disappear from the tissue if more than half the maximal enzyme content is allowed to form before synthesis is inhibited. If cycloheximide at a concentration 10-fold that needed to inhibit synthesis completely is added to disks after they have attained a maximal enzyme level, then subsequent loss of enzyme activity from the tissue is prevented. The initial stability of the enzyme in the absence of further synthesis and the inhibition of enzyme disappearance by high concentrations of cycloheximide suggest A) that early phases of induction involve synthesis of enzyme protein in the absence of turnover, B) that a system capable of degrading or inactivating the lyase subsequently forms in the tissue, and C) that the formation of the degrading or inactivating system requires protein synthesis.The effect of cycloheximide on uptake and incorporation of l-isoleucine-U-(14)C into soluble and insoluble proteins of tuber disks was also examined. During induction the rate of uptake increased 3 to 4-fold, and the rate of incorporation into protein, corrected for change in uptake, increased 25-fold. Cycloheximide inhibited incorporation of isoleucine-(14)C into proteins of fresh disks more than 80%. It did not prevent activation of general protein synthesis during induction and inhibited incorporation in induced disks only 20%. At all times incorporation of amino acid into the soluble, lyase-rich, protein fraction was more sensitive to cycloheximide than the insoluble fraction.