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/,M cycloheximide instead of water and almost completely in disks aged in the presence of 10 /uM inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 FAM 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 suffioient 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 disaippear 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 inacti-vating system requires protein synthesis. The effect of cycloheximide on uptake and incorporation of L-isoleucine-U-14C into soluble and insoluble proteins of tuber disks was also examined. During induction the rate of uptake inereased 3 to 4-fold, and the rate of incorporation into protein, corrected for change in uptake, increased 25-fold. Cycloheximide inhibited incorporation of isoleucine-14C into *proteins of fresh disks more than 80 %N. 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. Studties of enzyme induction in plant (11, 14) and animal (10, 26, 27) 'tissue's have shown that protein turnover may be intimately associated with the induction process. Changes in the rate of degradation lindependen.t of changes :in rate of synthesis can occur during induction of specific enzymes (15, 26). These studies 'suggest thait 'specific turnover systems exist and that their exilstence 'in the tisstue is regulated by mechanisms simil,ar to .those controlling the synthesis o'f inducible enzymes. Evidence of protein turnover in plants concern's the overall synthesis and degradation of the total bulk of protein 'in tissues under examinaition (5, 6, 13, 29). Little information exists on ithe formation or specificity of individual turnover systems. The induction of phenylalanine ammonia-lyase in disks of ' This work was supported in part bv Grant GB-2626 from the National Science Foundation. 365 tissue cut from potato tubers appears to involve both the synthesis and 'turnover or inactivation of the enzyme (33). Cycloheximide tinder suitable conditions will inhibit either the appearance or the di'sappearance o'f the. enzyme in the tiissue (34). Engelsma (9) has recently described a similar effect of cycloheximide on synthesis and disappearance of a phenylalanine ammonia-lyase in gherkin seedlings. This paper describes the conditionis under which the selective effects of cycloheximide on potato disks are obtained. The results presented stuggest that the inducti'on process involves the syn-thesi's of both lyase protein and the protein components of a system degrading or inactivating-the enzyme (inac,tivating system). Materials and Methods Kennebec potato tubers stored less than 6 month's at 40 were used for sltudies of the cycloheximide inhibition o'f enzyme synthesis. Incorporation stud